WO2022212574A1 - Compositions and methods for simultaneous genetic analysis of multiple libraries - Google Patents

Compositions and methods for simultaneous genetic analysis of multiple libraries Download PDF

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Publication number
WO2022212574A1
WO2022212574A1 PCT/US2022/022640 US2022022640W WO2022212574A1 WO 2022212574 A1 WO2022212574 A1 WO 2022212574A1 US 2022022640 W US2022022640 W US 2022022640W WO 2022212574 A1 WO2022212574 A1 WO 2022212574A1
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criterion
adaptor
disclosure
reads
kits
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PCT/US2022/022640
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English (en)
French (fr)
Inventor
Jiannan GUO
Carolyn Hutcheon
Mark Li
Lee Lim
Ira PEKKER
Chen-Hsun TSAI
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Resolution Bioscience Inc
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Resolution Bioscience Inc
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Priority to CA3214221A priority Critical patent/CA3214221A1/en
Priority to EP22782137.8A priority patent/EP4314340B1/en
Priority to BR112023020082A priority patent/BR112023020082A2/pt
Priority to KR1020237037356A priority patent/KR20240004397A/ko
Priority to IL307355A priority patent/IL307355A/en
Priority to JP2023560829A priority patent/JP2024512156A/ja
Priority to US18/553,503 priority patent/US20240191293A1/en
Priority to AU2022252299A priority patent/AU2022252299A1/en
Publication of WO2022212574A1 publication Critical patent/WO2022212574A1/en
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    • C12Q1/6844—Nucleic acid amplification reactions
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
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Definitions

  • the present invention relates to compositions and methods for genetic analysis.
  • NGS Next generation sequencing
  • Current applications of NGS approaches include targeted gene panels and whole genome sequencing, with most applications choosing to utilize only one of the two approaches, or proceeding with both approaches in parallel.
  • the ability to combine libraries specific to different NGS approaches in a single sequencing analysis will consolidate the benefits of each approach, thereby improving data quality, cost, and turnaround time. Therefore, compositions and methods for simultaneous genetic analysis of multiple libraries are highly desired.
  • the kit comprises one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the one or more capture probe modules have passed a probe Quality Control (QC) process.
  • the kit further comprises a set of adaptors, wherein each adaptor comprises an adaptor module.
  • the set of adaptors have passed an adaptor QC process.
  • the kit comprises a set of adaptors, wherein each adaptor comprises an adaptor module, wherein the set of adaptors have passed an adaptor QC process.
  • the kit further comprises one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample.
  • the adaptor QC process comprises a test for adaptor ligation, wherein the test for adaptor ligation comprises (a) ligating the set of adaptors to a pre-determined amount of end-repaired DNA fragments to generate a library of adaptor-tagged DNA fragments (LIBS); and (b) amplifying the LIBS to generate a Library Post Amplification (LPA); wherein the set of adaptors is considered to have passed the test for adaptor ligation when the concentration of the LPA is higher than a pre-determined concentration.
  • LIBS library of adaptor-tagged DNA fragments
  • LPA Library Post Amplification
  • the adaptor QC process comprises a test for adaptor distribution comprising (a) ligating the set of adaptors to a pre-determined amount of end-repaired DNA fragments to generate a library of adaptor-tagged DNA fragments (LIBS); (b) amplifying the LIBS using a primer pair comprising at least one primer comprising an index sequence to generate a Library Post Index Amplification (LPIA); and (c) performing a quantitative genetic analysis on the LPIA, wherein the set of adaptors is considered to have passed the test for adaptor distribution when one or more pre-determined acceptance criteria for the quantitative genetic analysis has been met.
  • LIBS library of adaptor-tagged DNA fragments
  • LPIA Library Post Index Amplification
  • the pre-determined acceptance criteria for the adaptor QC process comprise one or more of: (a) Barcode Crosstalk is present in no more than 0.05%-5% of reads; (b) unknown adaptors are present in no more than 1%-
  • the set of adaptors is considered to have passed the adaptor QC process when it has passed the test for adaptor ligation and/or the test for adaptor distribution. In some embodiments of the kits of the disclosure, the set of adaptors is considered to have passed the adaptor QC process when it has passed the test for adaptor ligation. In some embodiments of the kits of the disclosure, the set of adaptors is considered to have passed the adaptor QC process when it has passed the test for adaptor distribution. In some embodiments of the kits of the disclosure, the set of adaptors is considered to have passed the adaptor QC process when it has passed the test for adaptor ligation and the test for adaptor distribution.
  • the probe QC process comprises a test for capture probe modules comprising: (a) ligating a set of adaptors to a DNA sample comprising end-repaired DNA fragments to generate a library of adaptor-tagged DNA fragments (LIBS); (b) amplifying the LIBS to generate a Library Post Amplification (LPA); (c) splitting or diluting the LPA to generate a Target Capture LPA (TC LPA) and a Whole- Genome LPA (WG LPA); (d) amplifying the WG LPA to generate a Whole-Genome Library Amplified (WGLA); (e) hybridizing the one or more capture probe modules to be tested to the TC LPA to form adaptor-tagged DNA fragment-capture probe module complexes; (f) isolating the adaptor-tagged DNA fragment-capture probe module complexes to form isolated adaptor- tagged DNA fragment-capture probe module complexes; (g) enzymatically processing the isolated adaptor
  • the pre-determined acceptance criteria for the probe QC process comprise one or more of: (a) at least 60%-99.9% of capture probes have at least 1 total reads; (b) at least 60%-99.9% of capture probes have at least 10 to at least 200 on-target total reads; and (c) at least 60%-99.9% of expected SNPs within the DNA sample are detected.
  • the kit comprises a first primer pair comprising a first F primer and a first R primer, wherein each adaptor module comprises an amplification region; wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region; wherein the first R primer comprises a tail sequence binding region and a sequencing primer binding region.
  • the kit comprises a second primer pair comprising a second F primer and a second R primer, wherein each adaptor module comprises an amplification region; wherein each of the second F primer and the second R primer comprises an amplification region binding region and a sequencing primer binding region.
  • the tail sequence of each capture probe module comprises a Library Tag.
  • the kit comprises (a) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; (b) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the tail sequence of each capture probe module comprises a Library Tag; (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region; wherein the first R primer comprises a Library Tag binding region and a sequencing primer binding region; (d) a second primer pair comprising a second F primer and a second R primer, wherein each of the second F primer and the second R primer comprises an amplification region binding region and a sequencing primer binding region, wherein none of the primers of the second primer pair bind to the Library Tag.
  • the Library Tag comprises a nucleic acid sequence or an amino acid sequence.
  • the first primer pair is used to generate a first modified library and the second primer pair is used to generate a second modified library, wherein the first modified library and the second modified library are configured to be combined into a Sequence-Ready Library (SRL).
  • SRL Sequence-Ready Library
  • both the first modified library and the second modified library are generated from the test sample.
  • the first modified library or the second modified library comprises a Library Tag, wherein the Library Tag is configured to distinguish the first modified library from the second modified library.
  • each library fragment of the first modified library is an adaptor-tagged DNA fragment comprising an adaptor, a capture probe module, and at least a portion of a DNA sequence of the test sample; and each library fragment of the second modified library is an adaptor-tagged DNA fragment comprising an adaptor and at least a portion of a DNA sequence of the test sample, wherein none of the adaptor-tagged DNA fragments of the second modified library comprises a capture probe module.
  • the kit comprises one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample.
  • the kit comprises one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the one or more capture probe modules have passed a probe Quality Control (QC) process.
  • the kit comprises a set of adaptors, wherein each adaptor comprises an adaptor module.
  • the kit comprises a set of adaptors, wherein each adaptor comprises an adaptor module, wherein the set of adaptors have passed an adaptor Quality Control (QC) process.
  • QC adaptor Quality Control
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample; and (b) a set of adaptors, wherein each adaptor comprises an adaptor module.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample; and (b) a set of adaptors, wherein each adaptor comprises an adaptor module, wherein the set of adaptors have passed an adaptor Quality Control (QC) process.
  • QC adaptor Quality Control
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the one or more capture probe modules have passed a probe Quality Control (QC) process; and (b) a set of adaptors, wherein each adaptor comprises an adaptor module.
  • QC probe Quality Control
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the one or more capture probe modules have passed a probe Quality Control (QC) process; and (b) a set of adaptors, wherein each adaptor comprises an adaptor module, wherein the set of adaptors have passed an adaptor Quality Control (QC) process.
  • QC probe Quality Control
  • QC adaptor Quality Control
  • the adaptor QC process comprises a test for adaptor ligation, wherein the test for adaptor ligation comprises (a) ligating the set of adaptors to a pre-determined amount of end-repaired DNA fragments to generate a library of adaptor-tagged DNA fragments (LIBS); and (b) amplifying the LIBS to generate a Library Post Amplification (LPA); wherein the set of adaptors is considered to have passed the test for adaptor ligation when the concentration of the LPA is higher than a pre-determined concentration.
  • LIBS library of adaptor-tagged DNA fragments
  • LPA Library Post Amplification
  • the end-repaired DNA fragments of step (a) of the test for adaptor ligation is generated from a DNA sample.
  • said DNA sample is a blended DNA sample of at least two different cell lines.
  • said DNA sample is a blended DNA sample of at two different cell lines.
  • the two different cell lines are blended at a 50:50 ratio.
  • the two different cell lines are NA09596 and NA12878.
  • said DNA sample is the 50:50 blended sample described in Example 15.
  • the DNA sample used in the test for adaptor ligation is different from the DNA sample used in the test for adaptor distribution.
  • the pre-determined amount of end- repaired DNA fragments is about 5 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 10 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 15 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 20 ng.
  • the pre-determined amount of end-repaired DNA fragments is about 25 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 30 ng. In some embodiments of the kits of the disclosure, the pre determined amount of end-repaired DNA fragments is about 35 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 40 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end- repaired DNA fragments is about 50 ng.
  • the pre-determined amount of end- repaired DNA fragments is 5 ng. In some embodiments of the kits of the disclosure, the pre determined amount of end-repaired DNA fragments is 10 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is 15 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is 20 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is 25 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is 30 ng.
  • the pre-determined amount of end-repaired DNA fragments is 35 ng. In some embodiments of the kits of the disclosure, the pre-determined amount is 40 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is 50 ng.
  • the pre-determined amount of end- repaired DNA fragments is about 1 ng to about 100 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 1 ng to about 50 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end- repaired DNA fragments is about 50 ng to about 100 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 5 ng to about 90 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 10 ng to about 80 ng.
  • the pre-determined amount of end-repaired DNA fragments is about 20 ng to about 60 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 20 ng to about 50 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 20 ng to about 40 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 30 ng to about 50 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 40 ng to about 50 ng.
  • the pre-determined amount of end-repaired DNA fragments is about 30 ng to about 40 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 20 ng to about 30 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is greater than about 100 ng.
  • the pre-determined amount of end- repaired DNA fragments is 1 ng to 100 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is 1 ng to 50 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is 50 ng to 100 ng. In some embodiments of the kits of the disclosure, the pre determined amount of end-repaired DNA fragments is 5 ng to 90 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is 10 ng to 80 ng.
  • the pre-determined amount of end- repaired DNA fragments is 20 ng to 60 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is 20 ng to 50 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is 20 ng to 40 ng. In some embodiments of the kits of the disclosure, the pre determined amount of end-repaired DNA fragments is 30 ng to 50 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is 40 ng to 50 ng.
  • the pre-determined amount of end-repaired DNA fragments is 30 ng to 40 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is 20 ng to 30 ng. In some embodiments of the kits of the disclosure, the pre-determined amount of end-repaired DNA fragments is greater than 100 ng.
  • the pre-determined concentration of the LPA is about 1 ng/pL. In some embodiments of the kits of the disclosure, the pre determined concentration of the LPA is about 5 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 10 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 20 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 30 n g/pL. In some embodiments of the kits of the disclosure, the pre determined concentration of the LPA is about 40 ng/pL.
  • the pre-determined concentration of the LPA is about 50 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 60 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 70 ng/pL. In some embodiments of the kits of the disclosure, the pre determined concentration of the LPA is about 80 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 90 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 100 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 150 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 200 ng/pL.
  • the pre-determined concentration of the LPA is 1 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 5 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 10 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 20 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 30 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 40 ng/pL.
  • the pre-determined concentration of the LPA is 50 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 60 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 70 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 80 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 90 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 100 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 150 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 200 ng/pL.
  • the pre-determined concentration of the LPA is about 1 ng/pL to about 200 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is greater than about 200 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 10 ng/pL to about 100 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 10 ng/pL to about 90 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 20 ng/pL to about 80 ng/pL.
  • the pre determined concentration of the LPA is about 10 ng/pL to about 80 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 30 ng/pL to about 80 ng/pL. In some embodiments of the kits of the disclosure, the pre determined concentration of the LPA is about 40 ng/pL to about 80 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is about 50 ng/pL to about 80 ng/pL. In some embodiments of the kits of the disclosure, the pre determined concentration of the LPA is about 50 ng/pL to about 70 ng/pL.
  • the pre-determined concentration of the LPA is about 60 ng/pL to about 70 ng/pL. In some embodiments of the kits of the disclosure, the pre determined concentration of the LPA is about 50 ng/pL to about 60 ng/pL.
  • the pre-determined concentration of the LPA is 1 ng/pL to 200 ng/pL. In some embodiments of the kits of the disclosure, the pre determined concentration of the LPA is 10 ng/pL to 100 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 10 ng/pL to 90 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 20 ng/pL to 80 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 30 ng/pL to 80 ng/pL.
  • the pre-determined concentration of the LPA is 10 ng/pL to 80 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 40 ng/pL to 80 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 50 ng/pL to 80 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 50 ng/pL to 70 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 60 ng/pL to 70 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is 50 ng/pL to 60 ng/pL.
  • the pre-determined concentration of the LPA is greater than about 200 ng/pL. In some embodiments of the kits of the disclosure, the pre-determined concentration of the LPA is greater than 200 ng/pL. [0042] In some embodiments of the kits of the disclosure, the set of adaptors is considered to have passed the adaptor QC process when it has passed the test for adaptor ligation.
  • the adaptor QC process comprises a test for adaptor distribution comprising (a) ligating the set of adaptors to a pre-determined amount of end-repaired DNA fragments to generate a library of adaptor-tagged DNA fragments (LIBS); (b) amplifying the LIBS using a primer pair comprising at least one primer comprising an index sequence to generate a Library Post Index Amplification (LPIA); and (c) performing a quantitative genetic analysis on the LPIA, wherein the set of adaptors is considered to have passed the test for adaptor distribution when one or more pre-determined acceptance criteria for the quantitative genetic analysis has been met.
  • LIBS library of adaptor-tagged DNA fragments
  • LPIA Library Post Index Amplification
  • the end-repaired DNA fragments of step (a) of the test for adaptor distribution are generated from a DNA sample.
  • said DNA sample comprises wild-type (wt) cell-free DNA (cfDNA).
  • said DNA sample consists of wt cfDNA.
  • said DNA sample comprises wt cfDNA obtained from a healthy human.
  • said DNA sample is the wt cfDNA sample described in Example 16.
  • the DNA sample used in the test for adaptor distribution is different from the DNA sample used in the test for adaptor ligation.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (a).
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.05% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.1% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.2% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.3% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.4% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.6% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.7% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.8% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.9% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than about 1% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than about 2% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than about 3% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than about 4% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than about 5% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than 0.05% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.1% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.2% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 0.3% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 0.4% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.5% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than 0.6% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.7% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.8% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 0.9% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 1% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 2% of reads.
  • criterion (a) is Barcode Crosstalk present in no more than 3% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 4% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 5% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.05% to about 10% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.05% to about 5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.1% to about 5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.1% to about 2% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.05% to about 2% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.1% to about 1% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.05% to about 1% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.1% to about 0.5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.1% to about 0.4% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.1% to about 0.3% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.2% to about 0.3% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.1% to about 0.2% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than 0.05% to 10% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.05% to 5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.1% to 5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.1% to 2% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.05% to 2% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than 0.1% to 1% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.05% to 1% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.1% to 0.5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.1% to 0.4% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.1% to 0.3% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than 0.2% to 0.3% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.1% to 0.2% of reads.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (b).
  • criterion (b) is unknown adaptors are present in no more than about 1% of reads.
  • criterion (b) is unknown adaptors are present in no more than about 5% of reads.
  • criterion (b) is unknown adaptors are present in no more than about 10% of reads.
  • criterion (b) is unknown adaptors are present in no more than about 15% of reads.
  • criterion (b) is unknown adaptors are present in no more than about 20% of reads.
  • criterion (b) is unknown adaptors are present in no more than about 25% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 30% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 35% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 40% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 45% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 50% of reads.
  • criterion (b) is unknown adaptors are present in no more than 1% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 5% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 10% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 15% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 20% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 25% of reads.
  • criterion (b) is unknown adaptors are present in no more than 30% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 35% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 40% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 45% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 50% of reads. [0052] In some embodiments, criterion (b) is unknown adaptors are present in no more than about 1% to about 50% of reads.
  • criterion (b) is unknown adaptors are present in no more than about 1% to about 20% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 1% to about 15% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 1% to about 10% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 5% to about 20% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 5% to about 15% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 5% to about 10% of reads.
  • criterion (b) is unknown adaptors are present in no more than 1% to 50% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 1% to 20% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 1% to 15% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 1% to 10% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 5% to 20% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 5% to 15% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 5% to 10% of reads.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (c).
  • criterion (c) is no more than about 10% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than about 20% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than about 30% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 40% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 50% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than about 60% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 70% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 80% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 90% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (c).
  • criterion (c) is no more than 10% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than 20% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than 30% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than 40% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 50% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 60% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than 70% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 80% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 90% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than about 10% to about 90% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 10% to about 80% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 20% to about 80% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than about 30% to about 70% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 40% to about 60% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 40% to about 50% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 50% to about 60% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than 10% to 90% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 10% to 80% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 20% to 80% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than 30% to 70% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 40% to 60% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 40% to 50% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 50% to 60% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (d).
  • criterion (d) is at least about 60% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 65% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 70% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 75% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 80% of all unique adaptor sequences are present.
  • criterion (d) is at least about 85% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 90% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 95% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 96% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 97% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 98% of all unique adaptor sequences are present.
  • criterion (d) is at least about 99% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 99.5% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is about 100% of all unique adaptor sequences are present. [0059] In some embodiments, criterion (d) is at least 60% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 65% of all unique adaptor sequences are present.
  • criterion (d) is at least 70% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 75% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 80% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 85% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 90% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 95% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 96% of all unique adaptor sequences are present.
  • criterion (d) is at least 97% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 98% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 99% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 99.5% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is 100% of all unique adaptor sequences are present.
  • criterion (d) is at least about 60% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 70% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 80% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 90% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 95% to about 99.9% of all unique adaptor sequences are present.
  • criterion (d) is at least about 96% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 97% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 98% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 99% to about 99.9% of all unique adaptor sequences are present.
  • criterion (d) is at least about 60% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 70% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 80% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 90% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 95% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 96% to about 100% of all unique adaptor sequences are present.
  • criterion (d) is at least about 97% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 98% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 99% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 99.9% to about 100% of all unique adaptor sequences are present. [0062] In some embodiments, criterion (d) is at least 60% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 70% to 99.9% of all unique adaptor sequences are present.
  • criterion (d) is at least 80% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 90% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 95% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 96% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 97% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 98% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 99% to about 99.9% of all unique adaptor sequences are present.
  • criterion (d) is at least 60% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 70% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 80% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 90% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 95% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 96% to 100% of all unique adaptor sequences are present.
  • criterion (d) is at least 97% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 98% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 99% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 99.9% to 100% of all unique adaptor sequences are present.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (e).
  • criterion (e) is no more than about 1% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 5% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 10% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than about 15% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 20% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 25% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 30% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than about 35% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 40% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 45% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 50% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than 1% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 5% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 10% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 15% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than 20% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 25% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 30% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 35% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than 40% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 45% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 50% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than about 1% to about 50% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 5% to about 50% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 5% to about 40% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than about 10% to about 30% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 10% to about 20% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 20% to about 40% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 20% to about 30% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than about 15% to about 25% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 15% to about 20% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than 1% to 50% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 5% to 50% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 5% to 40% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 10% to 30% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than 10% to 20% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 20% to 40% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 20% to 30% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 15% to 25% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 15% to 20% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (a) and a criterion (b).
  • the pre-determined acceptance criteria comprise a criterion (c), a criterion (d), and a criterion (e).
  • the pre-determined acceptance criteria comprise a criterion (a), a criterion (b), and a criterion (c).
  • the pre-determined acceptance criteria comprise a criterion (a), a criterion (b), and a criterion (d).
  • the pre-determined acceptance criteria comprise a criterion (a), a criterion (b), and a criterion (e). In some embodiments, the pre-determined acceptance criteria comprise a criterion (a), a criterion (b), a criterion (c), and a criterion (d). In some embodiments, the pre determined acceptance criteria comprise a criterion (a), a criterion (b), a criterion (c), and a criterion (e). In some embodiments, the pre-determined acceptance criteria comprise a criterion (a), a criterion (b), a criterion (d), and a criterion (e).
  • the pre-determined acceptance criteria comprise a criterion (a), a criterion (b), a criterion (c), a criterion (d), and a criterion (e). In some embodiments, the pre-determined acceptance criteria comprise at least one criterion selected from criteria (a) (b) (c) (d) and (e).
  • the set of adaptors is considered to have passed the adaptor QC process when it has passed the test for adaptor distribution.
  • the set of adaptors is considered to have passed the adaptor QC process when it has passed both the test for adaptor ligation and the test for adaptor distribution.
  • the probe QC process comprises a test for capture probe modules comprising (a) ligating a set of adaptors to a DNA sample comprising end-repaired DNA fragments to generate a library of adaptor-tagged DNA fragments (LIBS); (b) amplifying the LIBS to generate a Library Post Amplification (LPA); (c) splitting or diluting the LPA to generate a Target Capture LPA (TC LPA) and a Whole- Genome LPA (WG LPA); (d) amplifying the WG LPA to generate a Whole-Genome Library Amplified (WGLA); (e) hybridizing the one or more capture probe modules to be tested to the TC LPA to form adaptor-tagged DNA fragment-capture probe module complexes; (f) isolating the adaptor-tagged DNA fragment-capture probe module complexes to form isolated adaptor- tagged DNA fragment-capture probe module complexes; (g) enzymatically processing the isolated adaptor-
  • LPA Library Post Amplification
  • TC LPA
  • the terms LPA and adaptor-tagged Parent Library are used interchangeably.
  • the terms WGLA and LPWG library are used interchangeably.
  • the terms SRL and combined library are used interchangeably.
  • the TC LPA is at least about 1 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 5 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 10 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 15 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 20 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 25 ng/pL.
  • the TC LPA is at least about 30 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 35 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 40 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 45 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 50 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 55 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 60 ng/pL.
  • the TC LPA is at least about 65 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 70 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 75 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 80 ng/pL. [0074] In some embodiments of the kits of the disclosure, the TC LPA is at least 1 n g/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least 5 ng/gL.
  • the TC LPA is at least 10 ng/gL In some embodiments of the kits of the disclosure, the TC LPA is at least 15 ng/gL In some embodiments of the kits of the disclosure, the TC LPA is at least 20 ng/gL In some embodiments of the kits of the disclosure, the TC LPA is at least 25 ng/gL In some embodiments of the kits of the disclosure, the TC LPA is at least 30 ng/gL In some embodiments of the kits of the disclosure, the TC LPA is at least 35 ng/gL In some embodiments of the kits of the disclosure, the TC LPA is at least 40 ng/gL In some embodiments of the kits of the disclosure, the TC LPA is at least 45 ng/gL In some embodiments of the kits of the disclosure, the TC LPA is at least 50 ng/gL In some embodiments of the kits of the disclosure, the TC LPA is at least 55 ng/gL In some embodiments of
  • the TC LPA is at least about 1 ng/gL to about 100 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 1 ng/gL to about 80 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 10 ng/gL to about 80 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 10 ng/gL to about 70 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 10 ng/gL to about 60 ng/gL.
  • the TC LPA is at least about 20 ng/gL to about 60 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 20 ng/gL to about 50 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 20 ng/gL to about 40 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 30 ng/gL to about 50 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 30 ng/gL to about 40 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least about 20 ng/gL to about 30 ng/gL.
  • the TC LPA is at least 1 ng/gL to 100 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least 1 ng/gL to 80 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least 10 ng/gL to 80 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least 10 ng/gL to 70 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least 10 ng/gL to 60 ng/gL.
  • the TC LPA is at least 20 ng/gL to 60 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least 20 ng/gL to 50 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least 20 ng/gL to 40 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least 30 ng/gL to 50 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least 30 ng/gL to 40 ng/gL. In some embodiments of the kits of the disclosure, the TC LPA is at least 20 ng/gL to 30 ng/gL.
  • the WG LPA is at least about 0.1 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 0.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 1 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 1.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 2 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 2.5 ng/gL.
  • the WG LPA is at least about 3 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 3.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 4 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 4.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 5.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 6 ng/gL.
  • the WG LPA is at least about 6.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 7 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 7.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 8 ng/gL.
  • the WG LPA is at least 0.1 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 0.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 1 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 1.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 2 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 2.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 3 ng/gL.
  • the WG LPA is at least 3.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 4 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 4.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 5.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 6 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 6.5 ng/gL.
  • the WG LPA is at least 7 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 7.5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 8 ng/gL.
  • the WG LPA is at least about 0.1 ng/gL to about 10 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 0.1 ng/gL to about 8 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 1 ng/gL to about 10 ng/gL. In some embodiments of the kits of the disclosure, the WGLPA is at least about 1 ng/gL to about 5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 2 ng/gL to about 10 ng/gL.
  • the WG LPA is at least about 2 ng/gL to about 6 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 3 ng/gL to about 5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 3 ng/gL to about 4 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least about 2 ng/gL to about 3 ng/gL.
