WO2014093330A1 - Methods for targeted genomic analysis - Google Patents

Methods for targeted genomic analysis Download PDF

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Publication number
WO2014093330A1
WO2014093330A1 PCT/US2013/074102 US2013074102W WO2014093330A1 WO 2014093330 A1 WO2014093330 A1 WO 2014093330A1 US 2013074102 W US2013074102 W US 2013074102W WO 2014093330 A1 WO2014093330 A1 WO 2014093330A1
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Prior art keywords
capture probe
multifunctional capture
sequence
library
genomic
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PCT/US2013/074102
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English (en)
French (fr)
Inventor
Christopher K. Raymond
Christopher D. Armour
Lee P. Lim
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CLEARFORK BIOSCIENCE Inc
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CLEARFORK BIOSCIENCE Inc
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Priority to JP2015545927A priority Critical patent/JP6410726B2/ja
Priority to CN202311824187.2A priority patent/CN117845337A/zh
Priority to SG11201504334WA priority patent/SG11201504334WA/en
Priority to CA2892646A priority patent/CA2892646A1/en
Priority to EP19153893.3A priority patent/EP3561072A1/en
Priority to EP23186301.0A priority patent/EP4293125A3/en
Priority to HK16101684.1A priority patent/HK1213944A1/zh
Priority to EP13862440.8A priority patent/EP2929056A4/en
Priority to CN201380072169.1A priority patent/CN105531375B/zh
Priority to CN202010078573.1A priority patent/CN111254500B/zh
Publication of WO2014093330A1 publication Critical patent/WO2014093330A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms

Definitions

  • CLFK_001_02WO_ST25.txt The text file is 188 KB, was created on December 10, 2013, and is being submitted electronically via EFS-Web.
  • the invention relates generally to a method for genetic analysis in individuals that reveals both the genetic sequences and chromosomal copy number of targeted and specific genomic loci in a single assay.
  • the present invention relates to methods that provide sensitive and specific detection of target gene sequences or gene transcripts and methods that reveal both variant sequences and overall gene copy number in a single assay.
  • Particular embodiments contemplated herein provide a method for generating a tagged DNA library comprising treating fragmented DNA with end-repair enzymes to generate fragmented end-repaired DNA; and ligating a random nucleic acid tag sequence, and optionally a sample code sequence and/or a PCR primer sequence to the fragmented end-repaired DNA to generate the tagged DNA library.
  • the random nucleic acid tag sequence is from about 2 to about 100 nucleotides. In some embodiments, the present invention provides that the random nucleic acid tag sequence is from about 2 to about 8 nucleotides.
  • the fragmented end-repaired DNA contains blunt ends.
  • the blunt ends are further modified to contain a single base pair overhang.
  • the ligating comprises ligating a multifunctional adaptor module to the fragmented end-repaired DNA to generate the tagged DNA library, wherein the multifunctional adaptor molecule comprises: i) a first region comprising a random nucleic acid tag sequence; ii) a second region comprising a sample code sequence; and iii) a third region comprising a PCR primer sequence.
  • the method further comprises hybridizing a tagged DNA library with at least one multifunctional capture probe module to form a complex, wherein the multifunctional capture probe module hybridizes to a specific target region in the DNA library.
  • the method further comprises isolating the tagged DNA library-multifunctional capture probe module complex.
  • the method further comprises 3 '-5' exonuclease enzymatic processing of the isolated tagged DNA library-multifunctional capture probe module complex to remove the single stranded 3' ends.
  • the enzyme for use in the 3 '-5' exonuclease enzymatic processing is T4 polymerase.
  • the method further comprises 5 '-3' DNA polymerase extension of the isolated tagged DNA library-multifunctional capture probe module complex from the 3 ' end of the multifunctional capture probe utilizing the isolated tagged DNA library fragments as template.
  • the method further comprises joining of the
  • the method further comprises performing PCR on the 3 '-5' exonuclease enzymatically processed complex, wherein the tail portion of the multifunctional capture probe molecule is copied in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the genomic target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence.
  • a method for targeted genetic analysis comprising: a) hybridizing a tagged DNA library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library-multifunctional capture probe module complex from a); c) performing 3 '-5' exonuclease enzymatic processing on the isolated tagged DNA library-multifunctional capture probe module complex from b) using an enzyme with 3 '-5' exonuclease activity to remove the single stranded 3' ends; d) performing PCR on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is copied in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence; and e) performing targeted genetic analysis on the hybrid nucleic acid
  • a method for targeted genetic analysis comprising: a) hybridizing a tagged DNA library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged genomic library-multifunctional capture probe module complex from a); c) performing 5 '-3' DNA polymerase extension of multifunctional capture probe utilizing the isolated tagged DNA library fragments as template; d) performing PCR on the enzymatically processed complex from c) wherein the complement of the isolated target region is copied in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the complement of the DNA target region, the target- specific region of the multifunctional capture probe and the multifunctional capture probe module tail sequence; and e) performing targeted genetic analysis on the hybrid nucleic acid molecule from d).
  • a method for targeted genetic analysis comprising: a) hybridizing a tagged DNA library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library-multifunctional capture probe module complex from a); c) performing the creation of a hybrid multifunctional capture probe-isolated tagged DNA target molecule through the concerted action of a 5' FLAP endonuclease, DNA polymerization and nick closure by a DNA ligase; d) performing PCR on the enzymatically processed complex from c) wherein the of the multifunctional capture probe molecule is joined to the isolated tagged DNA target clone in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the genomic target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module; and e) performing targeted genetic analysis on the hybrid nucle
  • a method for determining copy number of a specific target region comprising: a) hybridizing a tagged DNA library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library-multifunctional capture probe module complex from a); c) performing 3 '-5' exonuclease enzymatic processing on the isolated tagged DNA library-multifunctional capture probe module complex from b) using an enzyme with 3 '-5' exonuclease activity to remove the single stranded 3' ends; d) performing a PCR reaction on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is replicated in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence; e) performing
  • a method for determining copy number of a specific target region comprising: a) hybridizing a tagged DNA library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library-multifunctional capture probe module complex from a); c) performing 5 '-3' DNA polymerase extension of multifunctional capture probe utilizing the isolated tagged DNA library fragments as template; d) performing a PCR reaction on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is replicated in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence; e) performing PCR amplification of the hybrid nucleic acid in d); and e) quantitating the PCR reaction in d), wherein the quantitation
  • a method for determining copy number of a specific target region comprising: a) hybridizing a tagged DNA library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library-multifunctional capture probe module complex from a); c) performing the creation of a hybrid multifunctional capture probe-isolated tagged DNA target molecule through the concerted action of a 5 ' FLAP endonuclease, DNA polymerization and nick closure by a DNA ligase; d) performing a PCR reaction on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is replicated in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence; e) performing PCR amplification of
  • a method for targeted genetic analysis comprising: a) hybridizing a tagged DNA library with a multifunctional capture probe hybrid module complex, wherein the multifunctional capture probe hybrid module selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library-multifunctional capture probe hybrid module complex from a); c) performing PCR on the complex from b) to replicate a region that is 3' relative to the sequence of the multifunctional capture probe in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the multifunctional capture probe hybrid module and the complement of a region of the tagged DNA library sequence that is located 3' relative to the multifunctional capture probe; and d) performing targeted genetic analysis on the hybrid nucleic acid molecule from c).
  • a method for targeted genetic analysis comprising: a) hybridizing a tagged DNA library with a multifunctional capture probe hybrid module complex, wherein the multifunctional capture probe hybrid module selectively hybridizes to a specific target region in the genomic library; b) isolating the tagged DNA library-multifunctional capture probe hybrid module complex from a); c) performing 5 '-3' DNA polymerase extension of multifunctional capture probe utilizing the isolated tagged DNA library fragments as template, wherein the hybrid nucleic acid molecule comprises the multifunctional capture probe hybrid module and the complement of a region of the tagged DNA library sequence that is located 3 ' relative to the multifunctional capture probe; and d) performing targeted genetic analysis on the hybrid nucleic acid molecule from c).