  • the WG LPA is at least 0.1 ng/gL to 10 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 0.1 ng/gL to 8 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 1 ng/gL to 10 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 1 ng/gL to 5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 2 ng/gL to 10 ng/gL.
  • the WG LPA is at least 2 ng/gL to 6 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 3 ng/gL to 5 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 3 ng/gL to 4 ng/gL. In some embodiments of the kits of the disclosure, the WG LPA is at least 2 ng/gL to 3 ng/gL. [0081] In some embodiments of the kits of the disclosure, the TCLA is at least about 1.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 1.5 ng/gL.
  • the TCLA is at least about 2.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 2.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 3.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 3.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 4.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about at least 4.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 5.0 ng/gL.
  • the TCLA is at least about 5.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 6.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 6.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 7.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 7.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 8.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 8.5 ng/gL.
  • the TCLA is at least about 9.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 9.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 10 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 15 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 20 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 25 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 30 ng/gL.
  • the TCLA is at least about 35 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 40 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 45 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 50 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 55 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 60 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 65 ng/gL.
  • the TCLA is at least about 70 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 75 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 80 ng/gL. [0082] In some embodiments of the kits of the disclosure, the TCLA is at least 1.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 1.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 2.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 2.5 ng/gL.
  • the TCLA is at least 3.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 3.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 4.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about at 4.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 5.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 5.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 6.0 ng/gL.
  • the TCLA is at least 6.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 7.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 7.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 8.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 8.5 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 9.0 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 9.5 ng/gL.
  • the TCLA is at least 10 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 15 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 20 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 25 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 30 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 35 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 40 ng/gL.
  • the TCLA is at least 45 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 50 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 55 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 60 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 65 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 70 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 75 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 80 ng/gL.
  • the TCLA is at least about 1 ng/gL to about 200 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 100 ng/gL to about 200 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 1 ng/gL to about 100 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 1 ng/gL to about 80 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 1 ng/gL to about 50 ng/gL.
  • the TCLA is at least about 1 ng/gL to about 40 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 1 ng/gL to about 30 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 1 ng/gL to about 20 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 1 ng/gL to about 10 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least about 5 ng/gL to about 10 ng/gL.
  • the TCLA is at least 1 ng/gL to 200 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 100 ng/gL to 200 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 1 ng/gL to 100 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 1 ng/gL to 80 ng/gL. In some embodiments of the kits of the disclosure, the TCLA is at least 1 ng/gL to
  • the TCLA is at least 1 ng/gL to
  • the TCLA is at least 1 ng/gL to
  • the TCLA is at least 1 ng/gL to
  • the TCLA is at least 1 ng/gL to
  • the TCLA is at least 5 ng/gL to
  • the WGLA is at least about 1.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 1.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 2.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 2.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 3.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 3.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 4.0 ng/gL.
  • the WGLA is at least about at least 4.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 5.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 5.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 6.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 6.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 7.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 7.5 ng/gL.
  • the WGLA is at least about 8.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 8.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 9.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 9.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 10 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 15 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 20 ng/gL.
  • the WGLA is at least about 25 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 30 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 35 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 40 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 45 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 50 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 55 ng/gL.
  • the WGLA is at least about 60 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 65 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 70 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 75 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 80 ng/gL.
  • the WGLA is at least 1.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 1.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 2.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 2.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 3.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 3.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 4.0 ng/gL.
  • the WGLA is at least about at 4.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 5.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 5.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 6.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 6.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 7.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 7.5 ng/gL.
  • the WGLA is at least 8.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 8.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 9.0 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 9.5 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 10 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 15 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 20 ng/gL.
  • the WGLA is at least 25 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 30 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 35 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 40 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 45 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 50 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 55 ng/gL.
  • the WGLA is at least 60 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 65 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 70 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 75 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 80 ng/gL.
  • the WGLA is at least about 1 ng/gL to about 200 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 100 ng/gL to about 200 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 1 ng/gL to about 100 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 1 ng/gL to about 80 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 1 ng/gL to about 50 ng/gL.
  • the WGLA is at least about 1 ng/gL to about 40 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 1 ng/gL to about 30 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 1 ng/gL to about 20 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 1 ng/gL to about 10 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least about 5 ng/gL to about 10 ng/gL.
  • the WGLA is at least 1 ng/gL to 200 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 100 ng/gL to 200 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 1 ng/gL to 100 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 1 ng/gL to 80 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 1 ng/gL to 50 ng/gL.
  • the WGLA is at least 1 ng/gL to 40 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 1 ng/gL to 30 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 1 ng/gL to 20 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 1 ng/gL to 10 ng/gL. In some embodiments of the kits of the disclosure, the WGLA is at least 5 ng/gL to 10 ng/gL.
  • the one or more capture probe modules are considered to have passed the probe QC process if one or more pre-determined acceptance criteria for the quantitative genetic analysis has been met.
  • the one or more capture probe modules are considered to have passed the probe QC process if one or more pre-determined acceptance criteria for the quantitative genetic analysis have been met.
  • the pre-determined acceptance criteria for the probe QC process comprise a criterion (a).
  • criterion (a) is about 100% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 99.9% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 99.8% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 99.7% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 99.6% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 99.5% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99.4% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99.3% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99.2% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99.1% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 98.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.8% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.7% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.6% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.5% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.4% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 98.3% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.2% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.1% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 97% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 96% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 95% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least about 90% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least about 85% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least about 80% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least about 75% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 70% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least about 65% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least about 60% of capture probes have at least 1 total reads.
  • criterion (a) is 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.8% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.7% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.6% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.5% of capture probes have at least 1 total reads.
  • criterion (a) is at least 99.4% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.3% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.2% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.1% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.9% of capture probes have at least 1 total reads.
  • criterion (a) is at least 98.8% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.7% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.6% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.5% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.4% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.3% of capture probes have at least 1 total reads.
  • criterion (a) is at least 98.2% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.1% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 97% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 96% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion
  • criterion (a) is at least 95% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least 90% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least 85% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least 80% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least 75% of capture probes have at least 1 total reads.
  • criterion (a) is at least 70% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least 65% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least 60% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 60% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 70% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 80% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 90% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 95% to about 99.9% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 96% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 97% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99% to about 100% of capture probes have at least 1 total reads.
  • criterion (a) is at least 60% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 70% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 80% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 90% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 95% to 99.9% of capture probes have at least 1 total reads.
  • criterion (a) is at least 96% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 97% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99% to 100% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 60% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 70% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 80% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 90% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 95% to about 100% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 96% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 97% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99.5% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99.9% to about 100% of capture probes have at least 1 total reads.
  • criterion (a) is at least 60% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 70% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 80% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 90% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 95% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 96% to 100% of capture probes have at least 1 total reads.
  • criterion (a) is at least 97% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.5% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.9% to 100% of capture probes have at least 1 total reads.
  • the pre-determined acceptance criteria for the probe QC process comprise a criterion (b).
  • criterion (b) is about 100% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 99.9% of capture probes have at least 10-200 on- target total reads.
  • criterion (b) is at least about 99.5% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 99% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 98.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 98% of capture probes have at least 10- 200 on-target total reads. In some embodiments, criterion (b) is at least about 97.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 97% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 96.5% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 96% of capture probes have at least 10- 200 on-target total reads. In some embodiments, criterion (b) is at least about 95.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 95% of capture probes have at least 10-200 on-target total reads. In some embodiments, the pre-determined acceptance criteria for the probe QC process comprise criterion (b) at least about 90% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 85% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 80% of capture probes have at least 10- 200 on-target total reads. In some embodiments, criterion (b) is at least about 75% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 70% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 65% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 60% of capture probes have at least 10- 200 on-target total reads.
  • criterion (b) is 100% of capture probes have at least 10-200 on- target total reads. In some embodiments, criterion (b) is at least 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 99.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 99% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 98.5% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least 98% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 97.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 97% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 96.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 96% of capture probes have at least 10-200 on- target total reads.
  • criterion (b) is at least 95.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 95% of capture probes have at least 10-200 on-target total reads. In some embodiments of the kits of the disclosure, the pre-determined acceptance criteria for the probe QC process comprise criterion (b) at least 90% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 85% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 80% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least 75% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 70% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 65% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 60% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is about 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 99.9% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 99.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 99% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 98.5% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least about 98% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 97.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 97% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 96.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 96% of capture probes have at least 50 on- target total reads.
  • criterion (b) is at least about 95.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 95% of capture probes have at least 50 on-target total reads. In some embodiments, the pre determined acceptance criteria for the probe QC process comprise criterion (b) at least about 90% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 85% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 80% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least about 75% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 70% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 65% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 60% of capture probes have at least 50 on-target total reads. [0100] In some embodiments, criterion (b) is 100% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 99.9% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 99.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 99% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 98.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 98% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 97.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 97% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 96.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 96% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 95.5% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 95% of capture probes have at least 50 on-target total reads.
  • the pre-determined acceptance criteria for the probe QC process comprise criterion (b) at least 90% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 85% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 80% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 75% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 70% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 65% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 60% of capture probes have at least 50 on-target total reads.
  • criterion (b) is about 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 99.9% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 99.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 99% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 98.5% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least about 98% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 97.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 97% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 96.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 96% of capture probes have at least 60 on- target total reads.
  • criterion (b) is at least about 95.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 95% of capture probes have at least 60 on-target total reads. In some embodiments, the pre determined acceptance criteria for the probe QC process comprise criterion (b) at least about 90% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 85% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 80% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least about 75% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 70% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 65% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 60% of capture probes have at least 60 on-target total reads. [0102] In some embodiments, criterion (b) is 100% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 99.9% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 99.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 99% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 98.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 98% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 97.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 97% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 96.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 96% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 95.5% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 95% of capture probes have at least 60 on-target total reads.
  • the pre-determined acceptance criteria for the probe QC process comprise criterion (b) at least 90% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 85% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 80% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 75% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 70% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 65% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 60% of capture probes have at least 60 on-target total reads.
  • criterion (b) is about 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 99.9% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 99.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 99% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 98.5% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least about 98% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 97.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 97% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 96.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 96% of capture probes have at least 70 on- target total reads.
  • criterion (b) is at least about 95.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 95% of capture probes have at least 70 on-target total reads. In some embodiments, the pre determined acceptance criteria for the probe QC process comprise criterion (b) at least about 90% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 85% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 80% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least about 75% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 70% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 65% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 60% of capture probes have at least 70 on-target total reads. [0104] In some embodiments, criterion (b) is 100% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 99.9% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 99.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 99% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 98.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 98% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 97.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 97% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 96.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 96% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 95.5% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 95% of capture probes have at least 70 on-target total reads.
  • the pre-determined acceptance criteria for the probe QC process comprise criterion (b) at least 90% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 85% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 80% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 75% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 70% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 65% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 60% of capture probes have at least 70 on-target total reads.
  • criterion (b) is about 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 99.9% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 99.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 99% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 98.5% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least about 98% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 97.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 97% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 96.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 96% of capture probes have at least 80 on- target total reads.
  • criterion (b) is at least about 95.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 95% of capture probes have at least 80 on-target total reads. In some embodiments, the pre determined acceptance criteria for the probe QC process comprise criterion (b) at least about 90% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 85% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 80% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least about 75% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 70% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 65% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 60% of capture probes have at least 80 on-target total reads. [0106] In some embodiments, criterion (b) is 100% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 99.9% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 99.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 99% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 98.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 98% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 97.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 97% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 96.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 96% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 95.5% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 95% of capture probes have at least 80 on-target total reads.
  • the pre-determined acceptance criteria for the probe QC process comprise criterion (b) at least 90% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 85% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 80% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 75% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 70% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 65% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 60% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least about 60% to about 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 70% to about 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 80% to about 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 90% to about 99.9% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 95% to about 99.9% of capture probes have at least 10-200 on- target total reads. In some embodiments, criterion (b) is at least about 99% to about 99.9% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least 60% to 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 70% to 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 80% to 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 90% to 99.9% of capture probes have at least 10-200 on- target total reads. In some embodiments, criterion (b) is at least 95% to 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 99% to 99.9% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 60% to about 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 70% to about 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 80% to about 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 90% to about 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 95% to about 100% of capture probes have at least 10-200 on- target total reads. In some embodiments, criterion (b) is at least about 99% to about 100% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least 60% to 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 70% to 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 80% to 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 90% to 100% of capture probes have at least 10-200 on- target total reads. In some embodiments, criterion (b) is at least 95% to 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 99% to 100% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 60% to about 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 70% to about 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 80% to about 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 90% to about 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 95% to about 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 99% to about 100% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 60% to 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 70% to 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 80% to 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 90% to 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 95% to 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 99% to 100% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least about 60% to about 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 70% to about 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 80% to about 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 90% to about 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 95% to about 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 99% to about 100% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 60% to 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 70% to 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 80% to 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 90% to 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 95% to 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 99% to 100% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least about 60% to about 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 70% to about 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 80% to about 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 90% to about 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 95% to about 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 99% to about 100% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 60% to 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 70% to 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 80% to 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 90% to 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 95% to 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 99% to 100% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least about 60% to about 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 70% to about 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 80% to about 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 90% to about 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 95% to about 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 99% to about 100% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 60% to 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 70% to 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 80% to 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 90% to 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 95% to 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 99% to 100% of capture probes have at least 80 on-target total reads.
  • the pre-determined acceptance criteria for the probe QC process comprise a criterion (c).
  • criterion (c) is at least about 99.9% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 99.5% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 99% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 98% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 97% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 96% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 95% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 90% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 85% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 80% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 75% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 70% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 65% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 60% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 99.5% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 99% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 98% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 97% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 96% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least 95% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 90% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 85% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 80% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 75% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 70% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 65% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 60% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 60% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 70% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 80% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 90% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 95% to about 99.9% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 96% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 97% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 98% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 99% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 100% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least 60% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 70% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 80% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 90% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 95% to 99.9% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least 96% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 97% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 98% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 99% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 100% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 60% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 70% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 80% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 90% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 95% to about 100% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 96% to about 100% of expected SNPs within the DNA sample are detected. . In some embodiments, criterion (c) is at least about 97% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 98% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 99% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is about 100% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least 60% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 70% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 80% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 90% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 95% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 96% to 100% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least 97% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 98% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 99% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is 100% of expected SNPs within the DNA sample are detected.
  • the pre-determined acceptance criteria for the probe QC process comprise a criterion (a) and a criterion (b). In some embodiments of the kits of the disclosure, the pre-determined acceptance criteria for the probe QC process comprise a criterion (a) and a criterion (c). In some embodiments of the kits of the disclosure, the pre-determined acceptance criteria for the probe QC process comprise a criterion (b) and a criterion (c). In some embodiments of the kits of the disclosure, the pre determined acceptance criteria for the probe QC process comprise a criterion (a), a criterion (b), and a criterion (c).
  • the DNA sample used in the probe QC process comprises human wild type DNA. In some embodiments of the kits of the disclosure, the DNA sample used in the probe QC process comprises a plurality of single nucleotide polymorphisms (SNPs). In some embodiments of the kits of the disclosure, the DNA sample used in the probe QC process comprises any suitable DNA.
  • SNPs single nucleotide polymorphisms
  • the kit comprises a DNA sample used as a positive control sample specific to the capture probe modules of the same kit.
  • the positive control sample comprises human wild type DNA.
  • the positive control sample comprises a plurality of SNPs.
  • the positive control sample comprises the DNA sample used in the probe QC process of the capture probe modules of the kit.
  • the positive control sample comprises any suitable DNA.
  • the adaptor QC process is performed according to any one of the methods of performing an adaptor QC process of the disclosure.
  • the probe QC process is performed according to any one of the methods of performing a probe QC process of the disclosure.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample; (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; and (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region, wherein the first R primer comprises a tail sequence binding region and a sequencing primer binding region.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample; (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; and (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region, wherein the first R primer comprises a tail sequence binding region and a sequencing primer binding region; wherein the capture probe modules have passed a probe QC process.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample; (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; and (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region, wherein the first R primer comprises a tail sequence binding region and a sequencing primer binding region; wherein the set of adaptors has passed an adaptor QC process.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample; (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; and (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region, wherein the first R primer comprises a tail sequence binding region and a sequencing primer binding region; wherein the set of adaptors has passed an adaptor QC process and the capture probe modules have passed a probe QC process.
  • the kit comprises (a) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; and (b) a second primer pair comprising a second F primer and a second R primer, wherein each of the second F primer and the second R primer comprises an amplification region binding region and a sequencing primer binding region.
  • the kit comprises (a) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; and (b) a second primer pair comprising a second F primer and a second R primer, wherein each of the second F primer and the second R primer comprises an amplification region binding region and a sequencing primer binding region; wherein the set of adaptors has passed an adaptor QC process.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample; (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region, wherein the first R primer comprises a tail sequence binding region and a sequencing primer binding region; and (d) a second primer pair comprising a second F primer and a second R primer, wherein each of the second F primer and the second R primer comprises an amplification region binding region and a sequencing primer binding region.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample; (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region, wherein the first R primer comprises a tail sequence binding region and a sequencing primer binding region; and (d) a second primer pair comprising a second F primer and a second R primer, wherein each of the second F primer and the second R primer comprises an amplification region binding region and a sequencing primer binding region; wherein the capture probe modules have passed a probe QC process.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample; (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region, wherein the first R primer comprises a tail sequence binding region and a sequencing primer binding region; and (d) a second primer pair comprising a second F primer and a second R primer, wherein each of the second F primer and the second R primer comprises an amplification region binding region and a sequencing primer binding region; wherein the set of adaptors has passed an adaptor QC process.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample; (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region, wherein the first R primer comprises a tail sequence binding region and a sequencing primer binding region; and (d) a second primer pair comprising a second F primer and a second R primer, wherein each of the second F primer and the second R primer comprises an amplification region binding region and a sequencing primer binding region; wherein the set of adaptors has passed an adaptor QC process and the capture probe modules have passed a probe QC process.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the tail sequence of each capture probe module comprises a Library Tag; and (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the tail sequence of each capture probe module comprises a Library Tag; and (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; wherein the set of adaptors has passed an adaptor QC process.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the tail sequence of each capture probe module comprises a Library Tag; and (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; wherein the one or more capture probe modules have passed a probe QC process.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the tail sequence of each capture probe module comprises a Library Tag; and (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; wherein the one or more capture probe modules have passed a probe QC process and the set of adaptors has passed an adaptor QC process.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the tail sequence of each capture probe module comprises a Library Tag; (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region, wherein the first R primer comprises a Library Tag binding region and a sequencing primer binding region; and (d) a second primer pair comprising a second F primer and a second R primer, wherein each of the second F primer and the second R primer comprises an amplification region binding region and a sequencing primer binding region, and wherein none of the primers of the second primer pair bind to the Library Tag.
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the tail sequence of each capture probe module comprises a Library Tag; (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region, wherein the first R primer comprises a Library Tag binding region and a sequencing primer binding region; and (d) a second primer pair comprising a second F primer and a second R primer, wherein each of the second F primer and the second R primer comprises an amplification region binding region and a sequencing primer binding region, and wherein none of the primers of the second primer pair bind to the Library Tag; wherein the one or more capture probe modules have passed a
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the tail sequence of each capture probe module comprises a Library Tag; (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region, wherein the first R primer comprises a Library Tag binding region and a sequencing primer binding region; and (d) a second primer pair comprising a second F primer and a second R primer, wherein each of the second F primer and the second R primer comprises an amplification region binding region and a sequencing primer binding region, and wherein none of the primers of the second primer pair bind to the Library Tag; wherein the set of adaptors has passed an adaptor
  • the kit comprises (a) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the tail sequence of each capture probe module comprises a Library Tag; (b) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region, wherein the first R primer comprises a Library Tag binding region and a sequencing primer binding region; and (d) a second primer pair comprising a second F primer and a second R primer, wherein each of the second F primer and the second R primer comprises an amplification region binding region and a sequencing primer binding region, and wherein none of the primers of the second primer pair bind to the Library Tag; wherein the one or more capture probe modules have passed a
  • the first primer pair is used to generate a first modified library.
  • the second primer pair is used to generate a second modified library.
  • the first primer pair is used to generate a first modified library and the second primer pair is used to generate a second modified library.
  • the first modified library and the second modified library are configured to be combined into a Sequence-Ready Library (SRL).
  • the tail sequence of each capture probe module comprises a Library Tag.
  • the Library Tag comprises a nucleic acid sequence or an amino acid sequence.
  • the Library Tag comprises one or more of a DNA, an RNA, a PNA, or a non-naturally occurring nucleic acid, a synthetic nucleic acid, a modified nucleic acid, a non-naturally occurring amino acid, a synthetic amino acid, and a modified amino acid.
  • the Library Tag comprises a detectable label.
  • the detectable label comprises one or more of a fluorescent moiety, a magnetic or paramagnetic moiety, an enzymatic moiety, a binding moiety, an epitope, and a radioactive moiety.
  • the Library Tag selectively or specifically binds to a detectable moiety.
  • the detectable moiety comprises one or more of a fluorescent moiety, a magnetic or paramagnetic moiety, an enzymatic moiety, a binding moiety, an epitope, and a radioactive moiety.
  • the Library Tag comprises a unique polynucleotide sequence. In some embodiments, the unique polynucleotide sequence has no more than 70% sequence identity to any DNA fragment of the test sample.
  • the kit comprises (a) a set of adaptors, wherein each adaptor comprises an adaptor module comprising an amplification region; (b) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the tail sequence of each capture probe module comprises a Library Tag; (c) a first primer pair comprising a first F primer and a first R primer, wherein the first F primer comprises an amplification region binding region and a sequencing primer binding region; wherein the first R primer comprises a Library Tag binding region and a sequencing primer binding region; and (d) a second primer pair comprising a second F primer and a second R primer, wherein each of the second F primer and the second R primer comprises an amplification region binding region and a sequencing primer binding region, wherein none of the primers of the second primer pair bind to the Library Tag.
  • the kit is used for generating a first modified library and a second modified library, wherein the kit comprises (a) a set of adaptors, wherein each adaptor comprises an adaptor module; and (b) one or more capture probe modules, wherein each capture probe module comprises a tail sequence and a capture probe sequence capable of hybridizing to a target sequence in a test sample, wherein the tail sequence of each capture probe module comprises a Library Tag.
  • the first modified library comprises a DNA fragment comprising an adaptor, a capture probe module, and the capture probe module’s target sequence.
  • the second modified library comprises a DNA fragment comprising an adaptor and at least a portion of a DNA sequence of the test sample. In some embodiments, both the first modified library and the second modified library are generated from the same test sample.
  • the first primer pair is used to generate a first modified library and the second primer pair is used to generate a second modified library.
  • the first modified library comprises a first DNA fragment comprising an adaptor, a capture probe module, and the capture probe module’s target sequence.
  • the second modified library comprises a second DNA fragment comprising an adaptor and a at least a portion of a DNA sequence of the test sample.
  • the first modified library and the second modified library are configured to be combined into a Sequence-Ready Library (SRL).
  • SRL Sequence-Ready Library
  • the SRL is also referred to as the combined library.
  • each library fragment of the first modified library is an adaptor-tagged DNA fragment comprising an adaptor, a capture probe module, and at least a portion of a DNA sequence of the test sample.
  • each library fragment of the second modified library is an adaptor-tagged DNA fragment comprising an adaptor and at least a portion of a DNA sequence of the test sample.
  • none of the adaptor-tagged DNA fragments of the second modified library comprises a capture probe module.
  • the first modified library is a Target Capture Library (TCL) or amplified Target Capture Library (TCLA).
  • TCL Target Capture Library
  • TCLA amplified Target Capture Library
  • the second modified library is a Whole-Genome Library (WGL) or amplified Whole-Genome Library (WGLA).
  • the first modified library or the second modified library comprises the Library Tag, wherein the Library Tag is configured to distinguish the first modified library from the second modified library.
  • the first modified library comprises a first Library Tag and the second modified library comprises a second Library Tag, wherein the first Library Tag and the second Library Tag are not identical.
  • each library fragment of the first modified library comprises, from 5’ to 3’: a 5’ oligonucleotide (Al), a first 5’ adaptor module, at least a portion of a DNA sequence of the test sample, and a 3’ oligonucleotide (A2), or a 5’ oligonucleotide (Al), at least a portion of a DNA sequence of the test sample, a first 3’ adaptor module, and a 3’ oligonucleotide (A2); wherein each library fragment of the second modified library comprises, from 5’ to 3’: a 5’ oligonucleotide (Bl), a second 5’ adaptor module, at least a portion of a DNA sequence of the test sample, a second 3’ adaptor module, and a 3’ oligonucleotide (B2).
  • At least one of Al, A2, Bl, and B2 contains at least one Library Tag.
  • the Library Tag comprises a unique polynucleotide sequence, wherein the unique polynucleotide sequence has 70% identity, or less, to a sequence selected from the list consisting of Al, Bl, A2, B2, the first 5’ adaptor module, the target sequence, the first 3’ adaptor module, the second 5’ adaptor module, any DNA fragment of the test sample, and the second 3’ adaptor module.
  • each library fragment of the first modified library comprises, from 5’ to 3’: a 5’ oligonucleotide (Al), a first 5’ adaptor module, at least a portion of a DNA sequence of the test sample, and a 3’ oligonucleotide (A2), or a 5’ oligonucleotide (Al), at least a portion of a DNA sequence of the test sample, a first 3’ adaptor module, and a 3’ oligonucleotide (A2); wherein each library fragment of the second modified library comprises, from 5’ to 3’: a 5’ oligonucleotide (Bl), a second 5’ adaptor module, at least a portion of a DNA sequence of the test sample, a second 3’ adaptor module, and a 3’ oligonucleotide (B2); wherein at least one of Al, A2, Bl, and B2 comprises the Library Tag; wherein
  • each adaptor of the set of adaptors comprises a ligation strand oligonucleotide and a non-ligation strand oligonucleotide.
  • the non-ligation strand oligonucleotide is capable of hybridizing to a region at the 3’ end of the ligation strand oligonucleotide and forming a duplex therewith.
  • the ligation strand oligonucleotide comprises an adaptor module.
  • the ligation strand oligonucleotide comprises a dT, dA, dC, or dG overhang at the 3’ terminus.
  • the non-ligation strand oligonucleotide comprises a modification at its 3’ terminus that prevents ligation to the 5’ end of a dsDNA fragment and/or adaptor dimer formation, wherein the non-ligation strand is configured to be displaced from the duplex.
  • each adaptor of the set of adaptors comprises an ID region selected from a pool of unique ID regions, wherein the pool is selected from a plurality of pools, and wherein the selected pool is unique to the test sample.
  • the adaptor module comprises (a) an amplification region comprising a primer binding site; (b) an ID region; and (c) an anchor region.
  • the amplification region comprises a primer binding site, wherein the primer binding site allows for amplification using PCR (polymerase chain reaction), LAMP (loop-mediated isothermal amplification), NASBA (nucleic acid sequence-based amplification), SDA (standard displacement amplification), RCA (rolling circle replication), or LCR (ligase chain reaction).
  • PCR polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • NASBA nucleic acid sequence-based amplification
  • SDA standard displacement amplification
  • RCA rolling circle replication
  • LCR ligase chain reaction
  • the amplification region comprises or consists of between 10 and 50 nucleotides. In some embodiments, the amplification region comprises or consists of between 20 and 30 nucleotides. In some embodiments, the amplification region comprises or consists of 25 nucleotides.
  • the amplification region comprises or consists of about 10 to about 50 nucleotides. In some embodiments, the amplification region comprises or consists of about 20 to about 30 nucleotides. In some embodiments, the amplification region comprises or consists of about 25 nucleotides.
  • the anchor region comprises an overhang at the 3’ terminus.
  • the anchor region comprises or consists of between 1 and 50 nucleotides. In some embodiments, the anchor region comprises or consists of between 5 and 25 nucleotides. In some embodiments, the anchor region comprises or consists of 10 nucleotides.
  • the anchor region comprises or consists of about 1 to about 50 nucleotides. In some embodiments, the anchor region comprises or consists of about 5 to about 25 nucleotides. In some embodiments, the anchor region comprises or consists of about 10 nucleotides.
  • the ID region comprises or consists of between 3 and 50 nucleotides. In some embodiments, the ID region comprises or consists of between 3 and 15 nucleotides. In some embodiments, the ID region comprises or consists of 8 nucleotides.
  • the ID region comprises or consists of about 3 to about 50 nucleotides. In some embodiments, the ID region comprises or consists of about 3 to about 15 nucleotides. In some embodiments, the ID region comprises or consists of about 8 nucleotides.
  • the adaptor module further comprises a unique molecule identifier (UMI) multiplier.
  • UMI unique molecule identifier
  • the UMI multiplier is adjacent to or contained within the ID region.
  • the UMI multiplier comprises or consists of between 1 and 5 nucleotides.
  • the UMI multiplier comprises or consists of about 1 to about 5 nucleotides.
  • the UMI multiplier is 3 nucleotides in length, and comprises a nucleic acid sequence selected from a group of 64 unique nucleotide sequences.
  • the pool of ID regions comprises between 2 and 10,000 unique ID region sequences. In some embodiments, the pool of ID regions comprises between 10 and 500 unique ID region sequences. In some embodiments, the pool of ID regions comprises between 50 and 300 unique ID region sequences. In some embodiments, the pool of ID regions comprises 60 unique ID region sequences.
  • the pool of ID regions comprises about 2 to about 10,000 unique ID region sequences. In some embodiments, the pool of ID regions comprises about 10 to about 500 unique ID region sequences. In some embodiments, the pool of ID regions comprises about 50 to about 300 unique ID region sequences. In some embodiments, the pool of ID regions comprises about 60 unique ID region sequences.
  • each ID region of the pool of ID regions is 8 nucleotides in length.
  • each ID region of the pool of ID regions is about 8 nucleotides in length.
  • each ID region sequence is discrete from any other ID region sequence by Hamming distance of at least two.
  • each ID region is configured to identify the DNA fragment attached thereto.
  • each adaptor module of the set of adaptor modules is selected from a group consisting of between 64 and 2,560,000 unique nucleotide sequences.
  • each adaptor module of the set of adaptor modules is selected from a group consisting of about 64 to about 2,560,000 unique nucleotide sequences.
  • each adaptor module of the set of adaptor modules comprises a unique nucleotide sequence selected from 3840 unique nucleotide sequences, wherein each sequence of the 3840 unique nucleotide sequences is discrete from any other sequence by Hamming distance of at least two.
  • the anchor region of each adaptor of the set of adaptors comprises one of four nucleotide sequences, and wherein each ID region of a given sequence is paired to only one of the four anchor regions of a given sequence.
  • the amplification region of each adaptor of the set of adaptors comprises an identical primer binding site.
  • each ID region of the pool of ID regions is 8 nucleotides in length, each ID region sequence is discrete from any other ID region sequence by Hamming distance of at least two; each adaptor of the set of adaptors comprises a UMI multiplier that is adjacent to or contained within the ID region, wherein the UMI multiplier of each adaptor of the set of adaptors is three nucleotides in length, and wherein the UMI multiplier of a given sequence is paired to one ID region of a given sequence; the anchor region of each adaptor of the set of adaptors comprises one of four nucleotide sequences, and wherein each ID region of a given sequence is paired to only one of the four anchor regions of a given sequence; and the amplification region of each adaptor of the set of adaptors comprises an identical primer binding site.
  • the kit comprises one set of adaptors. In some embodiments, the kit comprises more than one set of adaptor modules.
  • the kit comprises between 2-1000 sets of adaptor modules. In some embodiments, the kit comprises between 2-500 sets of adaptor modules. In some embodiments, the kit comprises between 2-100 sets of adaptor modules. In some embodiments, the kit comprises between 2-50 sets of adaptor modules. In some embodiments, the kit comprises between 50-100 sets of adaptor modules. In some embodiments, the kit comprises between 5-50 sets of adaptor modules. In some embodiments, the kit comprises between 10- 50 sets of adaptor modules. In some embodiments, the kit comprises between 15-50 sets of adaptor modules. In some embodiments, the kit comprises between 20-50 sets of adaptor modules. In some embodiments, the kit comprises between 30-50 sets of adaptor modules. In some embodiments, the kit comprises between 40-50 sets of adaptor modules. In some embodiments, the kit comprises between 45-50 sets of adaptor modules. In some embodiments, the kit comprises 48 sets of adaptor modules.
  • the kit comprises about 2 to about 1000 sets of adaptor modules. In some embodiments, the kit comprises about 2 to about 500 sets of adaptor modules. In some embodiments, the kit comprises about 2 to about 100 sets of adaptor modules. In some embodiments, the kit comprises about 2 to about 50 sets of adaptor modules. In some embodiments, the kit comprises about 50 to about 100 sets of adaptor modules. In some embodiments, the kit comprises about 5 to about 50 sets of adaptor modules. In some embodiments, the kit comprises about 10 to about 50 sets of adaptor modules. In some embodiments, the kit comprises about 15 to about 50 sets of adaptor modules. In some embodiments, the kit comprises about 20 to about 50 sets of adaptor modules.
  • the kit comprises about 30 to about 50 sets of adaptor modules. In some embodiments, the kit comprises about 40 to about 50 sets of adaptor modules. In some embodiments, the kit comprises about 45 to about 50 sets of adaptor modules. In some embodiments, the kit comprises about 48 sets of adaptor modules.
  • each set of adaptor modules is unique to a given test sample.
  • the kit comprises one or more reagents for ligating adaptors of the adaptor set to the DNA fragments of the test sample to generate an adaptor-tagged DNA library.
  • the one or more reagents for ligating adaptors comprises a DNA ligase.
  • the DNA ligase is T4 DNA ligase.
  • the set of adaptors is configured to ligate to the DNA fragments of the test sample using a method comprising (a) ligating the set of adaptors with the DNA fragments to generate a plurality of adaptor/DNA fragment complexes; and (b) contacting the plurality of adaptor/DNA fragment complexes with one or more enzymes to form an adaptor-tagged DNA library comprising a plurality of adaptor-tagged DNA fragments.
  • each adaptor/DNA fragment complex comprises a ligation strand oligonucleotide ligated to each end of the DNA fragment.
  • the non-ligation strand oligonucleotide is displaced from the adaptor/DNA fragment complex in step (b).
  • the kit comprises one or more reagents for amplifying the adaptor-tagged DNA library to create a Library Post-Amplification (LPA).
  • the one or more reagents for amplifying the adaptor-tagged DNA library comprises a DNA polymerase.
  • one or more reagents for amplifying the adaptor-tagged DNA library comprises one or more primers for PCR.
  • the one or more primers comprise a single primer sequence that is complementary to the primer binding site of the adaptor amplification region.
  • the kit comprises one or more reagents for creating an amplified Whole-Genome Library (WGLA) from the LPA.
  • the one or more reagents for creating a WGLA comprises one or more primers that hybridize to the primer binding site of the adaptor amplification region.
  • the one or more primers comprise a sequencing adaptor capable of binding to a flow cell.
  • the sequencing adaptor comprises a sequencing primer binding site.
  • the one or more reagents for creating the WGLA comprises a DNA polymerase.
  • the WGLA is the sequence ready.
  • the tail sequence of the capture probe module comprises a primer binding site.
  • the tail sequence comprises a sequencing primer binding site.
  • the tail sequence is configured to hybridize to a partner oligonucleotide.
  • each capture probe module is selected from a capture probe panel comprising a plurality of capture probe modules.
  • At least one capture probe module is configured to hybridize downstream of a specific DNA target region and at least one capture probe module is configured to hybridize upstream of the specific DNA target region.
  • each capture probe of the plurality of capture probe modules is configured to hybridize to its target sequence within about 200 bp of any other capture probe.
  • each capture probe module of the capture probe panel hybridizes to an adaptor-tagged DNA fragment to form a plurality of adaptor-tagged DNA fragment-capture probe module complexes.
  • the partner oligonucleotide comprises a specific member of a binding pair to enable isolation of the adaptor-tagged DNA fragment-capture probe module complexes.
  • the partner oligonucleotide comprises a biotin molecule.
  • each capture probe module comprises a specific member of a binding pair to enable isolation of the adaptor-tagged DNA fragment-capture probe module complex.
  • the capture probe module comprises a biotin molecule.
  • each capture probe module comprises a capture probe less than about 60 nucleotides in length.
  • each capture probe module comprises a capture probe less than 60 nucleotides in length.
  • each capture probe module comprises a capture probe that is about 40 nucleotides in length.
  • each capture probe module comprises a capture probe that is 40 nucleotides in length.
  • the kit comprises components for isolating the adaptor-tagged DNA fragment-capture probe module complexes.
  • the components for isolating the adaptor-tagged DNA fragment-capture probe module complexes comprises streptavidin.
  • the kit comprises one of more enzymes for enzymatically processing the isolated adaptor-tagged DNA fragment-capture probe module complexes.
  • the one or more enzymes comprise a 5’ -3’ polymerase configured to extend each capture probe of the adaptor-tagged DNA fragment- capture probe module complexes using the adaptor-tagged DNA fragment as a template to create a plurality of Hybrid Molecules (Target Capture Library), wherein each Hybrid Molecule comprises a capture probe module and a complement of an adaptor-tagged DNA fragment.
  • the kit comprises one or more reagents for amplifying the Hybrid Molecules to create a Target Capture Library Amplified (TCLA), wherein the first modified library is the TCLA.
  • the one or more reagents for amplifying the Hybrid Molecules comprise one or more primers comprising a sequencing adaptor capable of binding to a flow cell.
  • the sequencing adaptor comprises a sequencing primer binding site.
  • the one or more reagents for amplifying the Hybrid Molecules comprise a DNA polymerase.
  • the SRL is configured to be sequenced on a single flow cell of a sequencing machine.
  • the kit comprises components for performing quantitative genetic analysis on the SRL.
  • the components for performing quantitative genetic analysis comprises one or more sequencing primers.
  • the quantitative genetic analysis is used to detect a nucleotide transition or transversion, a nucleotide insertion or deletion, a genomic rearrangement, or a change in copy number in the test sample DNA fragments.
  • the test sample is obtained from a tissue biopsy.
  • the tissue biopsy is obtained from a tumor or a tissue suspected of being a tumor.
  • the tissue biopsy is obtained from a malignant tumor or tumor suspected of being a malignant tumor.
  • test sample DNA fragments comprise cell free DNA (cfDNA), genomic DNA (gDNA), complementary DNA (cDNA), mitochondrial DNA, methylated DNA, demethylated DNA, or a combination thereof.
  • test sample DNA fragments comprise an epigenetic mark.
  • the test sample DNA fragments are obtained from a library selected from the list consisting of a whole genome library, an amplicon library, a whole exome library, a cDNA library, or a methylated DNA library.
  • the test sample is obtained from a biological sample selected from the group consisting of an amniotic fluid sample, a blood sample, a skin sample, a hair sample, a hair follicle sample, a saliva sample, a mucous sample, a sweat sample, a tear sample, an epithelial tissue sample, a urine sample, a semen sample, a seminal plasma sample, a serum sample, a prostatic fluid sample, a pre-ejaculatory fluid (Cowper's fluid) sample, an ocular fluid sample, an excreta sample, a biopsy sample, an ascites sample, a cerebrospinal fluid sample, a lymph sample, a tissue extract sample, a stool sample, and a formalin-fixed, paraffin embedded (FFPE) sample.
  • FFPE formalin-fixed, paraffin embedded
  • the DNA fragments of the test sample have been end repaired prior to ligation to adaptors.
  • the kit comprises one or more reagents for performing end repair.
  • the one or more reagents for performing end repair comprise one or more enzymes selected from a DNA polymerase, a kinase, and a Klenow fragment.
  • the one or more reagents for performing end repair comprise a DNA polymerase I, a T4 DNA polymerase, a T4 polynucleotide kinase, and a Klenow fragment.
  • the one or more reagents for performing end repair comprise an End Repair Buffer.
  • the End Repair Buffer comprises MgC12, NaCl, Tris-HCL, DTT, KCL, and dNTPs.
  • Some embodiments of the disclosure provide methods for performing an adaptor Quality Control (QC) process on a set of adaptors, wherein each adaptor comprises an adaptor module.
  • QC Quality Control
  • the adaptor QC process comprises a test for adaptor ligation, wherein the test for adaptor ligation comprises (a) ligating the set of adaptors to a pre-determined amount of end- repaired DNA fragments to generate a library of adaptor-tagged DNA fragments (LIBS); and (b) amplifying the LIBS to generate a Library Post Amplification (LPA); wherein the set of adaptors is considered to have passed the test for adaptor ligation when the concentration of the LPA is higher than a pre-determined concentration.
  • LIBS library of adaptor-tagged DNA fragments
  • LPA Library Post Amplification
  • the end-repaired DNA fragments of step (a) of the test for adaptor ligation is generated from a DNA sample.
  • said DNA sample is a blended DNA sample of at least two different cell lines.
  • said DNA sample is a blended DNA sample of at two different cell lines.
  • the two different cell lines are blended at a 50:50 ratio.
  • the two different cell lines are NA09596 and NA12878.
  • said DNA sample is the 50:50 blended sample described in Example 15.
  • the DNA sample used in the test for adaptor ligation is different from the DNA sample used in the test for adaptor distribution.
  • the pre-determined amount of end-repaired DNA fragments is about 5 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined amount of end-repaired DNA fragments is about 10 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 15 ng.
  • the pre-determined amount of end- repaired DNA fragments is about 20 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 25 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 30 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 35 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined amount of end-repaired DNA fragments is about 40 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 50 ng.
  • the pre-determined amount of end-repaired DNA fragments is 5 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined amount of end-repaired DNA fragments is 10 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is 15 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is 20 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is 25 ng.
  • the pre determined amount of end-repaired DNA fragments is 30 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is 35 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount is 40 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined amount of end-repaired DNA fragments is 50 ng. [0245] In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 1 ng to about 100 ng.
  • the pre-determined amount of end-repaired DNA fragments is about 1 ng to about 50 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 50 ng to about 100 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 5 ng to about 90 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 10 ng to about 80 ng.
  • the pre-determined amount of end-repaired DNA fragments is about 20 ng to about 60 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 20 ng to about 50 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 20 ng to about 40 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 30 ng to about 50 ng.
  • the pre-determined amount of end-repaired DNA fragments is about 40 ng to about 50 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 30 ng to about 40 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is about 20 ng to about 30 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is greater than about 100 ng.
  • the pre-determined amount of end-repaired DNA fragments is 1 ng to 100 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is 1 ng to 50 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is 50 ng to 100 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is 5 ng to 90 ng.
  • the pre-determined amount of end- repaired DNA fragments is 10 ng to 80 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is 20 ng to 60 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is 20 ng to 50 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is 20 ng to 40 ng.
  • the pre-determined amount of end-repaired DNA fragments is 30 ng to 50 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined amount of end-repaired DNA fragments is 40 ng to 50 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is 30 ng to 40 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end- repaired DNA fragments is 20 ng to 30 ng. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined amount of end-repaired DNA fragments is greater than 100 ng.
  • the pre-determined concentration of the LPA is about 1 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is about 5 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is about 10 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is about 20 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is about 30 ng/pL.
  • the pre-determined concentration of the LPA is about 40 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is about 50 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is about 60 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is about 70 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is about 80 ng/pL.
  • the pre-determined concentration of the LPA is about 90 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is about 100 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is about 150 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is about 200 ng/pL.
  • the pre-determined concentration of the LPA is 1 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 5 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 10 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 20 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 30 ng/pL.
  • the pre-determined concentration of the LPA is 40 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is 50 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 60 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 70 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is 80 ng/pL.
  • the pre-determined concentration of the LPA is 90 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 100 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is 150 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 200 ng/pL.
  • the pre-determined concentration of the LPA is about 1 ng/pL to about 200 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is greater than about 200 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is about 10 ng/pL to about 100 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is about 10 ng/pL to about 90 ng/pL.
  • the pre determined concentration of the LPA is about 20 ng/pL to about 80 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is about 10 ng/pL to about 80 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is about 30 ng/pL to about 80 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is about 40 ng/pL to about 80 ng/pL.
  • the pre determined concentration of the LPA is about 50 ng/pL to about 80 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is about 50 ng/pL to about 70 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is about 60 ng/pL to about 70 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is about 50 ng/pL to about 60 ng/pL.
  • the pre-determined concentration of the LPA is 1 ng/pL to 200 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is 10 ng/pL to 100 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 10 ng/pL to 90 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 20 n g/pL to 80 ng/pL.
  • the pre-determined concentration of the LPA is 10 ng/pL to 80 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 30 ng/pL to 80 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre determined concentration of the LPA is 40 ng/pL to 80 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 50 ng/pL to 80 ng/pL.
  • the pre-determined concentration of the LPA is 50 ng/pL to 70 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 60 ng/pL to 70 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is 50 ng/pL to 60 ng/pL.
  • the pre-determined concentration of the LPA is greater than about 200 ng/pL. In some embodiments of the methods for performing an adaptor QC process of the disclosure, the pre-determined concentration of the LPA is greater than 200 ng/pL.
  • the set of adaptors is considered to have passed the adaptor QC process when it has passed the test for adaptor ligation.
  • the adaptor QC process comprises a test for adaptor distribution comprising (a) ligating the set of adaptors to a pre-determined amount of end-repaired DNA fragments to generate a library of adaptor-tagged DNA fragments (LIBS); (b) amplifying the LIBS using at least one primer comprising an index sequence to generate a Library Post Index Amplification (LPIA); and (c) performing a quantitative genetic analysis on the LPIA, wherein the set of adaptors is considered to have passed the test for adaptor distribution when one or more pre determined acceptance criteria for the quantitative genetic analysis has been met.
  • LIBS library of adaptor-tagged DNA fragments
  • LPIA Library Post Index Amplification
  • the end-repaired DNA fragments of step (a) of the test for adaptor distribution are generated from a DNA sample.
  • said DNA sample comprises wild-type (wt) cell-free DNA (cfDNA).
  • said DNA sample consists of wt cfDNA.
  • said DNA sample comprises wt cfDNA obtained from a healthy human.
  • said DNA sample is the wt cfDNA sample described in Example 16.
  • the DNA sample used in the test for adaptor distribution is different from the DNA sample used in the test for adaptor ligation.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (a).
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.05% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.1% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.2% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.3% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.4% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.6% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.7% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.8% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.9% of reads.
  • criterion (a) is Barcode Crosstalk present in no more than about 1% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than about 2% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than about 3% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than about 4% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than about 5% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than 0.05% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.1% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.2% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 0.3% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 0.4% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.5% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than 0.6% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.7% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.8% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 0.9% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 1% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 2% of reads.
  • criterion (a) is Barcode Crosstalk present in no more than 3% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 4% of reads. In some embodiments, criterion (a) is Barcode Crosstalk present in no more than 5% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.05% to about 10% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.05% to about 5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.1% to about 5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.05% to about 2% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.1% to about 1% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than about 0.1% to about 0.5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.1% to about 0.4% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than about 0.1% to about 0.2% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than 0.05% to 10% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.05% to 5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.1% to 5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.05% to 2% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.1% to 1% of reads.
  • criterion (a) is Barcode Crosstalk is present in no more than 0.1% to 0.5% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.1% to 0.4% of reads. In some embodiments, criterion (a) is Barcode Crosstalk is present in no more than 0.1% to 0.2% of reads.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (b).
  • criterion (b) is unknown adaptors are present in no more than about 1% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 5% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 10% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 15% of reads.
  • criterion (b) is unknown adaptors are present in no more than about 20% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 25% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 30% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 35% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 40% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 45% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 50% of reads.
  • criterion (b) is unknown adaptors are present in no more than 1% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 5% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 10% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 15% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 20% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 25% of reads.
  • criterion (b) is unknown adaptors are present in no more than 30% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 35% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 40% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 45% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 50% of reads. [0262] In some embodiments, criterion (b) is unknown adaptors are present in no more than about 1% to about 50% of reads.
  • criterion (b) is unknown adaptors are present in no more than about 1% to about 20% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 1% to about 15% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 1% to about 10% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 5% to about 20% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 5% to about 15% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than about 5% to about 10% of reads.
  • criterion (b) is unknown adaptors are present in no more than 1% to 50% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 1% to 20% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 1% to 15% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 1% to 10% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 5% to 20% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 5% to 15% of reads. In some embodiments, criterion (b) is unknown adaptors are present in no more than 5% to 10% of reads.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (c).
  • criterion (c) is no more than about 10% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than about 20% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than about 30% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 40% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 50% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than about 60% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 70% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 80% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 90% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (c).
  • criterion (c) is no more than 10% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than 20% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than 30% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than 40% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 50% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 60% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than 70% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 80% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 90% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than about 10% to about 90% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 10% to about 80% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 20% to about 80% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than about 30% to about 70% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 40% to about 60% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 40% to about 50% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than about 50% to about 60% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than 10% to 90% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 10% to 80% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 20% to 80% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • criterion (c) is no more than 30% to 70% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 40% to 60% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 40% to 50% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments, criterion (c) is no more than 50% to 60% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (d).
  • criterion (d) is at least about 60% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 65% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 70% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 75% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 80% of all unique adaptor sequences are present.
  • criterion (d) is at least about 85% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 90% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 95% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 96% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 97% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 98% of all unique adaptor sequences are present.
  • criterion (d) is at least about 99% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 99.5% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is about 100% of all unique adaptor sequences are present.
  • criterion (d) is at least 60% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 65% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 70% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 75% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 80% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 85% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 90% of all unique adaptor sequences are present.
  • criterion (d) is at least 95% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 96% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 97% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 98% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 99% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 99.5% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is 100% of all unique adaptor sequences are present.
  • criterion (d) is at least about 60% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 70% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 80% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 90% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 95% to about 99.9% of all unique adaptor sequences are present.
  • criterion (d) is at least about 96% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 97% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 98% to about 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 99% to about 99.9% of all unique adaptor sequences are present. [0271] In some embodiments, criterion (d) is at least 60% to 99.9% of all unique adaptor sequences are present.
  • criterion (d) is at least 70% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 80% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 90% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 95% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 96% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 97% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 98% to 99.9% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 99% to 99.9% of all unique adaptor sequences are present.
  • criterion (d) is at least about 60% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 70% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 80% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 90% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 95% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 96% to about 100% of all unique adaptor sequences are present.
  • criterion (d) is at least about 97% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 98% to about 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least about 99% to about 100% of all unique adaptor sequences are present.
  • criterion (d) is at least 60% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 70% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 80% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 90% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 95% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 96% to 100% of all unique adaptor sequences are present.
  • criterion (d) is at least 97% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 98% to 100% of all unique adaptor sequences are present. In some embodiments, criterion (d) is at least 99% to 100% of all unique adaptor sequences are present.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (e).
  • criterion (e) is no more than about 5% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than about 10% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than about 15% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than about 20% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 25% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 30% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 35% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than about 40% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 45% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 50% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than 5% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 10% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 15% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 20% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than 25% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 30% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 35% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 40% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than 45% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 50% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than about 1% to about 50% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 5% to about 50% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 5% to about 40% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than about 10% to about 30% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 10% to about 20% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 20% to about 40% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 20% to about 30% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than about 15% to about 25% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than about 15% to about 20% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. [0277] In some embodiments, criterion (e) is no more than 1% to 50% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 5% to 50% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than 5% to 40% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 10% to 30% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 10% to 20% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 20% to 40% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • criterion (e) is no more than 20% to 30% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 15% to 25% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences. In some embodiments, criterion (e) is no more than 15% to 20% of unique adaptor sequences have reads greater than twice the average number of reads for all unique adaptor sequences.