  • a method for targeted genetic analysis comprising: a) hybridizing a tagged DNA library with a multifunctional capture probe hybrid module complex, wherein the multifunctional capture probe hybrid module selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library-multifunctional capture probe hybrid module complex from a); c) performing the creation of a hybrid multifunctional capture probe-isolated tagged DNA target molecule through the concerted action of a 5' FLAP endonuclease, DNA polymerization and nick closure by a DNA ligase, wherein the hybrid nucleic acid molecule comprises the complement of the multifunctional capture probe hybrid module and a region of the tagged DNA library sequence that is located 5' relative to the multifunctional capture probe; and d) performing targeted genetic analysis on the hybrid nucleic acid molecule from c).
  • a method for determining copy number of a specific target region comprising: a) hybridizing a tagged DNA library with a multifunctional capture probe hybrid module complex, wherein the multifunctional capture probe hybrid module selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library-multifunctional capture probe hybrid module complex from a); c) performing PCR on the complex from b) to replicate a region that is 3 ' relative to sequence of the multifunctional capture probe in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the multifunctional capture probe hybrid module and the complement of a region of the tagged DNA library sequence that is located 3 ' relative to the
  • multifunctional capture probe d) performing PCR amplification of the hybrid nucleic acid in c); and e) quantitating the PCR reaction in d), wherein the quantitation allows for a determination of copy number of the specific target region.
  • the targeted genetic analysis is sequence analysis.
  • the tagged DNA library is amplified by PCR to generate an amplified tagged DNA library.
  • the DNA is from a biological sample selected from the group consisting of blood, skin, hair, hair follicles, saliva, oral mucous, vaginal mucous, sweat, tears, epithelial tissues, urine, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid), excreta, biopsy, ascites, cerebrospinal fluid, lymph, and tissue extract sample or biopsy sample.
  • a biological sample selected from the group consisting of blood, skin, hair, hair follicles, saliva, oral mucous, vaginal mucous, sweat, tears, epithelial tissues, urine, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid), excreta, biopsy, ascites, cerebrospinal fluid, lymph, and tissue extract sample or biopsy sample.
  • a tagged DNA library comprises tagged DNA sequences, wherein each tagged DNA sequence comprises: i) fragmented end-repaired DNA; ii) a random nucleotide tag sequence; iii) a sample code sequence; and iv) a PCR primer sequence.
  • a hybrid tagged DNA library comprises hybrid tagged DNA sequences for use in targeted genetic analysis, wherein each hybrid tagged DNA sequence comprises: i) fragmented end-repaired DNA; ii) a random nucleotide tag sequence; iii) a sample code sequence; iv) a PCR primer sequence; and v) a multifunctional capture probe module tail sequence.
  • a multifunctional adaptor module comprises: i) a first region comprising a random nucleotide tag sequence; ii) a second region comprising a sample code sequence; and iii) a third region comprising a PCR primer sequence.
  • a multifunctional capture probe module comprises: i) a first region capable of hybridizing to a partner oligonucleotide; ii) a second region capable of hybridizing to a specific target region; and iii) a third region comprising a tail sequence.
  • the first region of the capture probe module is bound to a partner oligonucleotide.
  • the partner oligonucleotide is chemically modified.
  • compositions comprise a tagged DNA library, a
  • multifunctional adaptor module and a multifunctional capture probe module.
  • composition comprises a hybrid tagged genomic library according to the methods of the present invention.
  • a composition comprises a reaction mixture for performing the methods contemplated herein.
  • a reaction mixture capable of generating a tagged DNA library comprises: a) fragmented DNA and b) DNA end-repair enzymes to generate fragmented end-repaired DNA.
  • a reaction mixture further comprises a multifunctional adaptor module.
  • a reaction mixture further comprises a
  • a reaction mixture further comprises an enzyme with 3'- 5' exonuclease activity and PCR amplification activity.
  • the reaction mixture comprises a FLAP endonuclease, a DNA polymerase, and DNA ligase.
  • the DNA can be isolated genomic DNA or cDNA.
  • a method for generating a tagged genomic library comprising: treating fragmented genomic DNA with end-repair enzymes to generate fragmented end-repaired genomic DNA; and ligating a random nucleic acid tag sequence, and optionally a sample code sequence and/or a PCR primer sequence to the fragmented end-repaired genomic DNA to generate the tagged genomic library.