  • the pre-determined acceptance criteria for the adaptor QC process comprise a criterion (a) and a criterion (b).
  • the pre-determined acceptance criteria comprise a criterion (c), a criterion (d), and a criterion (e).
  • the pre determined acceptance criteria comprise a criterion (a), a criterion (b), and a criterion (c).
  • the pre-determined acceptance criteria comprise a criterion (a), a criterion (b), and a criterion (d).
  • the pre-determined acceptance criteria comprise a criterion (a), a criterion (b), and a criterion (e). In some embodiments, the pre-determined acceptance criteria comprise a criterion (a), a criterion (b), a criterion (c), and a criterion (d). In some embodiments, the pre-determined acceptance criteria comprise a criterion (a), a criterion (b), a criterion (c), and a criterion (e). In some embodiments, the pre-determined acceptance criteria comprise a criterion (a), a criterion (b), a criterion (d), and a criterion (e).
  • the pre-determined acceptance criteria comprise a criterion (a), a criterion (b), a criterion (c), a criterion (d), and a criterion (e).
  • the pre- determined acceptance criteria comprise at least one criterion selected from criteria (a) (b) (c) (d) and (e).
  • the set of adaptors is considered to have passed the adaptor QC process when it has passed the test for adaptor distribution.
  • the set of adaptors is considered to have passed the adaptor QC process when it has passed both the test for adaptor ligation and the test for adaptor distribution.
  • each adaptor of the set of adaptors comprises a ligation strand oligonucleotide and a non-ligation strand oligonucleotide.
  • the non-ligation strand oligonucleotide is capable of hybridizing to a region at the 3’ end of the ligation strand oligonucleotide and forming a duplex therewith.
  • the ligation strand oligonucleotide comprises an adaptor module.
  • the ligation strand oligonucleotide comprises a dT, dA, dC, or dG overhang at the 3’ terminus.
  • the non-ligation strand oligonucleotide comprises a modification at its 3’ terminus that prevents ligation to the 5’ end of a dsDNA fragment and/or adaptor dimer formation, wherein the non-ligation strand is configured to be displaced from the duplex.
  • each adaptor of the set of adaptors comprises an ID region selected from a pool of unique ID regions, wherein the pool is selected from a plurality of pools, and wherein the selected pool is unique to the test sample.
  • the adaptor module comprises (a) an amplification region comprising a primer binding site; (b) an ID region; and (c) an anchor region.
  • the amplification region comprises a primer binding site, wherein the primer binding site allows for amplification using PCR (polymerase chain reaction), LAMP (loop- mediated isothermal amplification), NASBA (nucleic acid sequence-based amplification), SDA (standard displacement amplification), RCA (rolling circle replication), or LCR (ligase chain reaction).
  • PCR polymerase chain reaction
  • LAMP loop- mediated isothermal amplification
  • NASBA nucleic acid sequence-based amplification
  • SDA standard displacement amplification
  • RCA rolling circle replication
  • LCR ligase chain reaction
  • the amplification region comprises or consists of between 10 and 50 nucleotides. In some embodiments, the amplification region comprises or consists of between 20 and 30 nucleotides. In some embodiments, the amplification region comprises or consists of 25 nucleotides.
  • the amplification region comprises or consists of about 10 to about 50 nucleotides. In some embodiments, the amplification region comprises or consists of about 20 to about 30 nucleotides. In some embodiments, the amplification region comprises or consists of about 25 nucleotides.
  • the anchor region comprises an overhang at the 3’ terminus.
  • the anchor region comprises or consists of between 1 and 50 nucleotides. In some embodiments, the anchor region comprises or consists of between 5 and 25 nucleotides. In some embodiments, the anchor region comprises or consists of 10 nucleotides.
  • the anchor region comprises or consists of about 1 to about 50 nucleotides. In some embodiments, the anchor region comprises or consists of about 5 to about 25 nucleotides. In some embodiments, the anchor region comprises or consists of about 10 nucleotides.
  • the ID region comprises or consists of between 3 and 50 nucleotides. In some embodiments, the ID region comprises or consists of between 3 and 15 nucleotides. In some embodiments, the ID region comprises or consists of 8 nucleotides.
  • the ID region comprises or consists of about 3 to about 50 nucleotides. In some embodiments, the ID region comprises or consists of about 3 to about 15 nucleotides. In some embodiments, the ID region comprises or consists of about 8 nucleotides. [0296] In some embodiments of the methods for performing an adaptor QC process of the disclosure, the adaptor module further comprises a unique molecule identifier (UMI) multiplier.
  • UMI unique molecule identifier
  • the UMI multiplier is adjacent to or contained within the ID region.
  • the UMI multiplier comprises or consists of between 1 and 5 nucleotides.
  • the UMI multiplier comprises or consists of about 1 to about 5 nucleotides.
  • the UMI multiplier is 3 nucleotides in length, and comprises a nucleic acid sequence selected from a group of 64 unique nucleotide sequences.
  • the pool of ID regions comprises between 2 and 10,000 unique ID region sequences. In some embodiments, the pool of ID regions comprises between 10 and 500 unique ID region sequences. In some embodiments, the pool of ID regions comprises between 50 and 300 unique ID region sequences. In some embodiments, the pool of ID regions comprises 60 unique ID region sequences.
  • the pool of ID regions comprises about 2 to about 10,000 unique ID region sequences. In some embodiments, the pool of ID regions comprises about 10 to about 500 unique ID region sequences. In some embodiments, the pool of ID regions comprises about 50 to about 300 unique ID region sequences. In some embodiments, the pool of ID regions comprises about 60 unique ID region sequences.
  • each ID region of the pool of ID regions is 8 nucleotides in length.
  • each ID region sequence is discrete from any other ID region sequence by Hamming distance of at least two.
  • each ID region is configured to identify the DNA fragment attached thereto.
  • each adaptor module of the set of adaptor modules is selected from a group consisting of between 64 and 2,560,000 unique nucleotide sequences.
  • each adaptor module of the set of adaptor modules is selected from a group consisting of about 64 to about 2,560,000 unique nucleotide sequences.
  • each adaptor module of the set of adaptor modules comprises a unique nucleotide sequence selected from 3840 unique nucleotide sequences, wherein each sequence of the 3840 unique nucleotide sequences is discrete from any other sequence by Hamming distance of at least two.
  • the anchor region of each adaptor of the set of adaptors comprises one of four nucleotide sequences, and wherein each ID region of a given sequence is paired to only one of the four anchor regions of a given sequence.
  • the amplification region of each adaptor of the set of adaptors comprises an identical primer binding site.
  • each ID region of the pool of ID regions is 8 nucleotides in length, each ID region sequence is discrete from any other ID region sequence by Hamming distance of at least two; each adaptor of the set of adaptors comprises a UMI multiplier that is adjacent to or contained within the ID region, wherein the UMI multiplier of each adaptor of the set of adaptors is three nucleotides in length, and wherein the UMI multiplier of a given sequence is paired to one ID region of a given sequence; the anchor region of each adaptor of the set of adaptors comprises one of four nucleotide sequences, and wherein each ID region of a given sequence is paired to only one of the four anchor regions of a given sequence; and the amplification region of each adaptor of the set of adaptors comprises an identical primer binding site.
  • the method comprises (a) ligating a set of adaptors to DNA fragments of a test sample to generate a plurality of adaptor/DNA fragment complexes; and (b) contacting the plurality of adaptor/DNA fragment complexes with one or more enzymes to form an adaptor- tagged DNA library comprising a plurality of adaptor-tagged DNA fragments.
  • each adaptor/DNA fragment complex comprises a ligation strand oligonucleotide ligated to each end of the DNA fragment.
  • the non ligation strand oligonucleotide is displaced from the adaptor/DNA fragment complex in step (b).
  • the set of adaptors is any adaptor set of the disclosure.
  • Some embodiments of the disclosure provide methods for performing a probe Quality Control (QC) process on one or more capture probe modules.
  • QC probe Quality Control
  • the method comprises a test for capture probe modules comprising (a) ligating a set of adaptors to a DNA sample comprising end-repaired DNA fragments to generate a library of adaptor-tagged DNA fragments (LIBS); (b) amplifying the LIBS to generate a Library Post Amplification (LPA); (c) splitting or diluting the LPA to generate a Target Capture LPA (TC LPA) and a Whole-Genome LPA (WG LPA); (d) amplifying the WG LPA to generate a Whole-Genome Library Amplified (WGLA); (e) hybridizing the one or more capture probe modules to be tested to the TC LPA to form adaptor-tagged DNA fragment-capture probe module complexes; (f) isolating the adaptor-tagged DNA fragment-capture probe module complexes to form isolated adaptor-tagged DNA fragment-capture probe module complexes; (g) enzymatically processing the
  • the TC LPA is at least about 1 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 15 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 20 ng/pL.
  • the TC LPA is at least about 25 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 30 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 35 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 40 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 45 ng/pL.
  • the TC LPA is at least about 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 55 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 60 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 65 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 70 ng/pL.
  • the TC LPA is at least about 75 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 80 ng/pL.
  • the TC LPA is at least 1 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 15 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 20 ng/pL.
  • the TC LPA is at least 25 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 30 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 35 ng/pL. In some embodiments of the kits of the disclosure, the TC LPA is at least 40 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 45 ng/pL.
  • the TC LPA is at least 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 55 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 60 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 65 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 70 ng/pL.
  • the TC LPA is at least 75 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 80 ng/pL.
  • the TC LPA is at least about 1 ng/pL to about 100 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 1 ng/pL to about 80 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 10 ng/pL to about 80 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 10 ng/pL to about 70 ng/pL.
  • the TC LPA is at least about 10 ng/pL to about 60 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 20 ng/pL to about 60 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 20 ng/pL to about 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 20 ng/pL to about 40 ng/pL.
  • the TC LPA is at least about 30 ng/pL to about 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 30 ng/pL to about 40 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least about 20 ng/pL to about 30 ng/pL.
  • the TC LPA is at least 1 ng/pL to 100 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 1 ng/pL to 80 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 10 ng/pL to 80 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 10 ng/pL to 70 ng/pL.
  • the TC LPA is at least 10 ng/pL to 60 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 20 ng/pL to 60 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 20 ng/pL to 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 20 ng/pL to 40 ng/pL.
  • the TC LPA is at least 30 ng/pL to 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 30 ng/pL to 40 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TC LPA is at least 20 ng/pL to 30 ng/pL.
  • the WG LPA is at least about 0.1 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 0.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 1 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 1.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 2 ng/pL.
  • the WG LPA is at least about 2.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 3 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 3.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 4 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 4.5 ng/pL.
  • the WG LPA is at least about 5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 5.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 6 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLPA is at least about 6.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 7 ng/pL.
  • the WG LPA is at least about 7.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 8 ng/pL.
  • the WG LPA is at least 0.1 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 0.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 1 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 1.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 2 ng/pL.
  • the WG LPA is at least 2.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 3 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 3.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 4 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 4.5 ng/pL.
  • the WG LPA is at least 5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 5.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 6 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 6.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 7 ng/pL.
  • the WG LPA is at least 7.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 8 ng/pL.
  • the WG LPA is at least about 0.1 ng/pL to about 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 0.1 ng/pL to about 8 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 1 ng/pL to about 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 1 ng/pL to about 5 ng/pL.
  • the WG LPA is at least about 2 ng/pL to about 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 2 ng/pL to about 6 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 3 ng/pL to about 5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 3 ng/pL to about 4 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least about 2 ng/pL to about 3 ng/pL.
  • the WGLPA is at least 0.1 ng/pL to 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 0.1 ng/pL to 8 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 1 ng/pL to 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 1 ng/pL to 5 ng/pL.
  • the WG LPA is at least 2 ng/pL to 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 2 ng/pL to 6 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 3 ng/pL to 5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 3 ng/pL to 4 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WG LPA is at least 2 ng/pL to 3 ng/pL.
  • the TCLA is at least about 1.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 1.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 2.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 2.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 3.0 ng/pL.
  • the TCLA is at least about 3.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 4.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about at least 4.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 5.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 5.5 ng/pL.
  • the TCLA is at least about 6.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 6.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 7.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 7.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 8.0 ng/pL.
  • the TCLA is at least about 8.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 9.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 9.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 15 ng/pL.
  • the TCLA is at least about 20 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 25 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 30 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 35 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 40 ng/pL.
  • the TCLA is at least about 45 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 55 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 60 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 65 ng/pL.
  • the TCLA is at least about 70 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 75 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 80 ng/pL.
  • the TCLA is at least 1.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 1.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 2.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 2.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 3.0 ng/pL.
  • the TCLA is at least 3.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 4.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about at least 4.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 5.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 5.5 ng/pL.
  • the TCLA is at least 6.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 6.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 7.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 7.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 8.0 ng/pL.
  • the TCLA is at least 8.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 9.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 9.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 15 ng/pL.
  • the TCLA is at least 20 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 25 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 30 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 35 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 40 ng/pL.
  • the TCLA is at least 45 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 55 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 60 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 65 ng/pL.
  • the TCLA is at least 70 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 75 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 80 ng/pL.
  • the TCLA is at least about 1 ng/pL to about 200 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 100 ng/pL to about 200 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 1 ng/pL to about 100 ng/pL. In some embodiments of the kits of the disclosure, the TCLA is at least about 1 ng/pL to about 80 ng/pL.
  • the TCLA is at least about 1 ng/pL to about 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 1 ng/pL to about 40 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 1 ng/pL to about 30 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 1 ng/pL to about 20 ng/pL.
  • the TCLA is at least about 1 ng/pL to about 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least about 5 ng/pL to about 10 ng/pL.
  • the TCLA is at least 1 ng/pL to 200 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 100 ng/pL to 200 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 1 ng/pL to 100 ng/pL. In some embodiments of the kits of the disclosure, the TCLA is at least 1 ng/pL to 80 ng/pL.
  • the TCLA is at least 1 ng/pL to 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 1 ng/pL to 40 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 1 ng/pL to 30 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 1 ng/pL to 20 ng/pL.
  • the TCLA is at least 1 ng/pL to 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the TCLA is at least 5 ng/pL to 10 ng/pL.
  • the WGLA is at least about 1.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 1.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 2.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 2.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 3.0 ng/pL.
  • the WGLA is at least about 3.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 4.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about at least 4.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 5.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 5.5 ng/pL.
  • the WGLA is at least about 6.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 6.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 7.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 7.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 8.0 ng/pL.
  • the WGLA is at least about 8.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 9.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 9.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 15 ng/pL.
  • the WGLA is at least about 20 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 25 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 30 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 35 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 40 ng/pL.
  • the WGLA is at least about 45 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 55 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 60 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 65 ng/pL.
  • the WGLA is at least about 70 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 75 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 80 ng/pL.
  • the WGLA is at least 1.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 1.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 2.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 2.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 3.0 ng/pL.
  • the WGLA is at least 3.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 4.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about at least 4.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 5.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 5.5 ng/pL.
  • the WGLA is at least 6.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 6.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 7.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 7.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 8.0 ng/pL.
  • the WGLA is at least 8.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 9.0 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 9.5 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 15 ng/pL.
  • the WGLA is at least 20 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 25 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 30 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 35 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 40 ng/pL.
  • the WGLA is at least 45 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 55 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 60 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 65 ng/pL.
  • the WGLA is at least 70 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 75 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 80 ng/pL.
  • the WGLA is at least about 1 ng/pL to about 200 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 100 ng/pL to about 200 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 1 ng/pL to about 100 ng/pL. In some embodiments of the kits of the disclosure, the WGLA is at least about 1 ng/pL to about 80 ng/pL.
  • the WGLA is at least about 1 ng/pL to about 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 1 ng/pL to about 40 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 1 ng/pL to about 30 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 1 ng/pL to about 20 ng/pL.
  • the WGLA is at least about 1 ng/pL to about 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least about 5 ng/pL to about 10 ng/pL.
  • the WGLA is at least 1 ng/pL to 200 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 100 ng/pL to 200 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 1 ng/pL to 100 ng/pL. In some embodiments of the kits of the disclosure, the WGLA is at least 1 ng/pL to 80 ng/pL.
  • the WGLA is at least 1 ng/pL to 50 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 1 ng/pL to 40 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 1 ng/pL to 30 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 1 ng/pL to 20 ng/pL.
  • the WGLA is at least 1 ng/pL to 10 ng/pL. In some embodiments of the methods for performing a probe QC process of the disclosure, the WGLA is at least 5 ng/pL to 10 ng/pL. [0331] In some embodiments of the methods for performing a probe QC process of the disclosure, the one or more capture probe modules are considered to have passed the probe QC process if one or more pre-determined acceptance criteria for the quantitative genetic analysis have been met.
  • the pre-determined acceptance criteria for the probe QC process comprise a criterion (a).
  • criterion (a) is about 100% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 99.9% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 99.8% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 99.7% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 99.6% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99.5% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99.4% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99.3% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99.2% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99.1% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 99% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.8% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.7% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.6% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.5% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 98.4% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.3% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.2% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98.1% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 97% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 96% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least about 95% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least about 90% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least about 85% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least about 80% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 75% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least about 70% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least about 65% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least about 60% of capture probes have at least 1 total reads.
  • criterion (a) is 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.8% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.7% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.6% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.5% of capture probes have at least 1 total reads.
  • criterion (a) is at least 99.4% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.3% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.2% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.1% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.9% of capture probes have at least 1 total reads.
  • criterion (a) is at least 98.8% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.7% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.6% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.5% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.4% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.3% of capture probes have at least 1 total reads.
  • criterion (a) is at least 98.2% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98.1% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 97% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 96% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least 95% of capture probes have at least 1 total reads.
  • criterion (a) is at least 90% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least 85% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least 80% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least 75% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least 70% of capture probes have at least 1 total reads.
  • criterion (a) is at least 65% of capture probes have at least 1 total reads. In some embodiments of the kits of the disclosure, criterion (a) is at least 60% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 60% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 70% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 80% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 90% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 95% to about 99.9% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 96% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 97% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99% to about 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99% to about 100% of capture probes have at least 1 total reads.
  • criterion (a) is at least 60% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 70% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 80% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 90% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 95% to 99.9% of capture probes have at least 1 total reads.
  • criterion (a) is at least 96% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 97% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99% to 99.9% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99% to 100% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 60% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 70% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 80% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 90% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 95% to about 100% of capture probes have at least 1 total reads.
  • criterion (a) is at least about 96% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 97% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 98% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99.5% to about 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least about 99.9% to about 100% of capture probes have at least 1 total reads.
  • criterion (a) is at least 60% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 70% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 80% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 90% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 95% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 96% to 100% of capture probes have at least 1 total reads.
  • criterion (a) is at least 97% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 98% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.5% to 100% of capture probes have at least 1 total reads. In some embodiments, criterion (a) is at least 99.9% to 100% of capture probes have at least 1 total reads.
  • the pre-determined acceptance criteria for the probe QC process comprise a criterion (b).
  • criterion (b) is about 100% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 99.9% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 99.5% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 99% of capture probes have at least 10-200 on- target total reads.
  • criterion (b) is at least about 98.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 98% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 97.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 97% of capture probes have at least 10- 200 on-target total reads. In some embodiments, criterion (b) is at least about 96.5% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 96% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 95.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 95% of capture probes have at least 10- 200 on-target total reads. In some embodiments, the pre-determined acceptance criteria for the probe QC process comprise criterion (b) at least about 90% of capture probes have at least 10- 200 on-target total reads. In some embodiments, criterion (b) is at least about 85% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 80% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 75% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 70% of capture probes have at least 10- 200 on-target total reads. In some embodiments, criterion (b) is at least about 65% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 60% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is 100% of capture probes have at least 10-200 on- target total reads. In some embodiments, criterion (b) is at least 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 99.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 99% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 98.5% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least 98% of capture probes have at least 10-200 on- target total reads. In some embodiments, criterion (b) is at least 97.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 97% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 96.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 96% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least 95.5% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 95% of capture probes have at least 10-200 on-target total reads. In some embodiments of the kits of the disclosure, the pre-determined acceptance criteria for the probe QC process comprise criterion (b) at least 90% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 85% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 80% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least 75% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 70% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 65% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 60% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is about 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 99.9% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 99.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 99% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 98.5% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least about 98% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 97.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 97% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 96.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 96% of capture probes have at least 50 on- target total reads.
  • criterion (b) is at least about 95.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 95% of capture probes have at least 50 on-target total reads. In some embodiments, the pre determined acceptance criteria for the probe QC process comprise criterion (b) at least about 90% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 85% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 80% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least about 75% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 70% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 65% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 60% of capture probes have at least 50 on-target total reads. [0341] In some embodiments, criterion (b) is 100% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 99.9% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 99.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 99% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 98.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 98% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 97.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 97% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 96.5% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 96% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 95.5% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 95% of capture probes have at least 50 on-target total reads.
  • the pre-determined acceptance criteria for the probe QC process comprise criterion (b) at least 90% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 85% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 80% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 75% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 70% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 65% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 60% of capture probes have at least 50 on-target total reads.
  • criterion (b) is about 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 99.9% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 99.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 99% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 98.5% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least about 98% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 97.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 97% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 96.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 96% of capture probes have at least 60 on- target total reads.
  • criterion (b) is at least about 95.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 95% of capture probes have at least 60 on-target total reads. In some embodiments, the pre determined acceptance criteria for the probe QC process comprise criterion (b) at least about 90% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 85% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 80% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least about 75% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 70% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 65% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 60% of capture probes have at least 60 on-target total reads. [0343] In some embodiments, criterion (b) is 100% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 99.9% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 99.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 99% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 98.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 98% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 97.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 97% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 96.5% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 96% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 95.5% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 95% of capture probes have at least 60 on-target total reads.
  • the pre-determined acceptance criteria for the probe QC process comprise criterion (b) at least 90% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 85% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 80% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 75% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 70% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 65% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 60% of capture probes have at least 60 on-target total reads.
  • criterion (b) is about 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 99.9% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 99.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 99% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 98.5% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least about 98% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 97.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 97% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 96.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 96% of capture probes have at least 70 on- target total reads.
  • criterion (b) is at least about 95.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 95% of capture probes have at least 70 on-target total reads. In some embodiments, the pre determined acceptance criteria for the probe QC process comprise criterion (b) at least about 90% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 85% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 80% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least about 75% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 70% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 65% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 60% of capture probes have at least 70 on-target total reads. [0345] In some embodiments, criterion (b) is 100% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 99.9% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 99.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 99% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 98.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 98% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 97.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 97% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 96.5% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 96% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 95.5% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 95% of capture probes have at least 70 on-target total reads.
  • the pre-determined acceptance criteria for the probe QC process comprise criterion (b) at least 90% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 85% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 80% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 75% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 70% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 65% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 60% of capture probes have at least 70 on-target total reads.
  • criterion (b) is about 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 99.9% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 99.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 99% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 98.5% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least about 98% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 97.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 97% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 96.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 96% of capture probes have at least 80 on- target total reads.
  • criterion (b) is at least about 95.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 95% of capture probes have at least 80 on-target total reads. In some embodiments, the pre determined acceptance criteria for the probe QC process comprise criterion (b) at least about 90% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 85% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 80% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least about 75% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 70% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 65% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 60% of capture probes have at least 80 on-target total reads. [0347] In some embodiments, criterion (b) is 100% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 99.9% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 99.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 99% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 98.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 98% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 97.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 97% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 96.5% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 96% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 95.5% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 95% of capture probes have at least 80 on-target total reads.
  • the pre-determined acceptance criteria for the probe QC process comprise criterion (b) at least 90% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 85% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 80% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 75% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 70% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 65% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 60% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least about 60% to about 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 70% to about 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 80% to about 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 90% to about 99.9% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 95% to about 99.9% of capture probes have at least 10-200 on- target total reads. In some embodiments, criterion (b) is at least about 99% to about 99.9% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least 60% to 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 70% to 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 80% to 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 90% to 99.9% of capture probes have at least 10-200 on- target total reads. In some embodiments, criterion (b) is at least 95% to 99.9% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 99% to 99.9% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 60% to about 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 70% to about 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 80% to about 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 90% to about 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least about 95% to about 100% of capture probes have at least 10-200 on- target total reads.
  • criterion (b) is at least about 99% to about 100% of capture probes have at least 10-200 on-target total reads. [0351] In some embodiments, criterion (b) is at least 60% to 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 70% to 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 80% to 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 90% to 100% of capture probes have at least 10-200 on- target total reads.
  • criterion (b) is at least 95% to 100% of capture probes have at least 10-200 on-target total reads. In some embodiments, criterion (b) is at least 99% to 100% of capture probes have at least 10-200 on-target total reads.
  • criterion (b) is at least about 60% to about 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 70% to about 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 80% to about 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 90% to about 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 95% to about 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least about 99% to about 100% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least 60% to 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 70% to 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 80% to 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 90% to 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 95% to 100% of capture probes have at least 50 on-target total reads. In some embodiments, criterion (b) is at least 99% to 100% of capture probes have at least 50 on-target total reads.
  • criterion (b) is at least about 60% to about 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 70% to about 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 80% to about 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 90% to about 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 95% to about 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least about 99% to about 100% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least 60% to 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 70% to 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 80% to 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 90% to 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 95% to 100% of capture probes have at least 60 on-target total reads. In some embodiments, criterion (b) is at least 99% to 100% of capture probes have at least 60 on-target total reads.
  • criterion (b) is at least about 60% to about 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 70% to about 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 80% to about 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 90% to about 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 95% to about 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least about 99% to about 100% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least 60% to 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 70% to 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 80% to 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 90% to 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 95% to 100% of capture probes have at least 70 on-target total reads. In some embodiments, criterion (b) is at least 99% to 100% of capture probes have at least 70 on-target total reads.
  • criterion (b) is at least about 60% to about 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 70% to about 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 80% to about 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 90% to about 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 95% to about 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least about 99% to about 100% of capture probes have at least 80 on-target total reads.
  • criterion (b) is at least 60% to 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 70% to 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 80% to 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 90% to 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 95% to 100% of capture probes have at least 80 on-target total reads. In some embodiments, criterion (b) is at least 99% to 100% of capture probes have at least 80 on-target total reads.
  • the pre-determined acceptance criteria for the probe QC process comprise a criterion (c).
  • criterion (c) is at least about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 99.5% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 99% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 98% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 97% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 96% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 95% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 90% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 85% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 80% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 75% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 70% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 65% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 60% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 99.5% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 99% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 98% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 97% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 96% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least 95% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 90% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 85% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 80% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 75% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 70% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 65% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 60% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 60% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 70% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 80% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 90% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 95% to about 99.9% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 96% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 97% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 98% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 99% to about 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is about 100% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least 60% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 70% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 80% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 90% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 95% to 99.9% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least 96% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 97% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 98% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 99% to 99.9% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is 100% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 60% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 70% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 80% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 90% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 95% to about 100% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least about 96% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 97% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 98% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 99% to about 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is about 100% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least 60% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 70% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 80% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 90% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 95% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 96% to 100% of expected SNPs within the DNA sample are detected.
  • criterion (c) is at least 97% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least 98% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is at least about 99% to 100% of expected SNPs within the DNA sample are detected. In some embodiments, criterion (c) is 100% of expected SNPs within the DNA sample are detected.
  • the pre-determined acceptance criteria for the probe QC process comprise a criterion (a) and a criterion (b). In some embodiments of the methods for performing a probe QC process of the disclosure, the pre-determined acceptance criteria for the probe QC process comprise a criterion (a) and a criterion (c). In some embodiments of the methods for performing a probe QC process of the disclosure, the pre-determined acceptance criteria for the probe QC process comprise a criterion (b) and a criterion (c). In some embodiments of the methods for performing a probe QC process of the disclosure, the pre-determined acceptance criteria for the probe QC process comprise a criterion (a), a criterion (b), and a criterion (c).
  • the DNA sample used in the probe QC process comprises human wild type DNA.
  • the DNA sample used in the probe QC process comprises a plurality of single nucleotide polymorphisms (SNPs).
  • the DNA sample used in the probe QC process comprises any suitable DNA.
  • the tail sequence of the capture probe module comprises a primer binding site.
  • the tail sequence comprises a sequencing primer binding site.
  • the tail sequence is configured to hybridize to a partner oligonucleotide.
  • each capture probe module is selected from a capture probe panel comprising a plurality of capture probe modules.
  • At least one capture probe module is configured to hybridize downstream of a specific DNA target region and at least one capture probe module is configured to hybridize upstream of the specific DNA target region.
  • each capture probe of the plurality of capture probe modules is configured to hybridize to its target sequence within about 200 bp of any other capture probe.
  • each capture probe module of the capture probe panel hybridizes to an adaptor- tagged DNA fragment to form a plurality of adaptor-tagged DNA fragment-capture probe module complexes.
  • the partner oligonucleotide comprises a specific member of a binding pair to enable isolation of the adaptor-tagged DNA fragment-capture probe module complexes.
  • the partner oligonucleotide comprises a biotin molecule.
  • each capture probe module comprises a specific member of a binding pair to enable isolation of the adaptor-tagged DNA fragment-capture probe module complex.
  • the capture probe module comprises a biotin molecule.
  • each capture probe module comprises a capture probe less than about 60 nucleotides in length.
  • each capture probe module comprises a capture probe less than 60 nucleotides in length.
  • each capture probe module comprises a capture probe that is about 40 nucleotides in length.
  • each capture probe module comprises a capture probe that is 40 nucleotides in length.
  • the step of enzymatically processing the isolated adaptor-tagged DNA fragment- capture probe module complexes to generate Hybrid Molecules comprises performing 5’-3’ polymerase extension on each capture probe of the adaptor-tagged DNA fragment-capture probe module complexes using the adaptor-tagged DNA fragment as a template to create a plurality of Hybrid Molecules (Target Capture Library), wherein each Hybrid Molecule comprises a capture probe module and a complement of an adaptor-tagged DNA fragment.
  • the SRL is sequenced on a single flow cell of a sequencing machine.
  • quantitative genetic analysis on the SRL comprises use of one or more sequencing primers.
  • the quantitative genetic analysis is used to detect a nucleotide transition or transversion, a nucleotide insertion or deletion, a genomic rearrangement, or a change in copy number in the test sample DNA fragments.
  • Some embodiments of the disclosure provide methods for simultaneous DNA analysis, comprising: (a) to a first adaptor-tagged library comprising a first genetic locus, performing a first process to generate a first modified library; (b) to a second adaptor-tagged library comprising a second genetic locus, performing a second process to generate a second modified library; wherein the first process and the second process are not identical; and (c) contacting the first modified library and the second modified library to generate a combined library; wherein the first adaptor-tagged library and the second adaptor-tagged library are from the same adaptor-tagged parent library.
  • the method comprises an initial step of splitting the adaptor-tagged parent library into at least two partitions, wherein a first partition is the first adaptor-tagged library and a second partition is the second adaptor-tagged library.
  • the first modified library comprises: a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a first 5’ adaptor module, a sequence of the first genetic locus, and a 3’ oligonucleotide (A2); or a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a sequence of the first genetic locus, a first 3’ adaptor module, and a 3’ oligonucleotide (A2); or a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a first 5’ adaptor module, a sequence of the first genetic locus, a first 3’ adaptor module, and a 3’ oligonucleotide (A2); wherein the first genetic locus comprises a DNA region; wherein
  • the first modified library comprises: a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a first 5’ adaptor module, a sequence of the first genetic locus, and a 3’ oligonucleotide (A2); or a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a sequence of the first genetic locus, a first 3’ adaptor module, and a 3’ oligonucleotide (A2); or a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a first 5’ adaptor module, a sequence of the first genetic locus, a first 3’ adaptor module, and a 3’ oligonucleotide (A2); wherein the first genetic locus comprises a DNA region; a 5’ oligonucleotide (Al), a first 5
  • the first modified library comprises: a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a first 5’ adaptor module, a sequence of the first genetic locus, and a 3’ oligonucleotide (A2); or a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a sequence of the first genetic locus, a first 3’ adaptor module, and a 3’ oligonucleotide (A2); or a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a first 5’ adaptor module, a sequence of the first genetic locus, a first 3’ adaptor module, and a 3’ oligonucleotide (A2); wherein the first genetic locus comprises a DNA region; wherein
  • the first test sample and the second test sample are not the same sample.
  • the first genetic locus and the second genetic locus are identical.
  • the first genetic locus and the second genetic locus are not identical.
  • the Library Tag distinguishes the first DNA sequence of the first modified library from the second DNA sequence of the second modified library.
  • At least one of Al, A2, Bl, and B2 comprises at least one Library Tag.
  • the Library Tag comprises a nucleic acid sequence or an amino acid sequence.
  • the Library Tag comprises one or more of a DNA, an RNA, a PNA, or a non-naturally occurring nucleic acid, a synthetic nucleic acid, a modified nucleic acid, a non-naturally occurring amino acid, a synthetic amino acid, and a modified amino acid.
  • the Library Tag comprises a detectable label.
  • the detectable label comprises one or more of a fluorescent moiety, a magnetic or paramagnetic moiety, an enzymatic moiety, a binding moiety, an epitope, and a radioactive moiety.
  • the Library Tag selectively or specifically binds to a detectable moiety.
  • the detectable moiety comprises one or more of a fluorescent moiety, a magnetic or paramagnetic moiety, an enzymatic moiety, a binding moiety, an epitope, and a radioactive moiety.
  • the Library Tag comprises a unique polynucleotide sequence.
  • the unique polynucleotide sequence comprises a sequence having 70% identity, or less, to a sequence selected from the list consisting of Al, Bl, A2, B2, the first 5’ adaptor module, the first genetic locus, the first 3 ’ adaptor module, the second 5 ’ adaptor module, the second genetic locus, and the second 3’ adaptor module.
  • the first test sample and the second test sample are not the same sample; Al, A2, Bl, or B2 contains a Library Tag; the Library Tag is capable of distinguishing the first DNA sequence of the first modified library from the second DNA sequence of the second modified library; the Library Tag comprises a unique polynucleotide sequence; and the unique polynucleotide sequence comprises a sequence having 70% identity, or less, to a sequence selected from the list consisting of Al, Bl, A2, B2, the first 5’ adaptor module, the first genetic locus, the first 3’ adaptor module, the second 5’ adaptor module, the second genetic locus, and the second 3’ adaptor module.
  • the first adaptor-tagged library and the second adaptor-tagged library each comprises an adaptor module.
  • each adaptor comprises a ligation strand oligonucleotide and a non-ligation strand oligonucleotide.
  • the non-ligation strand oligonucleotide is capable of hybridizing to a region at the 3’ end of the ligation strand oligonucleotide and forming a duplex therewith.
  • the ligation strand oligonucleotide comprises an adaptor module.
  • the ligation strand oligonucleotide comprises a dT, dA, dC, or dG overhang at the 3’ terminus.
  • the non-ligation strand oligonucleotide comprises a modification at its 3 ’ terminus that prevents ligation to the 5’ end of a dsDNA fragment and/or adaptor dimer formation, wherein the non ligation strand is configured to be displaced from the duplex.
  • each adaptor is selected from a set of adaptors, wherein each adaptor of the set of adaptors comprises an ID region selected from a pool of unique ID regions, wherein the pool is selected from a plurality of pools, and wherein the selected pool is unique to the test sample.
  • the adaptor module comprises (a) an amplification region comprising a primer binding site; (b) an ID region; and (c) an anchor region.
  • the adaptor module comprises (a) an amplification region comprising a polynucleotide comprising a primer binding site; (b) an ID region; and (c) an anchor region.
  • the amplification region comprises a primer binding site, wherein the primer binding site allows for amplification using PCR (polymerase chain reaction), LAMP (loop-mediated isothermal amplification), NASBA (nucleic acid sequence-based amplification), SDA (standard displacement amplification), RCA (rolling circle replication), or LCR (ligase chain reaction).
  • PCR polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • NASBA nucleic acid sequence-based amplification
  • SDA standard displacement amplification
  • RCA rolling circle replication
  • LCR ligase chain reaction
  • the amplification region comprises a polynucleotide sequence comprising a primer binding site, wherein the primer binding site allows for amplification of the nucleic acids of the first and/or second adaptor tagged library using PCR (polymerase chain reaction), LAMP (loop-mediated isothermal amplification), NASBA (nucleic acid sequence-based amplification), SDA (standard displacement amplification), RCA (rolling circle replication), or LCR (ligase chain reaction).
  • PCR polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • NASBA nucleic acid sequence-based amplification
  • SDA standard displacement amplification
  • RCA rolling circle replication
  • LCR ligase chain reaction
  • the amplification region comprises or consists of between 10 and 50 nucleotides. In some embodiments, the amplification region comprises or consists of between 20 and 30 nucleotides. In some embodiments, the amplification region comprises or consists of 25 nucleotides.
  • the amplification region comprises or consists of about 10 to about 50 nucleotides. In some embodiments, the amplification region comprises or consists of about 20 to about 30 nucleotides. In some embodiments, the amplification region comprises or consists of about 25 nucleotides. [0422] In some embodiments of the methods for simultaneous DNA analysis of the disclosure, the anchor region comprises an overhang at the 3’ terminus.
  • the anchor region comprises or consists of between 1 and 50 nucleotides. In some embodiments, the anchor region comprises or consists of between 5 and 25 nucleotides. In some embodiments, the anchor region comprises or consists of 10 nucleotides.
  • the anchor region comprises or consists of about 1 to about 50 nucleotides. In some embodiments, the anchor region comprises or consists of about 5 to about 25 nucleotides. In some embodiments, the anchor region comprises or consists of about 10 nucleotides.
  • the ID region comprises or consists of between 3 and 50 nucleotides. In some embodiments, the ID region comprises or consists of between 3 and 15 nucleotides. In some embodiments, the ID region comprises or consists of 8 nucleotides.
  • the ID region comprises or consists of about 3 to about 50 nucleotides. In some embodiments, the ID region comprises or consists of about 3 to about 15 nucleotides. In some embodiments, the ID region comprises or consists of about 8 nucleotides.
  • the adaptor module further comprises a unique molecule identifier (UMI) multiplier.
  • UMI unique molecule identifier
  • the UMI multiplier is adjacent to or contained within the ID region.
  • the UMI multiplier comprises or consists of between 1 and 5 nucleotides.
  • the UMI multiplier comprises or consists of about 1 to about 5 nucleotides.
  • the UMI multiplier is 3 nucleotides in length, and comprises a nucleic acid sequence selected from a group of 64 unique nucleotide sequences.
  • a plurality of adaptor modules comprises the adaptor molecule.
  • each adaptor module of the plurality of adaptor modules is unique.
  • the amplification region of each adaptor module of the plurality of adaptor modules comprises a custom primer binding site.
  • the custom primer binding site comprises a sequence having 100% sequence identity to a sequence of the custom primer binding site of each adaptor module of the plurality of adaptor modules.
  • the plurality of adaptor modules comprises a first subdivision and a second subdivision.
  • each adaptor module comprises an ID region comprising a polynucleotide sequence that is distinct from the polynucleotide sequence of the ID region of any other adaptor module.
  • the pool of ID regions comprises between 2 and 10,000 unique ID region sequences. In some embodiments, the pool of ID regions comprises between 10 and 500 unique ID region sequences. In some embodiments, the pool of ID regions comprises between 50 and 300 unique ID region sequences. In some embodiments, the pool of ID regions comprises 60 unique ID region sequences.
  • the pool of ID regions comprises about 2 to about 10,000 unique ID region sequences. In some embodiments, the pool of ID regions comprises about 10 to about 500 unique ID region sequences. In some embodiments, the pool of ID regions comprises about 50 to about 300 unique ID region sequences. In some embodiments, the pool of ID regions comprises about 60 unique ID region sequences.
  • each ID region of the pool of ID regions is 8 nucleotides in length.
  • each ID region sequence is discrete from any other ID region sequence by Hamming distance of at least two.
  • each ID region is configured to identify the DNA fragment attached thereto.
  • each adaptor module of the set of adaptor modules is selected from a group consisting of between 64 and 2,560,000 unique nucleotide sequences.
  • each adaptor module of the set of adaptor modules is selected from a group consisting of about 64 to about 2,560,000 unique nucleotide sequences.
  • each adaptor module of the set of adaptor modules comprises a unique nucleotide sequence selected from 3840 unique nucleotide sequences, wherein each sequence of the 3840 unique nucleotide sequences is discrete from any other sequence by Hamming distance of at least two.
  • the anchor region of each adaptor of the set of adaptors comprises one of four nucleotide sequences, and wherein each ID region of a given sequence is paired to only one of the four anchor regions of a given sequence.
  • the amplification region of each adaptor of the set of adaptors comprises an identical primer binding site.
  • each ID region of the pool of ID regions is 8 nucleotides in length, each ID region sequence is discrete from any other ID region sequence by Hamming distance of at least two; each adaptor of the set of adaptors comprises a UMI multiplier that is adjacent to or contained within the ID region, wherein the UMI multiplier of each adaptor of the set of adaptors is three nucleotides in length, and wherein the UMI multiplier of a given sequence is paired to one ID region of a given sequence; the anchor region of each adaptor of the set of adaptors comprises one of four nucleotide sequences, and wherein each ID region of a given sequence is paired to only one of the four anchor regions of a given sequence; and the amplification region of each adaptor of the set of adaptors comprises an identical primer binding site.
  • the first subdivision comprises a first set of unique ID regions and the second subdivision comprises a second set of unique ID regions.
  • each adaptor module further comprises a sample tag comprising a polynucleotide sequence that is not identical to the polynucleotide sequence of the ID region.
  • each adaptor module in the first subdivision comprises a sample tag comprising a first polynucleotide sequence; wherein each adaptor module in the second subdivision comprises a sample tag comprising a second polynucleotide sequence; wherein the first and second polynucleotide sequences are not identical to each other.
  • the first subdivision of adaptor modules identifies each DNA molecule within the first test sample and the second subdivision of adaptor modules identifies each DNA molecule within the second test sample.
  • the ID region of each adaptor module of the plurality of adaptor modules is selected from a group consisting of between 2 and 10,000 unique nucleotide sequences.
  • the ID region of each adaptor module of the plurality of adaptor modules is selected from a group consisting of between 50 and 500 unique nucleotide sequences.
  • the ID region of each adaptor module of the plurality of adaptor modules is selected from a group consisting of between 100 and 400 unique nucleotide sequences.
  • the ID region of each adaptor module of the plurality of adaptor modules is selected from a group consisting of 60 unique nucleotide sequences.
  • the ID region of each adaptor module of the plurality of adaptor modules is 8 nucleotides in length
  • each adaptor module of the plurality of adaptor modules is selected from a group consisting of between 64 and 2,560,000 unique nucleotide sequences.
  • each adaptor module of the plurality of adaptor modules comprises one of 3840 unique nucleotide sequences, and each nucleotide sequence is discrete from any other sequence of the 3840 unique nucleotide sequences by Hamming distance of at least two.
  • the anchor region of each adaptor module of the plurality of adaptor modules is selected from a group consisting of four nucleotide sequences.
  • the amplification regions of each adaptor module of the plurality of adaptor modules comprise a universal primer sequence; the ID region of each adaptor module of the plurality of adaptor modules comprises or consists of 8 nucleotides; the plurality of adaptor modules is divided into two or more subdivisions, wherein each subdivision of adaptor modules comprises a set of unique ID regions; the polynucleotide sequence of each ID region is discrete from the nucleotide sequence of any other ID regions of the plurality of adaptor modules by Hamming distance of at least two; and each adaptor module of the plurality of adaptor modules comprises a UMI multiplier that is adjacent to or contained within the ID region, wherein the UMI multiplier of each adaptor module of the plurality of adaptor modules comprises or consists of three nucleotides.
  • the method comprises (a) ligating a set of adaptors to DNA fragments of a test sample to generate a plurality of adaptor/DNA fragment complexes; and (b) contacting the plurality of adaptor/DNA fragment complexes with one or more enzymes to form an adaptor-tagged DNA library comprising a plurality of adaptor-tagged DNA fragments.
  • each adaptor/DNA fragment complex comprises a ligation strand oligonucleotide ligated to each end of the DNA fragment.
  • the non-ligation strand oligonucleotide is displaced from the adaptor/DNA fragment complex in step (b).
  • the set of adaptors is any adaptor set of the disclosure.
  • the first process comprises: (a) contacting the first adaptor-tagged library with one or more capture probes under conditions suitable for hybridization to form one or more capture probe/adaptor-tagged DNA complexes, wherein each capture probe comprises: a first region comprising a primer binding site; and a second region capable of hybridizing to a target region in the first genetic locus of the first adaptor-tagged library; (b) isolating the one or more capture probe/adaptor-tagged DNA complexes from step (a), wherein each isolated capture probe/adaptor-tagged DNA complex comprises a capture probe and an adaptor-tagged DNA molecule; and (c) enzymatically processing the one or more isolated capture probe/adaptor- tagged DNA complexes from step (b) to generate one or more adaptor-tagged hybrid nucleic acid molecules (Hybrid Molecules), wherein each Hybrid Molecule comprises: at least a portion of the capture probe or a complement thereof; and
  • the first region of the capture probe further comprises a sequence having complementarity to a partner oligonucleotide.
  • the enzymatic processing of one or more isolated capture probe/adaptor-tagged DNA complexes to generate one or more adaptor-tagged Hybrid Molecules comprises performing 5 '-3' DNA polymerase extension of the capture probe using the adaptor-tagged DNA molecule in the complex as a template.
  • At least one capture probe hybridizes downstream of the specific region in the target region and at least one capture probe hybridizes upstream of the specific region in the target region.
  • the capture probe comprises a sequencing primer binding site.
  • the Hybrid Molecule comprises at least one Library Tag.
  • the first region of the capture probe comprises a Library Tag.
  • the Library Tag comprises a sequencing primer binding site.
  • the method comprises (a) contacting the first adaptor-tagged library with one or more capture probes under conditions suitable for hybridization to form one or more capture probe/adaptor- tagged DNA complexes, wherein each capture probe comprises: a first region comprising a primer binding site; and a second region capable of hybridizing to a target region in the first genetic locus of the first adaptor-tagged library; (b) isolating the one or more capture probe/adaptor-tagged DNA complexes from step (a), wherein each isolated capture probe/adaptor-tagged DNA complex comprises a capture probe and an adaptor-tagged DNA molecule; (c) enzymatically processing the one or more isolated capture probe/adaptor-tagged DNA complexes from step (b) to generate one or more adaptor-tagged hybrid nucleic acid molecules (Hybrid Molecules), and (d) amplifying the one or more Hybrid Molecules; wherein each Hybrid Molecule comprises
  • the amplifying comprises a first primer comprising a 5’ oligonucleotide (Al) and a second primer comprising a 3’ oligonucleotide (A2), and wherein each of the amplified Hybrid Molecule(s) comprise Al and A2.
  • the target region comprises a genetic lesion.
  • the target region comprises an epigenetic mark.
  • the capture probe binds to the epigenetic mark.
  • the epigenetic mark comprises a methylation mark.
  • the first process comprises: (a) contacting the first adaptor-tagged library with one or more capture probes under conditions suitable for hybridization to form one or more capture probe/adaptor-tagged DNA complexes, wherein each capture probe comprises: a first region comprising a Library Tag comprising a sequencing primer binding site, and a sequence having complementarity to a partner oligonucleotide; and a second region capable of hybridizing to a target region in the first genetic locus of the first adaptor-tagged library; (b) isolating the one or more capture probe/adaptor-tagged DNA complexes from step (a), wherein each isolated capture probe/adaptor-tagged DNA complex comprises a capture probe and an adaptor-tagged
  • step (c) enzymatically processing the one or more isolated capture probe/adaptor- tagged DNA complexes from step (b) to generate one or more adaptor-tagged hybrid nucleic acid molecules (Hybrid Molecules), wherein the enzymatic processing comprises performing
  • each Hybrid Molecule comprises the capture probe and a complement of the adaptor-tagged DNA molecule that is 3’ from where the capture probe hybridized to the target region in the first adaptor-tagged library; and (d) amplifying the one or more Hybrid Molecules in step (c), wherein the amplifying comprises hybridizing a first primer comprising a 5’ oligonucleotide (Al) and a second primer comprising a 3’ oligonucleotide (A2) to a Hybrid Molecule, and wherein each of the amplified Hybrid Molecule(s) comprise Al and A2.
  • the second process comprises amplifying or extending the second genetic locus of the second adaptor-tagged library.
  • the amplifying or extending comprises hybridizing a first primer comprising a 5’ oligonucleotide (B 1) and a second primer comprising a 3’ oligonucleotide (B2) to an adaptor- tagged DNA molecule, and wherein the amplified or extended second genetic locus comprises B1 and B2.
  • At least one of B1 and B2 comprises a Library Tag.
  • the second process comprises amplifying or extending the second genetic locus of the second adaptor-tagged library, wherein the amplifying or extending comprises hybridizing a first primer comprising a 5’ oligonucleotide (Bl) and a second primer comprising a 3’ oligonucleotide (B2) to an adaptor-tagged DNA molecule, wherein at least one of Bl and B2 comprises a Library Tag, and wherein the amplified or extended second genetic locus comprises Bl and B2.
  • the amplifying or extending comprises hybridizing a first primer comprising a 5’ oligonucleotide (Bl) and a second primer comprising a 3’ oligonucleotide (B2) to an adaptor-tagged DNA molecule, wherein at least one of Bl and B2 comprises a Library Tag, and wherein the amplified or extended second genetic locus comprises Bl and B2.
  • the method further comprises a step of genetic analysis.
  • the genetic analysis comprises detecting the presence of at least one Library Tag in at least one modified library.
  • the genetic analysis comprises sequencing of the combined library to generate a plurality of sequencing reads and performing bioinformatics analysis on the plurality of sequencing reads.
  • the first modified library comprises a first Library Tag and the second modified library comprises a second Library Tag.
  • the first Library Tag generates a first signal and the second Library Tag generates a second signal.
  • both the first and the second signals are detected or detectable.
  • the first signal and the second signal are not identical.
  • the first signal is detected or detectable and the second signal is not detected or detectable.
  • the second signal is detected or detectable and the first signal is not detected or detectable.
  • only one of the first modified library and the second modified library comprises a Library Tag.
  • the Library Tag generates a first signal and the lack of the Library Tag generates a second signal.
  • both the first and the second signals are detected or detectable.
  • the first signal and the second signal are not identical.
  • the first signal is detected or detectable and the second signal is not detected or detectable.
  • the second signal is detected or detectable and the first signal is not detected or detectable.
  • the first signal is a sequencing read and the second signal is an empty sequencing read.
  • the empty sequencing read (the second signal) is detected as a series of two or more G nucleotides.
  • the detected first signal and the detected second signal can be differentiated from one another.
  • the detected first signal and the non-detected second signal can be differentiated from one another.
  • the non-detected first signal and the detected second signal can be differentiated from one another.
  • the genetic analysis is used to identify one or more genetic lesions.
  • the one or more genetic lesions comprise a nucleotide transition, a nucleotide transversion, a nucleotide insertion, a nucleotide deletion, a genomic rearrangement, or a change in copy number.
  • the genetic analysis is used to detect one or more genetic lesions that cause or are associated with a genetic disease.
  • the genetic disease is cancer.
  • the one or more genetic lesions are chromosomal rearrangements.
  • the first modified library and second modified library are demultiplexed after sequencing by identification of at least one Library Tag or a portion thereof.
  • Some embodiments of the disclosure provide a composition comprising a first modified library and a second modified library, wherein the first modified library comprises: a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a first 5’ adaptor module, a sequence of the first genetic locus, and a 3’ oligonucleotide (A2); or a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a sequence of the first genetic locus, a first 3’ adaptor module, and a 3’ oligonucleotide (A2); or a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), afirst 5’ adaptor module, a sequence of the first genetic locus, a first 3’ adaptor module, and a 3’ oligonucleotide (A2); wherein the first modified library comprises
  • Some embodiments of the disclosure provide a composition comprising a first modified library and a second modified library, wherein the first modified library comprises: a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a first 5’ adaptor module, a sequence of the first genetic locus, and a 3’ oligonucleotide (A2); or a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a sequence of the first genetic locus, a first 3’ adaptor module, and a 3’ oligonucleotide (A2); or a first DNA molecule comprising, from 5’ to 3’, a 5’ oligonucleotide (Al), a first 5’ adaptor module, a sequence of the first genetic locus, a first 3’ adaptor module, and a 3’ oligonucleotide (A2); wherein the first modified library comprises
  • the first test sample and the second test sample are the same sample.
  • the first test sample and the second test sample are not the same sample.
  • the first genetic locus and the second genetic locus are identical.
  • the first genetic locus and the second genetic locus are not identical.
  • the Library Tag distinguishes the first DNA sequence of the first modified library from the second DNA sequence of the second modified library.
  • At least one of Al, A2, Bl, and B2 contains at least one Library Tag.
  • the Library Tag comprises a nucleic acid sequence or an amino acid sequence.
  • the Library Tag comprises one or more of a DNA, an RNA, a PNA, or a non-naturally occurring nucleic acid, a synthetic nucleic acid, a modified nucleic acid, a non-naturally occurring amino acid, a synthetic amino acid, and a modified amino acid.
  • the Library Tag comprises a detectable label.
  • the detectable label comprises one or more of a fluorescent moiety, a magnetic or paramagnetic moiety, an enzymatic moiety, a binding moiety, an epitope, and a radioactive moiety.
  • the Library Tag selectively or specifically binds to a detectable moiety.
  • the detectable moiety comprises one or more of a fluorescent moiety, a magnetic or paramagnetic moiety, an enzymatic moiety, a binding moiety, an epitope, and a radioactive moiety.
  • the Library Tag comprises a unique polynucleotide sequence.
  • the unique polynucleotide sequence comprises a sequence having 70% identity, or less, to a sequence selected from the list consisting of Al, Bl, A2, B2, the first 5 adaptor module, the first genetic locus, the first 3 adaptor module, the second 5 adaptor module, the second genetic locus, and the second 3 adaptor module.
  • the first test sample and the second test sample are not the same sample; Al, A2, Bl, or B2 contains a Library Tag; the Library Tag is capable of distinguishing the first DNA sequence of the first modified library from the second DNA sequence of the second modified library; the Library Tag comprises a unique polynucleotide sequence; and the unique polynucleotide sequence comprises a sequence having 70% identity, or less, to a sequence selected from the list consisting of Al, Bl, A2, B2, the first 5 adaptor module, the first genetic locus, the first 3 adaptor module, the second 5 adaptor module, the second genetic locus, and the second 3 adaptor module.
  • each adaptor of the set of adaptors comprises a ligation strand oligonucleotide and a non-ligation strand oligonucleotide.
  • the non-ligation strand oligonucleotide is capable of hybridizing to a region at the 3’ end of the ligation strand oligonucleotide and forming a duplex therewith.
  • the ligation strand oligonucleotide comprises an adaptor module.
  • the ligation strand oligonucleotide comprises a dT, dA, dC, or dG overhang at the 3’ terminus.
  • the non-ligation strand oligonucleotide comprises a modification at its 3’ terminus that prevents ligation to the 5’ end of a dsDNA fragment and/or adaptor dimer formation, wherein the non-ligation strand is configured to be displaced from the duplex.
  • each adaptor of the set of adaptors comprises an ID region selected from a pool of unique ID regions, wherein the pool is selected from a plurality of pools, and wherein the selected pool is unique to the test sample.
  • the adaptor module comprises (a) an amplification region comprising a primer binding site; (b) an ID region; and (c) an anchor region.
  • the adaptor module comprises: (a) an amplification region comprising a polynucleotide sequence comprising a primer binding site; (b) an ID region; and (c) an anchor region.
  • the amplification region comprises a primer binding site, wherein the primer binding site allows for amplification using PCR (polymerase chain reaction), LAMP (loop-mediated isothermal amplification), NASBA (nucleic acid sequence-based amplification), SDA (standard displacement amplification), RCA (rolling circle replication), or LCR (ligase chain reaction).
  • PCR polymerase chain reaction
  • LAMP loop-mediated isothermal amplification
  • NASBA nucleic acid sequence-based amplification
  • SDA standard displacement amplification
  • RCA rolling circle replication
  • LCR ligase chain reaction
  • the amplification region comprises a polynucleotide sequence comprising a primer binding site, wherein the primer binding site allows for amplification of the nucleic acids of the first and/or second adaptor- tagged library using for PCR (polymerase chain reaction), LAMP (loop-mediated isothermal amplification), NASBA (nucleic acid sequence-based amplification), SDA (standard displacement amplification), RCA (rolling circle replication), or LCR (ligase chain reaction).
  • the amplification region comprises or consists of between 10 and 50 nucleotides. In some embodiments, the amplification region comprises or consists of between 20 and 30 nucleotides. In some embodiments, the amplification region comprises or consists of 25 nucleotides.
  • the amplification region comprises or consists of about 10 to about 50 nucleotides. In some embodiments, the amplification region comprises or consists of about 20 to about 30 nucleotides. In some embodiments, the amplification region comprises or consists of about 25 nucleotides.
  • the anchor region comprises an overhang at the 3’ terminus.
  • the anchor region comprises or consists of between 1 and 50 nucleotides. In some embodiments, the anchor region comprises or consists of between 5 and 25 nucleotides. In some embodiments, the anchor region comprises or consists of 10 nucleotides.
  • the anchor region comprises or consists of about 1 to about 50 nucleotides. In some embodiments, the anchor region comprises or consists of about 5 to about 25 nucleotides. In some embodiments, the anchor region comprises or consists of about 10 nucleotides.
  • the ID region comprises or consists of between 3 and 50 nucleotides. In some embodiments, the ID region comprises or consists of between 3 and 15 nucleotides. In some embodiments, the ID region comprises or consists of 8 nucleotides.
  • the ID region comprises or consists of about 3 to about 50 nucleotides. In some embodiments, the ID region comprises or consists of about 3 to about 15 nucleotides. In some embodiments, the ID region comprises or consists of about 8 nucleotides.
  • the adaptor module further comprises a unique molecule identifier (UMI) multiplier.
  • UMI unique molecule identifier
  • the UMI multiplier is adjacent to or contained within the ID region.
  • the UMI multiplier comprises or consists of between 1 and 5 nucleotides. [0545] In some embodiments of the compositions of the disclosure, the UMI multiplier comprises or consists of about 1 to about 5 nucleotides.
  • the UMI multiplier is 3 nucleotides in length, and comprises a nucleic acid sequence selected from a group of 64 unique nucleotide sequences.
  • the pool of ID regions comprises between 2 and 10,000 unique ID region sequences. In some embodiments, the pool of ID regions comprises between 10 and 500 unique ID region sequences. In some embodiments, the pool of ID regions comprises between 50 and 300 unique ID region sequences. In some embodiments, the pool of ID regions comprises 60 unique ID region sequences.
  • the pool of ID regions comprises about 2 to about 10,000 unique ID region sequences. In some embodiments, the pool of ID regions comprises about 10 to about 500 unique ID region sequences. In some embodiments, the pool of ID regions comprises about 50 to about 300 unique ID region sequences. In some embodiments, the pool of ID regions comprises about 60 unique ID region sequences.
  • each ID region of the pool of ID regions is 8 nucleotides in length.
  • each ID region sequence is discrete from any other ID region sequence by Hamming distance of at least two.
  • each ID region is configured to identify the DNA fragment attached thereto.
  • each adaptor module of the set of adaptor modules is selected from a group consisting of between 64 and 2,560,000 unique nucleotide sequences.
  • each adaptor module of the set of adaptor modules is selected from a group consisting of about 64 to about 2,560,000 unique nucleotide sequences.
  • each adaptor module of the set of adaptor modules comprises a unique nucleotide sequence selected from 3840 unique nucleotide sequences, wherein each sequence of the 3840 unique nucleotide sequences is discrete from any other sequence by Hamming distance of at least two.
  • the anchor region of each adaptor of the set of adaptors comprises one of four nucleotide sequences, and wherein each ID region of a given sequence is paired to only one of the four anchor regions of a given sequence.
  • the amplification region of each adaptor of the set of adaptors comprises an identical primer binding site.
  • each ID region of the pool of ID regions is 8 nucleotides in length, each ID region sequence is discrete from any other ID region sequence by Hamming distance of at least two; each adaptor of the set of adaptors comprises a UMI multiplier that is adjacent to or contained within the ID region, wherein the UMI multiplier of each adaptor of the set of adaptors is three nucleotides in length, and wherein the UMI multiplier of a given sequence is paired to one ID region of a given sequence; the anchor region of each adaptor of the set of adaptors comprises one of four nucleotide sequences, and wherein each ID region of a given sequence is paired to only one of the four anchor regions of a given sequence; and the amplification region of each adaptor of the set of adaptors comprises an identical primer binding site.
  • a plurality of adaptor modules comprises the adaptor module.
  • each adaptor module of the plurality of adaptor modules is unique.
  • compositions of the disclosure wherein the amplification region of each adaptor module of the plurality of adaptor modules comprises a custom primer binding site.
  • the custom primer binding site comprises a sequence having 100% sequence identity to a sequence of the custom primer binding site of each adaptor module of the plurality of adaptor modules.
  • the plurality of adaptor modules comprises a first subdivision and a second subdivision.
  • each adaptor module comprises an ID region comprising a polynucleotide sequence that is distinct from the polynucleotide sequence of the ID region of any other adaptor module.
  • the first subdivision comprises a first set of unique ID regions and the second subdivision comprises a second set of unique ID regions.
  • each adaptor module further comprises a sample tag comprising a polynucleotide sequence that is not identical to the polynucleotide sequence of the ID region.
  • each adaptor module in the first subdivision comprises a sample tag comprising a first polynucleotide sequence and each adaptor module in the second subdivision comprises a sample tag comprising a second polynucleotide sequence; wherein the first and second polynucleotide sequences are not identical to each other.
  • the first subdivision of adaptor modules identifies each DNA molecule within the first test sample and the second subdivision of adaptor modules identifies each DNA molecule within the second test sample.
  • the first subdivision of adaptor modules identifies the first test sample and the second subdivision of adaptor modules identifies the second test sample.
  • the ID region of each adaptor module of the plurality of adaptor modules is selected from a group consisting of between 2 and 10,000 unique nucleotide sequences.
  • the ID region of each adaptor module of the plurality of adaptor modules is selected from a group consisting of between 50 and 500 unique nucleotide sequences.
  • the ID region of each adaptor module of the plurality of adaptor modules is selected from a group consisting of between 100 and 400 unique nucleotide sequences.
  • the ID region of each adaptor module of the plurality of adaptor modules is selected from a group consisting of 60 unique nucleotide sequences.
  • each adaptor module of the plurality of adaptor modules comprises or consists of 8 nucleotides.
  • each adaptor module of the plurality of adaptor modules is selected from a group consisting of between 64 and 2,560,000 unique nucleotide sequences.
  • each adaptor module of the plurality of adaptor modules comprises a unique nucleotide sequence selected from among 3840 unique nucleotide sequences, and each nucleotide sequence is discrete from any other sequence of the 3840 unique nucleotide sequences by Hamming distance of at least two.
  • the anchor region of each adaptor module of the plurality of adaptor modules is selected from a group consisting of four nucleotide sequences.
  • the amplification regions of each adaptor module of the plurality of adaptor modules comprise a universal primer sequence; the ID region of each adaptor module of the plurality of adaptor modules comprises or consists of 8 nucleotides; the plurality of adaptor modules is divided into two or more subdivisions, wherein each subdivision of adaptor modules comprises a set of unique ID regions, wherein the polynucleotide sequence of each ID region is discrete from the nucleotide sequence of any other ID regions of the plurality of adaptor modules by Hamming distance of at least two; and each adaptor module of the plurality of adaptor modules comprises a UMI multiplier that is adjacent to or contained within the ID region, wherein the UMI multiplier of each adaptor module of the plurality of adaptor modules comprises or consists of three nucleotides.
  • the test sample is a tissue biopsy.
  • the tissue biopsy is obtained from a tumor or a tissue suspected of being a tumor.
  • the tissue biopsy is obtained from a malignant tumor or tumor suspected of being a malignant tumor.
  • the DNA molecule is cell free DNA (cfDNA), genomic DNA (gDNA), complementary DNA (cDNA), mitochondrial DNA, methylated DNA, or demethylated DNA.
  • the DNA molecule comprises an epigenetic mark.
  • the DNA molecule is obtained from a library selected from the list consisting of a whole genome library, an amplicon library, a whole exome library, a cDNA library, or a methylated DNA library.
  • the DNA molecule is isolated or generated from the test sample.
  • the test sample comprises a biological sample selected from the group consisting of an amniotic fluid sample, a blood sample, a skin sample, a hair sample, a hair follicle sample, a saliva sample, a mucous sample, a sweat sample, a tear sample, an epithelial tissue sample, a urine sample, a semen sample, a seminal plasma sample, a serum sample, a prostatic fluid sample, a pre-ejaculatory fluid (Cowper's fluid) sample, an ocular fluid sample, an excreta sample, a biopsy sample, an ascites sample, a cerebrospinal fluid sample, a lymph sample, a tissue extract sample, a stool sample, and a formalin-fixed, paraffin embedded (FFPE) sample.
  • FFPE formalin-fixed, paraffin embedded
  • the DNA molecule is obtained by the steps comprising (a) isolating cellular DNA from the test sample; or (b) fragmenting the cellular DNA to obtain the genomic DNA fragment.
  • step (b) fragmenting the cellular DNA is performed by contacting the cellular DNA with at least one digestion enzyme.
  • step (b) fragmenting the cellular DNA is performed by applying mechanical stress to the cellular DNA.
  • step (b) fragmenting the cellular DNA is performed by contacting the cellular DNA with one or more compounds to chemically disrupt one or more bonds of the cellular DNA.
  • FIGURE l is a schematic diagram depicting 3 different types of adaptor modules.
  • FIGURE 2 is a schematic diagram depicting an illustrative process for matching anchor regions to ID regions of adaptors (adaptor elements are illustrative and length is not to-scale), as well as the generation of adaptor sets assigned to different samples.
  • black boxes represent identical amplification regions
  • patterned boxes represent distinct ID regions, with each pattern representing a different ID region.
  • Grey boxes represent anchor regions, with each type of anchor region sequence represented by a different shade of grey.
  • FIGURE 3 is a schematic diagram depicting one embodiment of the methods of the disclosure. Patterns indicate compositions of a library, wherein the same pattern indicates an identical composition (e.g ., the 1 st adaptor-tagged library and the 2 nd adaptor-tagged library), and two patterns indicate a mixture of different compositions (e.g., the Combined Library).
  • FIGURE 4 is a schematic diagram depicting an illustrative workflow of some embodiments of the methods of the disclosure. In this embodiment, an amplified Target Capture Library (TCLA) and an LPWG library are separately generated then combined for sequencing.
  • TCLA Target Capture Library
  • LPWG library an amplified Target Capture Library
  • FIGURE 5 is a schematic diagram depicting an illustrative process for generating an adaptor-tagged Parent Library.
  • the Parent Library is generating in the following order: in a first step, dsDNA fragments are end-repaired, providing fragments with 5 phosphate groups and 3’-dA(deoxyribonucleic acid adenine) tails at both strand ends; in a second step, the ligation strand of adaptors are ligated to the 5 ends of the fragments, tagging the fragments with ID regions and PCR primer binding sites; in a third step, the tagged DNA is filled-in (i.e., extended) and the non-ligation strand is dissociated from the adaptor, generating an adaptor-tagged Parent Library comprising adaptor-tagged DNA fragments.
  • the adaptor-tagged Parent Library and/or the collection of adaptor-tagged DNA fragments is referred as the LIB or LIBS.
  • the extended adaptor-tagged DNA fragments are amplified by PCR to generate an amplified library.
  • the amplified library is referred to as the Library Post- Amplification (LPA).
  • the third and fourth step may be performed continuously (i.e., without adding additional reagents) in a thermal cycler without adding additional reagents to the reaction mixture.
  • FIGURE 6 is a schematic diagram depicting an illustrative process for generating a Target Capture Library (TCL).
  • a capture probe module in a first step, is hybridized to a denatured adaptor-tagged DNA fragment (e.g ., a LIBS fragment or an LPA fragment).
  • the capture probe module comprises a tail sequence (green) and a capture probe sequence (dark blue).
  • the tail sequence is hybridized to a partner oligonucleotide (grey) comprising a biotin label at the 3’ end of the partner oligonucleotide.
  • the capture probe module in a second step, is extended at its 3’ end using the adaptor-tagged DNA fragment as template, generating a Target Capture Library (TCL).
  • TCL Target Capture Library
  • the TCL in a third step, is amplified using primers FP and RP, where FP binds to at least a portion of the amplification region of the adaptor and RP binds to at least a portion of the tail sequence of the capture probe module.
  • FP comprises a binding site (red) for a first sequencing primer and RP comprises a binding site (green) for a second sequencing primer.
  • a Library Tag is embedded in the tail region of the capture probe (green).
  • the resulting amplified library is referred to as the amplified Target Capture Library (TCLA) and is ready for sequencing.
  • FIGURE 7 is a schematic diagram depicting an illustrative embodiment of a method for the generation of a Low-pass Whole Genome (LPWG) library.
  • the LPWG is referred to as the Amplified Whole Genome Library or Whole Genome Library Amplified (WGLA).
  • adaptor-tagged DNA fragments of the adaptor- tagged Parent Library, the amplified adaptor-tagged Parent Library, the LIBS, or the LPA are amplified with primers comprising sequencing adaptors (e.g., oligo 1 (red) and oligo 2 (blue)) and an amplification region binding region (yellow).
  • the amplification region binding regions is referred to as the ACA sequence.
  • oligo 1 and oligo 2 may be Illumina ® P5 and P7 sequences; amplification with these primers produces a library of DNA fragments with oligo 1 and oligo 2 sequences at both ends, ready for sequencing.
  • the LPWG library (WGLA) does not possess a Read 2 sequencing primer binding domain.
  • the WGLA does not posses a Library Tag.
  • FIGURE 8 is a graph showing library concentrations after 8, 12, or 16 cycles of LPWG amplification with either LIB or LPA input.
  • FIGURE 9 is a graph showing concentrations of LPWG libraries measured by qPCR or by a standard assay used to determine concentration of DNA (Assay), amplified for 8, 12, or 16 cycles with annealing temperature of either 58°C or 69°C.
  • FIGURE 10 is a graph showing library concentrations measured by Assay, by qPCR with oligo 1 and/or oligo 2 primers, or by qPCR with Primer Mix 1 S (“AC A”) after 4, 6, 8, 12, 14, or 16 cycles. Error bars represent standard deviation.
  • FIGURE 11 is a graph showing the ratio of measured concentration by Assay to measured concentration by qPCR with oligo 1/oligo 2 primers after 8, 12, 14, or 18 cycles of amplification.
  • FIGURE 12A is a graph showing the overlay of Bioanalzyer traces of LPWG libraries amplified for 4 (blue), 6 (green), or 8 (red) cycles.
  • FIGURE 12B is a graph showing the overlay of Bioanalzyer traces of LPWG libraries amplified for 8 (blue), 12 (green), 14 (turquoise), or 16 (red) cycles.
  • FIGURE 13 is a graph showing overlay of Bioanalzyer traces of LPWG libraries amplified from 145 bp size-selected fragment for 0 (blue), 6 (green), 8 (turquoise), 12 (pink), or 16 (red) cycles.
  • FIGURE 14 is a graph showing concentration of LPWG libraries amplified from 5, 10, 25, 50, 75, or 100 ng wild type LPA for 8 cycles, measured by either Assay or by qPCR.
  • FIGURE 15 is a graph showing concentration of LPWG libraries amplified from 10, 20, 50, 100, or 200 ng wild type LPA for 16 cycles, measured by either Assay or by qPCR.
  • FIGURE 16 is a graph showing the number of sequencing reads for LPWG libraries amplified from 10, 20, 50, 100, or 200 ng wild type LPA for 16 cycles, normalized before loading onto the flow cell. (Sequencing run number 16)
  • FIGURE 17 is a graph showing the average number of reads from 2 replicate samples with varying inputs into the LPWG PCR. Error bars denote standard deviation between the replicates.
  • FIGURE 18 is a graph showing the concentrations of LPWG libraries amplified for 16 cycles in either 0.5x (50 pL), lx (100 pL) or 2x (200 pL) reaction volumes.
  • FIGURE 19 is a graph showing the overlay of Bioanalzyer traces of LPWG libraries amplified in 0.5x, lx, or 2x reaction volumes.
  • FIGURE 20 is a graph showing concentrations of LPWG libraries amplified for 16 cycles in either 0.5x (0.5 pM), lx (1 pM) or 2x (2 pM) primers.
  • FIGURE 21 is a graph showing the overlay of Bioanalzyer traces of LPWG libraries amplified with 0.5x, lx, or 2x primer concentrations.
  • FIGURE 22 is a graph showing concentrations of LPWG libraries amplified for 8, 12, 14, or 16 cycles with Enzyme 1 or Enzyme 2.
  • FIGURE 23 is a graph showing the overlay of Bioanalzyer traces of LPWG libraries amplified with Enzyme 1.
  • FIGURE 24 is a graph showing the distribution of co-loaded LPWG and Target Capture Library Amplified (TCLA).
  • FIGURE 25 is a graph showing the average depth for two capture libraries co-loaded with LPWG libraries. Target coverage and number of LPWG libraries are described along the x-axis.
  • FIGURE 26 is a graph showing the coverage of 16 clinical and wild type samples.
  • FIGURE 27 is a graph showing the calculated coverage for wild type LPWG libraries, co-loaded with target capture libraries.
  • the diagonal stripes boxplots show libraries loaded to a target of 0.5x coverage while the vertical stripes boxplot shows libraries targeted to lx coverage.
  • FIGURE 28 is a schematic diagram depicting an illustrative embodiment of de multiplexing (z.e., differentiating between) the TCLA and the WGLA library using the Read 2 sequence.
  • the Library Tag comprises or consists of the Read 2 sequence.
  • the FC sequence represents a sequence capable of binding to a flow cell of a sequencing machine.
  • Read 1 and Read 2 sequences that bind to the DNA molecules to be sequenced represent sequencing primers; the barcode sequence represents at least a portion of the adaptor module comprising a unique sequence.
  • FIGURE 29 is a diagram showing variant calls to TCLA libraries with or without the addition of LPWG Libraries.
  • Some embodiments of the disclosure provide compositions and methods that combine the benefits of different genetic analysis approaches by performing simultaneous genetic analysis on a single pool of different libraries, where each of the different libraries is specific to a different genetic analysis approach.
  • Genetic analysis approaches that utilize next- generation sequencing (NGS) methods currently used in the art include, but are not limited to, targeted sequencing of target-enriched libraries, whole genome sequencing of whole-genome libraries, and epigenetic analysis of chemically transformed libraries.
  • Genetic analysis approaches are generally chosen based on several factors, including the final application ( e.g ., clinical vs research), the required quality of results, technical efficiency, and cost, while also taking into consideration the benefits and disadvantages of each approach.
  • a targeted genetic analysis approach focuses on pre-determined DNA target regions, therefore it may not identify additional DNA abnormalities in other regions (non-target regions) of the genome.
  • LPWG sequencing provides coverage for the entire genome and may be performed at lower sequencing depth but may result in less sensitivity for specific DNA regions of interest.
  • compositions and methods that may be used in combining libraries specific to different genetic analysis approaches into a single Sequence-Ready Library (SRL), where the different libraries within the SRL may be analyzed simultaneously in a single sequencing analysis experiment (e.g ., on a single flow cell of a sequencing machine).
  • SRL Sequence-Ready Library
  • this approach increases sequencing efficiency, expands sequencing coverage, and maintains necessary sensitivity.
  • targeted genetic analysis at specific loci may be performed by deep sequencing to detect low frequency genetic variants, and simultaneous whole-genome sequencing may provide more accurate assessment of copy number variations.
  • compositions and methods of the disclosure provide numerous benefits, some of which are described below:
  • an adaptor-tagged Parent Library or a Library Post-Amplification (LPA) generated from a test sample is split into two parts — a first adaptor- tagged library and a second adaptor-tagged library — prior to each adaptor-tagged library undergoing a different modification process.
  • the first adaptor-tagged library may generate a first modified library
  • the second adaptor-tagged library may generate a second modified library.
  • the first modified library is a Target Capture Library (TCL) or amplified Target Capture Library (TCLA) and the second modified library is a Whole-Genome Library (WGL) or Whole-Genome Library Amplified (WGLA).
  • WGLA and low-pass whole- genome (LPWG) library may be used interchangeably in the disclosure.
  • Targeted genetic analysis on the TCL or TCLA may provide higher specificity and sensitivity for targeted regions, and it may be less robust in identifying larger genomic variations that may span a section of the chromosome.
  • TCL or TCLA and the WGL or WGLA into a single combined library, both targeted genetic analysis and broad-coverage whole genome analysis may be performed on the results of one sequencing run.
  • the frequency of one or more genetic lesions associated with cancer is determined using methods and compositions of the disclosure.
  • somatic mutations that reside within the tumor and not within the germline of the individual may be used to quantify the amount of circulating DNA that is shed from the tumor.
  • the combination of, for example, a TCLA and a WGLA library will promote the discovery of mutations originating from the tumor while retaining the ability to interrogate focused areas of interest.
  • the discovery of new mutations regardless of their biological significance, also improves quantification of tumor content in admixed cfDNA.
  • genetic analysis performed using compositions and methods of the disclosure may reduce cost.
  • genetic analysis may be done by sequencing, which may be expensive for each individual sequencing run.
  • multiple libraries e.g ., a Target Capture library and a LPWG library
  • costs may be lower due to the use of fewer reagents, lower facility costs, as well as reduced personnel time.
  • kits that may be used to generate DNA libraries for genetic analysis, for example, target-captured libraries for use in targeted genetic analysis.
  • Some embodiments of the disclosure provide kits that may be used to generate different libraries that may be combined into an SRL, for example, a combined library comprising a target-captured library and a whole-genome library.
  • an SRL generated by a kit of the disclosure may be used in a single sequencing analysis experiment.
  • a kit of the disclosure may be used in both targeted genetic analysis and whole-genome analysis in a single sequencing analysis experiment, where the targeted genetic analysis at specific loci detects low frequency genetic variants and the simultaneous whole-genome sequencing provides an assessment of copy number variations.
  • kits of the disclosure may increase sequencing efficiency, expand sequencing coverage, and/or maintain sensitivity. In some embodiments, kits of the disclosure may increase sequencing efficiency. In some embodiments, kits of the disclosure may expand sequencing coverage. In some embodiments, kits of the disclosure may maintain sensitivity.
  • At least one component of a kit of the disclosure has passed a QC process of the disclosure. In some embodiments, at least one component of a kit of the disclosure has passed multiple QC processes of the disclosure. In some embodiments, multiple components of a kit of the disclosure have passed a QC process of the disclosure. In some embodiments, multiple components of a kit of the disclosure have passed multiple QC processes of the disclosure.
  • the adaptors of a kit of the disclosure have passed an adaptor QC process of the disclosure. In some embodiments, the adaptor sets of a kit of the disclosure have passed an adaptor QC process of the disclosure. In some embodiments, the adaptors of a kit of the disclosure have passed an adaptor QC process as described in Example 16. In some embodiments, the adaptor sets of a kit of the disclosure have passed an adaptor QC process as described in Example 16.
  • the capture probe modules of a kit of the disclosure have passed a probe QC process of the disclosure. In some embodiments, the capture probe modules of a kit of the disclosure have passed a probe QC process as described in Example 18.
  • a kit of the disclosure comprises one or more reagents and/or primers selected from an End Repair Buffer, an End Repair Enzyme, a Ligation Mix, a Library PCR Mix, a Library Primer, a Post Amp PCR Mix, a Post Hyb Primer mix, a Genomic Mix, a FWD Seq Primer and a REV Seq Primer.
  • Each of these reagents and primers may be the corresponding composition described elsewhere in the disclosure, or as described in Examples 11-19.
  • one or more reagents and/or primers of a kit of the disclosure have passed a QC process of the disclosure.
  • one or more reagents and/or primers of a kit of the disclosure have passed a QC process as described in Example 15.
  • the positive control DNA sample of a kit of the disclosure has passed a QC process of the disclosure. In some embodiments, the positive control DNA sample of a kit of the disclosure has passed a QC process as described in Example 17.
  • the disclosure provides compositions and methods comprising an adaptor or adaptor module.
  • the disclosure provides a kit comprising a set of adaptors.
  • a set of adaptors refers to a collection of two or more adaptors, each comprising an adaptor module.
  • the term “adaptor module” refers to a polynucleotide sequence that is attached to a DNA fragment to form an adaptor-tagged DNA molecule.
  • the term “adaptor module” refers to a polynucleotide that comprises at least three elements: (i) an amplification region comprising a primer binding site; (ii) an ID region; and (iii) an anchor region.
  • the term “adaptor” may refer to a DNA molecule comprising the adaptor module or the complement thereof.
  • the term “adaptor” refers to a partially double-stranded DNA structure comprising an adaptor module.
  • the term “adaptor” refers to a single-stranded DNA molecule comprising an adaptor module, for example the ligation strand oligonucleotide.
  • FIGURE 1 provides illustrative compositions of an adaptor module of the disclosure, including three different types of adaptors.
  • a first type of adaptor module may include an ID region comprising distinct sections, wherein one section corresponds to a sample tag and another section corresponds to a fragment tag.
  • a second type of adaptor module may include an ID region that does not comprise distinct sections as in the first type; in other words, for the second type adaptor, the sample tag consists of the entirety of the ID region and the fragment tag also consists of the entirety of the ID region. Additionally, the second type adaptor may also include a UMI multiplier.
  • a third type of adaptor module may include an ID region, a UMI multiplier, and a T nucleotide overhang.
  • the adaptor comprises one or more amplification regions, one or more ID regions, one or more UMI multipliers, and one or more anchor regions.
  • the adaptor comprises, in order from 5’ to 3’, an amplification region, an ID region, a UMI multiplier, and an anchor region.
  • the UMI is contained within the ID region, and the adaptor comprises, in order from 5’ to 3’, an amplification region, an integrated ID region /UMI multiplier region, and an anchor region.
  • the adaptor comprises one or more amplification regions, one or more ID regions, one or more UMI multipliers, one or more anchor regions, and one or more nucleotides in the 3 ' overhang that are efficient ligation substrates.
  • the adaptor module further comprises one or more sequencing primer binding sites.
  • a partially double-stranded DNA adaptor structure is coupled to the DNA molecule prior to ligation to form an adaptor/DNA complex.
  • Said partially double- stranded adaptor structure comprises a ligation strand oligonucleotide and a non-ligation strand oligonucleotide.
  • the ligation strand oligonucleotide comprises an adaptor module, and the non-ligation strand oligonucleotide is capable of hybridizing to a region at the 3’ end of the ligation strand oligonucleotide and forming a duplex therewith.
  • the non-ligation strand is complementary to at least a portion of the ligation strand in order to form said duplex.
  • the adaptor further comprises a 3’ terminal overhang
  • the DNA molecule comprises a 3’ terminal overhang that is complementary to the 3’ terminal overhang of the adaptor.
  • the 3’ terminal overhang e.g ., a dT tail
  • one or more sets of adaptors are provided, where each set of adaptors is uniquely assigned to a given sample, and each adaptor is distinct from any other adaptor in any of the one or more adaptor sets.
  • FIGURE 2 provides a schematic diagram of adaptor sets in one embodiment; a total of 16 different adaptors are used in this embodiment.
  • compositions of the disclosure are assembled into a kit comprising one or more adaptor sets.
  • a kit of the disclosure is used to generate a combined sequence-ready DNA library, where the combined library is any of the combined libraries described in the disclosure.
  • the kit is used to generate a TCL.
  • the kit is used to generate a TCLA.
  • the kit is used to generate adaptor-tagged DNA fragments or LIBS.
  • the kit is used to generate an LPA.
  • the kit is used to generate a LPWG.
  • the kit is used to generate a WGLA.
  • the kit is used to generate a LIBS and/or an LPA and a WGLA.
  • the kit is used to generate a LIBS and/or an LPA, a WGLA, and a TCL. In some embodiments, the kit is used to generate a LIBS and/or an LPA, a WGLA, and a TCLA. In some embodiments, the kit is used to generate a LIBS and/or an LPA and a TCL. In some embodiments, the kit is used to generate a LIBS and/or an LPA and a TCLA. In some embodiments, the TCL, TCLA, LIBS, LPA, LPWG and WGLA are any one of the DNA libraries of the disclosure.
  • a kit used to generate a LPWG comprises adaptors that have passed an adaptor QC process of the disclosure.
  • a kit used to generate a WGLA comprises adaptors that have passed an adaptor QC process of the disclosure.
  • a kit used to generate a TCLA comprises adaptors that have passed an adaptor QC process of the disclosure and capture probes that have passed a capture probe QC process of the disclosure.
  • a kit used to generate TCL comprises adaptors that have passed an adaptor QC process of the disclosure and capture probes that have passed a capture probe QC process of the disclosure.
  • the kit comprises a set of adaptors.
  • adaptor sets of the kit may comprise any of the adaptor modules described in the disclosure. To assure high performance of the kit, each of adaptor sets may be subject to a Quality Control (QC) process prior to inclusion in the kit.
  • QC Quality Control
  • the QC process for adaptor sets tests adaptor ligation efficiency.
  • the QC process for adaptor sets comprises a test for adaptor ligation.
  • the efficiency of adaptor attachment refers to the conversion rate or percentage of input DNA fragments to adaptor-tagged DNA library molecules.
  • the test for adaptor ligation is performed using a DNA sample.
  • said DNA sample is blended DNA sample of at least two different cell lines.
  • said DNA sample is a blended DNA sample of at two different cell lines.
  • the two different cell lines are blended at a 50:50 ratio.
  • the two different cell lines are NA09596 and NA12878.
  • the test for adaptor ligation is performed using the 50:50 blended sample described in Example 15.
  • the DNA sample used in the test for adaptor ligation is different from the DNA sample used in the test for adaptor distribution.
  • a unique adaptor set is assigned for each test sample in the QC process for adaptor ligation efficiency.
  • the QC process for adaptor ligation efficiency may be performed using any of the end repair, adaptor ligation, and library amplification methods of the disclosure to generate an LPA (Library Post Amplification).
  • the QC process for adaptor ligation efficiency is performed according to the methods of Example 11 to generate an LPA.
  • one or more acceptance criteria is set for the QC process for adaptor attachment efficiency, where only the adaptor sets that pass such criteria will be included in a kit of the disclosure.
  • An illustrative acceptance criterion for adaptor attachment efficiency is the LPA has a concentration of at least 1 ng/pL to at least 200 ng/pL with 5 ng- 50 ng input DNA.
  • the QC process for adaptor sets tests adaptor distribution.
  • adaptor distribution may refer to the number of sequencing reads assigned to a specific adaptor sequence in comparison to an average number of sequencing reads for all adaptor sequences with an adaptor set.
  • adaptor QC processes used in the art do not test adaptor distribution. Because a random index sequence is commonly used as an adaptor tag in many adaptor ligation processes in the art, randomness and/or distribution evenness of the adaptor tag is assumed but not tested during QC.
  • the adaptor QC processes of the disclosure are designed to measure and quantify the actual distribution of the adaptors of the disclosure, thereby ensuring high quality and performance of the adaptors of the disclosure.
  • the distribution of the adaptors is uniform.
  • adaptor distribution is considered to be uniform when the number of sequencing reads for a unique adaptor sequence is not significantly different from the average number of sequencing reads for all unique adaptor sequences.
  • adaptor distribution is measured according to the methods of Example 16. In some embodiments, adaptor distribution is considered to have passed the acceptance criteria no more than 10%-80% of unique adaptor sequences have a number of reads that is 0% to 50% of the average number of reads for all unique adaptor sequences. In some embodiments of the disclosure, the distribution of the adaptors is improved compared to the distribution of a method or kit that do not use adaptors of the disclosure.
  • the QC process to test adaptor distribution may be performed using a DNA sample.
  • said DNA sample comprises WT cfDNA.
  • said DNA sample consists of WT cfDNA.
  • said DNA sample comprises WT cfDNA obtained from a healthy human.
  • said DNA sample is the WT cfDNA sample described in Example 16.
  • An illustrative WT cfDNA sample has a concentration of at least about 0.2 ng/pL and is obtained from a healthy human or a number of healthy humans.
  • the DNA sample used in the test for adaptor distribution is different from the DNA sample used in the test for adaptor ligation.
  • the QC process for adaptor distribution comprises assigning each test sample to a unique adaptor set.
  • the QC process for adaptor ligation efficiency may be performed using any of the end repair and adaptor ligation methods of the disclosure to generate a collection of adaptor-tagged DNA fragments (LIBS) for each test sample.
  • end repair and adaptor ligation is performed according to the methods of Example 11 to generate LIBS for each test sample.
  • the QC process for adaptor distribution comprises amplification of the LIBS of each test sample using Index Primers, where one or more unique Index Primers are assigned to each LIBS of each test sample.
  • one primer pair comprising an Index Primer is assigned to each LIBS of each test sample.
  • the primer pair comprises an Index Primer and a Primer F.
  • the Primer F comprises the sequence of SEQ ID NO: 7.
  • two Index Primers are assigned to each LIBS of each test sample.
  • the Index Primers comprise Illumina ® P5 and P7 sequences.
  • the Index Primers comprise the sequences of SEQ ID NO: 13 and SEQ ID NO: 14.
  • the QC process for adaptor distribution comprises a library amplification process according to any of the library amplification methods of the disclosure. In some embodiments, the QC process for adaptor distribution comprises a library amplification process according to the methods of Example 16.
  • the QC process for adaptor distribution comprises isolating or purifying the amplified libraries.
  • the isolating or purifying is performed using DNA purification beads.
  • the isolating or purifying is performed according to the methods of Example 16. Libraries at this stage (after isolation or purification) may be referred to as Library Post Index Amplification (LPIA).
  • LPIA Library Post Index Amplification
  • the QC process for adaptor distribution comprises sequencing the LPIA of each test sample. In some embodiments, the QC process for adaptor distribution comprises sequencing a combined LPIA pool by combining a certain amount of LPIA from each test sample. In some embodiments, the combined LPIA pool may be sequenced according to the methods of Example 16. In some embodiments, the combined LPIA pool may be sequenced using sequencing primers below:
  • the Index Sequencing Primer comprises: TGTATTCGAATTCTCTGGTCCCGTGCCAGTCAC (SEQ ID NO: 83).
  • one or more acceptance criteria is provided in the QC process to determine whether the adaptor sets meet the quality requirements to enable high performance of the kit.
  • cross-contamination of adaptor sets are analyzed. Cross contamination of adaptor sets may occur when an adaptor that is not assigned to a given sample appears in the sequencing reads of this sample.
  • acceptance criteria for a QC process of adaptor set cross-contamination may include: a. for each sample, no more than 0.05%-2% of “Barcode Crosstalk”; and b. for each sample, no more than l%-20% of reads are from unknown adaptors (i.e., not included in the adaptor set).
  • Barcode Crosstalk refers to the reads of one barcode (adaptor) being mislabeled as adaptor A when they are actually generated from adaptor B.
  • one or more acceptance criteria for the QC process of adaptor distribution is selected from Table 13.
  • the adaptor includes the following features: (i) one-step attachment; (ii) high efficiency attachment; (iii) uniform adaptor distribution; (iv) accommodation of sample multiplexing and sample identification; and/or (v) high number of unique molecule identifiers (UMIs).
  • UMIs unique molecule identifiers
  • the full-length adaptor may be attached to the DNA fragment in one step.
  • a “full length” adaptor comprises at least 4 regions comprising: an amplification region comprising a primer binding site, an ID region comprising a unique molecule identifier (UMI), a UMI multiplier, and an anchor region. Attaching a full-length adapter eliminates the need for adapter ligation in a stepwise fashion where the anchor is attached first, then the remaining regions of the adaptor are attached (for example, see the stepwise manner of adaptor ligation in the kinase/ligase ligation method described below).
  • the adapters may be attached to the DNA molecules with high efficiency.
  • the efficiency of adaptor attachment refers to the conversion rate or percentage of input DNA fragments to adaptor-tagged DNA library molecules.
  • a DNA fragment may be identified by the ID region of an attached adaptor, and a DNA fragment would not be identifiable using the ID region if it was not attached to an adaptor. Accordingly, a higher efficiency of adaptor attachment may lower the number of input DNA fragments lost in the library conversion process.
  • DNA alterations e.g., single nucleotide variants (SNVs), indels, copy number changes, DNA rearrangements, optionally related to tumors/cancers
  • SNVs single nucleotide variants
  • Highly efficient attachment of adaptors of the disclosure to these DNA molecules may facilitates capture of such infrequent variations.
  • at least 50% of input DNA molecules are converted into DNA library molecules by attachment of the adapters.
  • Uniform adaptor distribution Bioinformatics analysis may analyze intra-sample adaptor performance and inter-sample adaptor performance. Performance fluctuation between adaptor sets across samples may negatively impact the sensitivity of the analysis. Uniform adaptor distribution in the adaptor-tagged DNA libraries as measured by sequence reads is desirable. In some embodiments, there is the possibility of bias in the distribution of adaptors in the adaptor-tagged DNA libraries, where some adaptors may be less efficient in ligating to the DNA molecules or less efficiently amplified compared to the others in the adapter pool. This may result in fewer amplicons, fewer reads of those less efficient adapters, and fewer reads of the DNA fragments ligated to those less efficient adaptors during sequencing.
  • compositions and methods of the disclosure provide the option of eliminating such compensation.
  • the compositions and methods of the disclosure can provide the benefit of achieving uniform adaptor distribution, wherein each adaptor is represented at roughly the same ratio in sequencing results. This uniform adaptor distribution provides increased sensitivity.
  • the uniform adaptor distribution may be achieved by having multiple types of anchor regions that are all represented in each set of adapters.
  • adaptor module sequences that have poor performance in ligation or amplification are not selected as a candidate adaptor module sequence to be used in the methods of the disclosure.
  • the uniform adaptor distribution may be achieved by having unique ID regions (each ID region identifies both the sample and the DNA fragment attached thereto) randomly selected for each set of adaptors.
  • each adaptor set is uniquely assigned to one sample.
  • Adaptor-tagged DNA fragments are constructed where a given adaptor within the same adaptor set is attached to a DNA molecule of the same test sample. From a sequence counting perspective, it is beneficial for each unique adaptor of the same set of adaptors to possess essentially identical behavior to all other adaptors in this set.
  • each ID region has a Hamming distance of at least 2 between any other ID region, thus reducing the chance for a read to be spuriously assigned to the wrong sample.
  • Hamming distance refers to the number of nucleotides that are different between two nucleotide sequences.
  • each set of adaptors is split into further groups that are paired with specific anchor regions, allowing for further reduction in the possibility of an error in sample de-multiplexing. For example, in an 8mer sequence with Hamming distance of 2, the total number of possible sequences is 16,384.
  • paired or “matched” when used with respect to two different polynucleotide sequences or regions of DNA comprising different polynucleotide sequences means that the two different polynucleotide sequences or regions of DNA comprising different polynucleotide sequences are present on the same molecule. For example, if a particular ID region of DNA is said to be paired to a particular amplification region of DNA, it is meant that the ID region and the amplification tag are present on the same DNA polynucleotide molecule.
  • UMIs Unique Molecule Identifiers
  • Each genomic clone fragment has a particular pair of fragmentation sites corresponding to the position in the genomic sequence where the double-strand DNA was cleaved.
  • this cleavage site may be used to differentiate unique genomic clones, because each clone is likely to possess a different cleavage site.
  • uniquely derived fragments may often possess the exact same cleavage sites.
  • Genomic clones sharing the same cleavage site may be classified as either unique or as redundant (/. ., generated through amplification) with respect to other clone sequences derived from the same sample.
  • the ID region is the UMI.
  • the UMI is created by the combination of the ID region with the anchor region.
  • the UMI is created by a combination of the ID region with the UMI multiplier.
  • the UMI is created by a combination of the ID region, the UMI multiplier, and the anchor region.
  • the combination of the UMI and the cleavage site create a unique molecular identifier element (UMIE), which facilitates the classification of sequence reads as redundant reads or unique reads.
  • UMIE unique molecular identifier element
  • ligation strand oligonucleotide and “ligation strand” may be used interchangeably.
  • a ligation strand oligonucleotide comprising (i) a 3’ terminal overhang; (ii) an amplification region comprising a primer binding site; (iii) a unique ID region; (iv) a unique molecule identifier (UMI) multiplier; and (v) an anchor region comprising a polynucleotide sequence that is at least partially complementary to the non-ligation strand oligonucleotide.
  • the ligation strand comprises (i) a 3’ terminal overhang; (ii) an amplification region comprising a primer binding site; (iii) a unique ID region; and (iv) an anchor region comprising a polynucleotide sequence that is at least partially complementary to the non-ligation strand oligonucleotide.
  • the ligation strand of an adaptor may comprise the following structure: AMP-ID Region/UMI Multiplier- ACGTATGCCA-3’dT (SEQ ID NO: 1). In some embodiments, the ligation strand of an adaptor may comprise the following structure: AMP-ID Region/UMI Multiplier-CTAGCGTTAC-3’dT (SEQ ID NO: 2). In some embodiments, the ligation strand of an adaptor may comprise the following structure: AMP -ID Region/UMI Multiplier-GATCGACATG-3’dT (SEQ ID NO: 3). In some embodiments, the ligation strand of an adaptor may comprise the following structure: AMP-ID Region/UMI Multiplier- T GC AT C AGGT -3 ’ dT (SEQ ID NO: 4).
  • an adaptor may comprise a ligation strand with a 3’ dT overhang.
  • the ligation strand with a 3’ dT overhang may comprise any one of the sequences shown in Table 9.
  • the ligation strand with a 3’ dT overhang may comprise a sequence of any one of SEQ ID NO: 22 to 81.
  • the “NNN” within these ligation strand sequences represents a 3 -nucleotide UMI multiplier wherein each N may be selected from any one of A, G, C, or T.
  • the ligation strand with a 3’ dT overhang may comprise a sequence of any one of SEQ ID NO: 22 to 81 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide substitutions.
  • the ligation strand oligonucleotide upon contacting with a DNA molecule is capable of ligating to the 5’ end of each strand of the DNA molecule.
  • the ligation strand oligonucleotide comprises a dT overhang at the 3 terminus and the DNA molecule comprises a dA overhang at the 3 terminus of each strand.
  • the term 3 terminal overhang refers to one or more nucleotide overhangs or tails at the 3 terminus of a polynucleotide.
  • the 3 terminal overhang (e.g ., a dT tail) aids in the ligation of the ligation strand to ligate to the 5’ end of the DNA fragment, in order to drive the efficient ligation of the multifunctional adaptor to the DNA fragment having a complementary overhang (e.g. dA-overhang/tail).
  • a complementary overhang e.g. dA-overhang/tail
  • the ligation strand oligonucleotide comprises a dA overhang at the 3 terminus and the DNA molecule comprises a dT overhang at the 3 terminus of each strand. In some embodiments, the ligation strand oligonucleotide comprises a dC overhang at the 3 terminus and the DNA molecule comprises a dG overhang at the 3 terminus of each strand. In certain embodiments, the ligation strand oligonucleotide comprises a dG overhang at the 3 terminus and the DNA molecule comprises a dC overhang at the 3 terminus of each strand.
  • the ligation strand oligonucleotide is not phosphorylated at the 5’ terminus.
  • the ligation strand oligonucleotide comprises an adaptor module, wherein the anchor region of the ligation strand oligonucleotide comprises a polynucleotide sequence that is at least partially complementary to the non-ligation strand oligonucleotide.
  • the non-ligation strand oligonucleotide is capable of hybridizing to a region at the 5’ end of the ligation strand oligonucleotide and forming a duplex therewith.
  • the ligation strand oligonucleotide is between about 30 nucleotides and about 70 nucleotides in length. In some embodiments, the ligation strand oligonucleotide is between about 35 and about 65 nucleotides, between about 40 and about 60 nucleotides, or between about 40 and about 50 nucleotides in length. In some embodiments, the ligation strand oligonucleotide is about 47 nucleotides in length.
  • the ligation strand oligonucleotide is between 30 nucleotides and 70 nucleotides in length. In some embodiments, the ligation strand oligonucleotide is between 35 and 65 nucleotides, between 40 and 60 nucleotides, or between 40 and 50 nucleotides in length. In some embodiments, the ligation strand oligonucleotide is 47 nucleotides in length.
  • non-ligation strand oligonucleotide and “non-ligation strand” may be used interchangeably.
  • the non-ligation strand of an adaptor may comprise the sequence TGGCATACGT (SEP ID NO: 18). In some embodiments, the non-ligation strand of an adaptor may comprise the sequence GTAACGCTAG (SEP ID NO: 19). In some embodiments, the non-ligation strand of an adaptor may comprise the sequence CATGTCGATC (SEQ ID NO: 20). In some embodiments, the non-ligation strand of an adaptor may comprise the sequence ACCTGATGCA_(SEQ ID NO: 21).
  • the non-ligation strand is not phosphorylated. Lack of phosphorylation of the non-ligation strand may prevent the non-ligation strand from attaching to the 3’ end of the DNA fragment and may reduce the formation of adapter dimers.
  • the non-ligation strand oligonucleotide comprises a modification at its 3’ terminus that prevents ligation to the 5’ end of the DNA molecule and/or adaptor dimer formation.
  • said modification is a chemical modification.
  • amplification region refers to an element of the adaptor molecule that comprises a primer recognition site or primer binding site.
  • the primer binding site can be for any primer that is suitable for any amplification known in the art, such as methods disclosed in Fakruddin et al. “Nucleic acid amplification: Alternative methods of polymerase chain reaction” J Pharm Bioallied Sci. 2013 Oct-Dec; 5(4): 245-252.
  • amplification methods may include PCR (polymerase chain reaction), LAMP (loop-mediated isothermal amplification), NASBA (nucleic acid sequence-based amplification), SDA (strand displacement amplification), RCA (rolling circle amplification), or LCR (ligase chain reaction).
  • an adaptor comprises an amplification region that comprises one or more custom primer binding sites.
  • the custom primer binding site comprises a sequence having 100% sequence identity to a sequence of the custom primer binding site of each adaptor module of the plurality of adaptor modules.
  • the custom primer binding sites facilitate single-primer amplification of a DNA library.
  • the amplification region comprises one, two, three, four, five, six, seven, eight, nine, ten, or more primer binding sites for single-primer amplification of a DNA library.
  • the amplification region comprises a PCR primer binding site for Library Primer (SEQ ID NO: 5).
  • the Library Primer may be a single amplification primer, also referred to herein as the AKA primer.
  • sequence identity is determined by aligning two polynucleotide sequences.
  • Various methods exist in the art to align sequences and determine sequence identity or sequence percent identity including but not limited to, using the BLAST suite from the National Center for Biotechnology Information (NCBI).
  • sequence identity refers to sequence identity determined using BLAST version 2.12.0 under default parameters.
  • the amplification region is between about 5 and about 50 nucleotides, between about 10 and about 45 nucleotides, between about 15 and about 40 nucleotides, or between about 20 and about 30 nucleotides in length.
  • the amplification region is about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, or about 40 nucleotides
  • the amplification region is between 5 and 50 nucleotides, between 10 and 45 nucleotides, between 15 and 40 nucleotides, or between 20 and 30 nucleotides in length. In some embodiments, the amplification region is 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleo
  • ID Resion As used in the disclosure, the terms “multifunctional ID region” and “ID region” may be used interchangeably and refer to an element of the adaptor that comprises a polynucleotide sequence that uniquely identifies the particular DNA fragment and/or the sample from which it was derived. In some embodiments, the ID region uniquely identifies both the DNA fragment and the sample from which it was derived.
  • the multifunctional ID region is between about 3 and about 50 nucleotides, between about 3 and about 25 nucleotides, or between about 5 and about 15 nucleotides in length. In some embodiments, the multifunctional ID region is about 3 nucleotides, 4 nucleotides, about 5 nucleotides, about 6 nucleotides, about 7 nucleotides, about 8 nucleotides, about 9 nucleotides, about 10 nucleotides, about 11 nucleotides, about 12 nucleotides, about 13 nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, or about 20 nucleotides or more in length. In some embodiments, the multifunctional ID region is about 8 nucleotides in length.
  • the multifunctional ID region is between 3 and 50 nucleotides, between 3 and 25 nucleotides, or between 5 and 15 nucleotides in length. In some embodiments, the multifunctional ID region is 3 nucleotides, 4 nucleotides, 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides or more in length. In some embodiments, the multifunctional ID region is 8 nucleotides in length.
  • the ID region comprises one of between about 2 and about 10,000 unique nucleotide sequences, between about 50 and about 500 unique nucleotide sequences, or between about 100 and about 400 unique nucleotide sequences. In some embodiments, the ID region of each adaptor of the plurality of adaptors comprises one of about 60 unique nucleotide sequences.
  • the ID region comprises one of between 2 and 10,000 unique nucleotide sequences, between 50 and 500 unique nucleotide sequences, or between 100 and 400 unique nucleotide sequences. In some embodiments, the ID region of each adaptor of the plurality of adaptors comprises one of 60 unique nucleotide sequences.
  • the adaptor comprises one of between 64 and 2,560,000 unique nucleotide sequences.
  • one or more sets of adaptors are provided, where each adaptor set is uniquely assigned to a given sample, and wherein each adaptor has a unique ID region that is distinct from the ID region of any other adaptor of the one or more adaptor sets.
  • a pre-specified first adaptor set and a pre-specified second adaptor set are provided. Such pre-specified adaptor sets are each used to represent a single sample.
  • the first adaptor set comprises a first set of unique ID regions and the second adaptor set comprises a second set of unique ID regions. That is, each adapter module sequence in the first set is distinct from each adapter module sequence in the second set; additionally, each adaptor module sequence within a set is also distinct from every other adaptor module sequence within the same set.
  • the uniqueness of each adaptor module sequence allows identification of each uniquely derived library fragment.
  • the ID region serves to identify both the DNA fragment attached thereto and the individual sample from which the DNA fragment was derived, for example, a genomic library source.
  • each sample is assigned an adaptor set comprising a plurality ofbetween about 64 and about 2.5 million unique adapters. In some embodiments, each sample is assigned an adaptor set comprising a plurality ofbetween 64 and 2.5 million unique adapters. In some embodiments, each sample is assigned an adaptor set comprising a plurality of about 3,840 unique adaptors. In some embodiments, each sample is assigned an adaptor set comprising a plurality of 3,840 unique adaptors.
  • each sample is assigned an adaptor set comprising a plurality ofbetween about 1 and about 100 unique ID regions. In some embodiments, each sample is assigned an adaptor set comprising between about 1 and about 60 unique ID regions. In some embodiments, each sample is assigned an adaptor set comprising between 1 and 60 unique ID regions. In some embodiments, each sample is assigned an adaptor set comprising 60 unique ID regions, wherein each set of 60 unique ID regions is further divided into 4 groups (each group comprising 15 unique adaptors), wherein each ID region of one group is paired to one of 4 anchor sequences. Therefore, in such embodiments, the sample can be identified by the combination of the ID region and the anchor region.
  • the nucleotide sequence of each ID region is discrete from the nucleotide sequence of any other ID regions of the plurality of adaptor modules by a Hamming distance of at least two (meaning at least two base changes are required to change one ID region into another).
  • each sample may be assigned a set of adaptors ranging from 1 ⁇ 15,360 unique adaptors for DNA fragment identification.
  • each nucleotide sequence is discrete from any other sequence of the 15,360 unique nucleotide sequences by Hamming distance of at least two.
  • the multifunctional ID region is 8 nucleotides in length and comprises one of 60 unique nucleotide sequences
  • each sample may be assigned a set of adaptors ranging from 1 ⁇ 3840 unique adaptors for DNA fragment identification.
  • each nucleotide sequence is discrete from any other sequence of the 3840 unique nucleotide sequences by Hamming distance of at least two.
  • the ID region is capable of identifying both the sample and the DNA fragment.
  • the ID region is used to identify the individual DNA fragment attached thereto.
  • the ID region can also serve as a fragment tag that can enumerate clone diversity for sensitive variant detection.
  • FIGURE 1 One embodiment of such an adaptor module comprising separate barcodes (tags) is depicted in FIGURE 1 (1 st type), wherein the ID region comprises a separate sample tag and fragment tag.
  • each adaptor module further comprises a sample tag comprising a polynucleotide sequence that is not identical to the polynucleotide sequence of the ID region.
  • each adaptor module in the a first adaptor set comprises a sample tag comprising a first polynucleotide sequence and each adaptor module in a second adaptor set comprises a sample tag comprising a second polynucleotide sequence; wherein the first and second polynucleotide sequences are not identical to each other.
  • UMI multipliers are included in the adaptor modules.
  • a UMI multiplier is a short sequence of bases which, when combined with a UMI, increases the diversity of and total number of adaptor sequences in an adaptor pool.
  • the adaptor module comprises a UMI multiplier, wherein the UMI multiplier is adjacent to or contained within the ID region.
  • the adaptor comprises an ID region that is eight nucleotides in length and a UMI multiplier that is three nucleotides in length.
  • the UMI multiplier comprises one of 64 possible sequences.
  • the UMI multiplier is adjacent to or contained within the ID region.
  • each nucleotide position of the UMI multiplier can comprise any one of adenine, guanine, cytosine, or thymine.
  • a UMI multiplier comprising n number of nucleotides can comprise any of 4 n possible nucleotide sequences.
  • the UMI multiplier is one nucleotide in length and comprises one of four possible sequences.
  • the UMI multiplier is two nucleotides in length and comprises one of sixteen possible sequences.
  • the UMI multiplier is three nucleotides in length and comprises one of 64 possible sequences.
  • the UMI multiplier is four nucleotides in length and comprises one of 256 possible sequences. In some embodiments, the UMI multiplier is five nucleotides in length and comprises one of 1,024 possible sequences. In some embodiments, the UMI multiplier is six nucleotides in length and comprises one of 4,096 possible sequences. In some embodiments, the UMI multiplier is seven nucleotides in length and comprises one of 16,384 possible sequences. In some embodiments, the UMI multiplier is eight nucleotides in length and comprises one of 65,536 possible sequences. In some embodiments, the UMI multiplier is nine nucleotides in length and comprises one of 262,144 possible sequences. In some embodiments, the UMI multiplier is ten or more nucleotides in length and comprises one of 1,048,576 or more possible sequences.
  • the UMI multiplier is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 nucleotides in length. In some embodiments, the UMI multiplier is between 1 and 5 nucleotides in length. In some embodiments, the UMI multiplier is 3 nucleotides in length.
  • anchor region and “anchor sequence” may be used interchangeably and refer to a polynucleotide sequence that is at least partially complementary to the non-ligation strand oligonucleotide.
  • the anchor region may, in some embodiments, comprise one or more of the following properties:
  • Each anchor sequence may be part of a set of two or more unique anchor types that collectively represent each of the four possible DNA bases at each site within extension; this feature, balanced base representation, is useful to calibrate proper base calling in sequencing reads in some embodiments.
  • the number of total types of anchor sequences should match the total number of detection modes. For example, four colors are detected in Illumina® sequencing, therefore four types of anchor sequences may be used. To achieve maximum sensitivity, each detection mode may be utilized.
  • the methods and compositions of the disclosure may be used in any mode of detection known in the art, including but not limited to light-based detection, enzyme-based detection, and magnetic detection.
  • Each anchor sequence may be composed of only two of four possible bases, which are specifically chosen to be either an equal number of A + C or an equal number of G + T; an anchor sequence formed from only two bases reduces the possibility that the anchor sequence will participate in secondary structure formation that would preclude proper adaptor function.
  • each anchor sequence may be composed of equal numbers of A + C or G + T, each anchor sequence may share roughly the same melting temperature and duplex stability as every other anchor sequence in the set.
  • Each type of anchor sequence (ending in either A/T/G/C) may be approximately equally distributed in the sequencing reads, for example in approximately equimolar amounts (i.e. about 25% of the set have adapter sequences ending in A, about 25% ending in T, about 25% ending in G, and about 25% ending in C).
  • adapter modules are mixed with DNA fragments in equimolar amounts of adapters containing different anchor types (e.g . equimolar amounts of anchor 1, anchor 2, anchor 3, anchor 4) to provide a more even adapter distribution.
  • anchor sequences include but are not limited to: Anchor 1 ACGTATGCCA (SEQ ID NO: 1); Anchor 2 CTAGCGTTAC (SEQ ID NO: 2); Anchor 3 GATCGACATG (SEQ ID NO: 3); and Anchor 4 TGCATCAGGT (SEQ ID NO: 4).
  • adapter sequences end with a T nucleotide at the 3’ terminus (3’ T overhang).
  • some adaptors have TT as the last 2 nucleotides of the 3’ terminus, others may have AT, CT, or GT as the last 2 nucleotides of the 3’ terminus.
  • the plurality of adapters in an adaptor set comprises a 3’ dT overhang that does not hybridize to the non-ligation strand.
  • each anchor type having an identical distribution percentage (i.e., 100% divided by the number of anchor types), resulting in a “uniform” distribution of different adaptors comprising different anchors among all DNA fragments.
  • distribution of anchor types refers to the distribution of different anchor sequences in a DNA library that is sequence-ready or has been sequenced.
  • an ideal distribution of four anchor types would result in about 25% distribution of each anchor type.
  • the anchor sequences of a given adaptor set have a distribution percentage of between about 5% to about 75% (i.e., the distribution % of the most infrequent anchor type is about 5% and the distribution % of the most frequent anchor type is about 75%).
  • each anchor sequence of a given set has a distribution % of about 50%, about 34%, about 28%, about 27%, about 23%, about 14%, or about 9%.
  • adapters having anchors of a specific sequence or type may be present in higher amounts.
  • Such adapters may be IX, 2X, 4X, 5X, 6X, 7X, 8X, 9X, or more than 1 OX the amount of other anchor types in the adaptor set, resulting in more even distribution of adaptors in sequencing reads.
  • the adaptor set can comprise more than one anchor sequence.
  • a set of adaptors may contain 4 different anchor sequences are used simultaneously. These anchor sequences may also be used during sample de-multiplexing to lower errors.
  • sequencing reads need to pass inclusion filters for downstream consideration. In such embodiments, because the position of sequences within a sequencing read is fixed, the ID regions and anchors should have a fixed position within a sequencing read in order to pass the inclusion filters.
  • the anchor region of each adaptor of a given set of adaptors comprises one of four sequences.
  • a total of 16 distinct ID regions is split into four anchor groups (one group per each nucleotide A, C, T and G), with each anchor group comprising 4 ID regions and each ID region is matched with one of four anchor sequences.
  • a total of 240 ID regions may be split into four anchor groups (one group per each nucleotide A, C, T and G), with each anchor group comprising 60 ID regions and each ID region matched with one of four anchor sequences.
  • the 240 ID regions are divided into four sets of 60 sequences, with each set paired to a specific anchor region.
  • each adaptor set has an equimolar amount of each anchor sequence. Therefore, in some embodiments, identification involves not only the sequence information from the ID region, but also the sequence information from the associated anchor region. This may result in each adaptor having a unique sequence and the plurality of adaptors in each adaptor set having a balanced anchor composition. In some embodiments, a total of 60 ID regions may be split into four sets, with each set comprising 15 ID regions and each ID region matched with one of four anchor sequences.
  • the anchor region is between 1 and 50 nucleotides in length. In some embodiments, the anchor region is between 4 and 40 nucleotides in length. In certain embodiments, the anchor region is between 5 and 25 nucleotides in length. In some embodiments, the anchor region is at least 4 nucleotides, at least six nucleotides, at least 8 nucleotides, at least 10 nucleotides, at least 12 nucleotides, at least 14 nucleotides, or at least 16 nucleotides in length. In some embodiments, the anchor region is 10 nucleotides in length.
  • compositions and methods comprising one or more capture probe modules.
  • the disclosure provides a kit comprising one or more capture probe modules.
  • compositions and methods of the disclosure can be used for targeted genetic analysis of one or more target genetic loci of the DNA library clones.
  • targeted genetic analysis comprises one or more of the following steps: capturing and/or isolating DNA clones comprising a target genetic locus; amplification of the captured targeted genetic locus; sequencing of the amplified captured targeted genetic locus; and bioinformatic analysis of the resulting sequence reads.
  • DNA library clone refer to a DNA library fragment wherein the combination of the adaptor and the genomic DNA fragment result in a unique DNA sequence ( e.g ., a DNA sequence that can be distinguished from that of another DNA library clone).
  • the disclosure provides, in part, a capture probe module designed to retain the efficiency and reliability of larger probes but that minimizes uninformative sequence generation in a genomic DNA library that comprises smaller DNA fragments, e.g., a cfDNA clone library.
  • the capture probe module comprises a tail sequence and a capture probe sequence.
  • the capture probe module comprises a first region comprising the tail sequence and a second region capable of hybridizing to a target sequence in the tagged DNA library.
  • the tail sequence comprises a PCR primer binding site.
  • the first region is referred to as the tail region or tail sequence.
  • tail sequence refers to a polynucleotide at the 5' end of the capture probe module, which in some embodiments can serve as a primer binding site. In some embodiments, the tail sequence comprises a sequencing primer binding site.
  • the tail sequence is capable of hybridizing to a partner oligonucleotide.
  • an exemplary partner oligonucleotide can be: GTGAAAACCAGGATCAACTCCCGTGCCAGTCACATCTCAGATGAGCT/3BioTEG/ (SEQ ID NO: 6).
  • “3BioTEG” may be a Biotin-TEG label that binds to streptavidin coated magnetic beads.
  • the “3BioTEG” label may be any type of label known in the art capable of binding to a solid support.
  • the tail sequence is about 5 to about 100 nucleotides, about 10 to about 100 nucleotides, about 5 to about 75 nucleotides, about 5 to about 50 nucleotides, about 5 to about 25 nucleotides, or about 5 to about 20 nucleotides. In some embodiments, the tail sequence is about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, or about 40 nucleotides.
  • the tail sequence is 5 to 100 nucleotides, 10 to 100 nucleotides,
  • the tail sequence is 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, or 40 nucleotides.
  • the contiguous adaptor-tagged DNA fragments (unamplified) and tagged DNA library (amplified) are each useful for a variety of sequencing-based genetic analyses including the preparation of libraries containing Hybrid Molecules enriched for one or more genetic loci of interest and may be unamplified or amplified libraries.
  • the unamplified adaptor-tagged DNA fragments and/or amplified tagged DNA libraries, prepared as described above, can be hybridized to capture probes modules to generate libraries targeted to specific genetic loci, i.e., targeted libraries.
  • the adaptor-tagged DNA fragments can be hybridized with one or more capture probes. Each capture probe can target the same genetic loci in the adapter-tagged DNA fragments or they may target different genetic loci in the adapter-tagged DNA fragments. In some embodiments, a plurality of genetic loci in the amplified tagged DNA library fragments are targeted.
  • the capture probe modules are used with genomic DNA library constructed from cellular DNA. In some embodiments, the capture probe modules are used with genomic DNA library constructed from cfDNA. Because the average size of cfDNA is about 150 to about 170 bp and is highly fragmented, certain compositions and methods of the disclosure comprise the use of high density and relatively short capture probes to interrogate DNA target regions of interest. In some embodiments, the capture probes are capable of hybridizing to DNA target regions that are distributed across all chromosomal segments at a uniform density.
  • Chromosomal stability probes are used to interrogate copy number variations on a genome-wide scale in order to provide a genome-wide measurement of chromosomal copy number (e.g ., chromosomal ploidy).
  • Base composition refers to the relative amounts of various purines and pyrimidines (e.g., A, G, C, T) in a DNA composition.
  • purines and pyrimidines e.g., A, G, C, T
  • the lack of flexibility in capture probe design rules may not substantially impact probe performance of capture probe modules of the disclosure.
  • capture probes of the disclosure chosen strictly by positional constraint, without considering the limitations of base composition, may provide on-target sequence information; exhibit very little off-target and unmappable read capture; and yield uniform, useful, on-target reads with only few exceptions. Moreover, the high redundancy at close probe spacing may compensate for potentially poor-performing capture probes.
  • the capture probe modules of the disclosure are configured to be used in a high-density capture probe strategy wherein a plurality of capture probes hybridize to a target region or target genetic locus, wherein each of the plurality.
  • any two or more capture probes are designed to bind to the target region within about 5 nucleotides of each other, within aboutlO nucleotides of each other, within about 15 nucleotides of each other, within about 20 nucleotides of each other, within about 25 nucleotides of each other, within about 30 nucleotides of each other, within about 35 nucleotides of each other, within about 40 nucleotides of each other, within about 45 nucleotides of each other, within about 50 nucleotides of each other, within about 100 nucleotides of each other, within about 200 nucleotides or more of each other, as well as all intervening nucleotide lengths.
  • the target region comprises a target sequence to which a capture probe hybridizes. In some embodiments, the target region comprises a plurality of target sequences to which a plurality of capture probes hybridizes.
  • the capture probe is about 20 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides.
  • nucleotides 52 nucleotides, 53 nucleotides, 54 nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, 58 nucleotides, 59 nucleotides, or 60 nucleotides.
  • the capture probe is about 60 nucleotides. In some embodiments, the capture probe is about 65 nucleotides. In some embodiments, the capture probe is about 70 nucleotides. In some embodiments, the capture probe is about 75 nucleotides. In some embodiments, the capture probe is about 80 nucleotides. In some embodiments, the capture probe is about 85 nucleotides. In some embodiments, the capture probe is about 90 nucleotides. In some embodiments, the capture probe is about 95 nucleotides. In some embodiments, the capture probe is about 100 nucleotides, about 200 nucleotides, about 300 nucleotides, about 400 nucleotides, or about 100 nucleotides.
  • the capture probe is from about 10 nucleotides to about 20 nucleotides, about 20 nucleotides to about 60 nucleotides, about 60 nucleotides to about 100 nucleotides, about 100 nucleotides to about 500 nucleotides, about 200 nucleotides to about 500 nucleotides, about 300 nucleotides to about 500 nucleotides, or about 400 nucleotides to about 500 nucleotides, or any intervening range thereof.
  • the capture probe is 60 nucleotides, 65 nucleotides, 70 nucleotides, 75 nucleotides, 80 nucleotides, 85 nucleotides, 90 nucleotides, 95 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, or 100 nucleotides.
  • the capture probe is from 10 nucleotides to 20 nucleotides, 20 nucleotides to 60 nucleotides, 60 nucleotides to 100 nucleotides, 100 nucleotides to 500 nucleotides, 200 nucleotides to 500 nucleotides, 300 nucleotides to 500 nucleotides, or 400 nucleotides to 500 nucleotides, or any intervening range thereof.
  • the capture probe is substantially smaller than 60 nucleotides but hybridizes comparably, as well as, or better than a 60-nucleotide capture probe targeting the same DNA target region. In some embodiments, the capture probe is 40 nucleotides.
  • the capture probe module comprises a specific member of a binding pair to enable isolation and/or purification of one or more captured fragments of a tagged and or amplified genomic DNA library (e.g . , a cellular or cfDNA library) that hybridizes to the capture probe.
  • the capture probe module comprises a biotin molecule.
  • the capture probe module is conjugated to biotin or another suitable hapten, e.g., dinitrophenol, digoxigenin.
  • the partner oligonucleotide comprises a specific member of a binding pair to enable isolation and/or purification of one or more captured fragments of a tagged and or amplified genomic DNA library (e.g. , a cellular or cfDNA library) that hybridizes to the capture probe.
  • a tagged and or amplified genomic DNA library e.g. , a cellular or cfDNA library
  • the partner oligonucleotide comprises a biotin molecule.
  • the partner oligonucleotide is conjugated to biotin or another suitable hapten, e.g, dinitrophenol, digoxigenin.
  • the capture probe is hybridized to a tagged and optionally amplified DNA library to form a complex.
  • the capture probe substantially hybridizes to a specific genomic target region or target sequence in the DNA library.
  • Hybridization or hybridizing conditions can include any reaction conditions where two nucleotide sequences form a stable complex; for example, in some embodiments, the adaptor- tagged DNA library fragment and capture probe forms a stable adaptor-tagged DNA library fragment — capture probe complex.
  • reaction conditions are well known in the art and those of skill in the art will appreciate that such conditions can be modified as appropriate, e.g., decreased annealing temperatures with shorter length capture probes, and within the scope of the disclosure.
  • Substantial hybridization can occur when the second region of the capture probe complex exhibits 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92% 91%, 90%, 89%, 88%, 85%, 80%, 75%, or 70% sequence identity, homology or complementarity to a region of the tagged DNA library.
  • the capture probe is about 40 nucleotides and has an optimal annealing temperature of about 44° C to about 47° C.
  • the capture probe is 40 nucleotides and has an optimal annealing temperature of 44° C to 47° C.
  • compositions of the disclosure are assembled into a kit comprising one or more capture probe modules.
  • kits of the disclosure may be used to generate a combined sequence-ready DNA library, where the combined library is any of the combined libraries described in the disclosure.
  • a kit of the disclosure comprises a set of capture probe modules designed to target a specific collection of genes, e.g. , a set of lung cancer genes as described in Table 11. Such a set of capture probe modules may be referred to as a capture probe panel. These capture probe modules or capture probe panels may comprise any capture probe module of the disclosure. To assure high performance of the kit, each of the capture probe modules or capture probe panels may be subject to a Quality Control (QC) process prior to inclusion in the kit.
  • QC Quality Control
  • a unique adaptor set is assigned for each test sample in the QC process for capture probe modules.
  • the QC process for capture probe modules may be performed using any of the end repair, adaptor ligation, and library amplification methods of the disclosure to generate an LPA.
  • the QC process for capture probe modules comprises performing end repair, adaptor ligation, and library amplification according to the methods of Example 11 to generate an LPA.
  • the QC process for capture probe modules comprises creating a TC LPA and a WG LPA from an LPA.
  • the TC LPA and WG LPA are created according to the methods of Example 11.
  • a QC process on the TC LPA and WG LPA may be performed and libraries that passed QC are selected for downstream use.
  • a TC LPA of at least 1-80 ng / pL and a WG LPA of at least 0 1-8 ng / pL are used in downstream processes.
  • the QC process for capture probe modules comprises using a TC LPA to create a TCL using a specific capture probe panel.
  • the QC process for capture probe modules comprises creating a TCL according to any of the methods of the disclosure.
  • the QC process for capture probe modules comprises creating a TCL according to the methods of Example 12 In some embodiments, the QC process for capture probe modules comprises creating a TCLA from a TCL according to any of the methods of the disclosure. In some embodiments, the QC process for capture probe modules comprises creating a TCLA from a TCL according to the methods of Example 12 In some embodiments, the capture probe panel tested in the QC process for capture probe modules comprises a set of Lung Probes. In some embodiments, each capture probe module within the set of Lung Probes binds to a specific target sequence in a gene associated with lung cancer. In some embodiments, each capture probe module within the set of Lung Probes binds to a specific target sequence in a gene listed in Table 11.
  • the QC process for capture probe modules comprises using a WG LPA to create a WGLA according to any of the methods of the disclosure. In some embodiments, the QC process for capture probe modules comprises using a WG LPA to create a WGLA according to the methods of Example 13 In some embodiments, each TCLA and WGLA is required to have a concentration of at least 10 ng/pL to be used in downstream processes of the QC process for capture probe modules.
  • the QC process for capture probe modules comprises combining the TCLA and WGLA to create a sequence-ready library.
  • the TCLA and WGLA are combined according to the methods of Example 14.
  • the QC process for capture probe modules comprises sequencing the combined TCLA and WGLA library according to any of the sequencing methods of the disclosure.
  • one or more acceptance criteria is provided in the QC process to determine whether the capture probe modules meet the quality requirements to enable high performance of the kit.
  • one or more acceptance criteria for the QC process for capture probe modules is selected from Table 15.
  • only capture probe modules that have passed one or more of the QC processes of the disclosure is included in a kit of the disclosure.

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US12203127B2 (en) 2014-08-22 2025-01-21 Resolution Bioscience, Inc. Methods for quantitative genetic analysis of cell free DNA
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WO2025228840A1 (en) * 2024-05-02 2025-11-06 Medicover Biotech Ltd. Method for somatic mutation detection
WO2026038176A1 (en) * 2024-08-14 2026-02-19 Sophia Genetics S.A. Systems and methods for assaying various regions of a genome at different resolutions

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