  • the random nucleic acid tag sequence is from about 2 to about 100 nucleotides.
  • the random nucleic acid tag sequence is from about 2 to about 8 nucleotides.
  • the fragmented end-repaired genomic DNA contains blunt ends.
  • blunt ends are further modified to contain a single base pair overhang.
  • the ligating comprises ligating a multifunctional adaptor module to the fragmented end-repaired genomic DNA to generate the tagged genomic library, wherein the multifunctional adaptor molecule comprises: a first region comprising a random nucleic acid tag sequence; a second region comprising a sample code sequence; and a third region comprising a PCR primer sequence.
  • the methods contemplated herein comprise hybridizing a tagged genomic library with a multifunctional capture probe module to form a complex, wherein the multifunctional capture probe module hybridizes to a specific genomic target region in the genomic library.
  • the methods contemplated herein comprise isolating the tagged genomic library-multifunctional capture probe module complex.
  • the methods contemplated herein comprise 3 '-5' exonuclease enzymatic processing of the isolated tagged genomic library-multifunctional capture probe module complex to remove the single stranded 3' ends.
  • the enzyme for use in the 3 '-5' exonuclease enzymatic processing is T4 DNA polymerase.
  • the methods contemplated herein comprise performing PCR on the 3 '-5' exonuclease enzymatically processed complex from the preceding claims, wherein the tail portion of the multifunctional capture probe molecule is copied in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the genomic target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence.
  • a method for targeted genetic analysis comprising: (a) hybridizing a tagged genomic library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module selectively hybridizes to a specific genomic target region in the genomic library; (b) isolating the tagged genomic library-multifunctional capture probe module complex from a); (c) performing 3 '-5' exonuclease enzymatic processing on the isolated tagged genomic library-multifunctional capture probe module complex from b) using an enzyme with 3 '-5' exonuclease activity to remove the single stranded 3' ends; (d) performing PCR on the enzymatically processed complex from c) wherein the tail portion of the
  • multifunctional capture probe molecule is copied in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the genomic target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence; and (e) performing targeted genetic analysis on the hybrid nucleic acid molecule from d).
  • steps a) through d) are repeated at least about twice and the targeted genetic analysis of e) comprises a sequence alignment of the hybrid nucleic acid molecule sequences obtained from the at least two d) steps.
  • At least two different multifunctional capture probe modules are used in the at least two a) steps, wherein the at least two a) steps employ one multifunctional capture probe module each.
  • at least one multifunctional capture probe module hybridizes downstream of the genomic target region and at least one multifunctional capture probe module hybridizes upstream of the genomic target region.
  • a method for determining copy number of a specific genomic target region comprising: (a) hybridizing a tagged genomic library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module complex selectively hybridizes to a specific genomic target region in the genomic library; (b) isolating the tagged genomic library-multifunctional capture probe module complex from a); (c) performing 3 '-5' exonuclease enzymatic processing on the isolated tagged genomic library-multifunctional capture probe module complex from b) using an enzyme with 3 '-5' exonuclease activity to remove the single stranded 3' ends; (d) performing a PCR reaction on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is replicated in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the genomic target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence;
  • the methods contemplated herein comprise obtaining the sequences of the hybrid nucleic acid molecules from step e).
  • steps a) through e) are repeated at least about twice and a sequence alignment is performed using the hybrid nucleic acid molecule sequences obtained from the at least two e) steps.
  • At least two different multifunctional capture probe modules are used in the at least two a) steps, wherein the at least two a) steps employ one multifunctional capture probe module each.
  • At least one multifunctional capture probe module hybridizes downstream of the genomic target region and at least one multifunctional capture probe module hybridizes upstream of the genomic target region.
  • a method for determining copy number of a specific genomic target region comprising: (a) hybridizing a tagged genomic library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module complex selectively hybridizes to a specific genomic target region in the genomic library; (b) isolating the tagged genomic library-multifunctional capture probe module complex from a); (c) performing 3 '-5' exonuclease enzymatic processing on the isolated tagged genomic library-multifunctional capture probe module complex from b) using an enzyme with 3 '-5' exonuclease activity to remove the single stranded 3' ends; (d) performing a PCR reaction on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is replicated in order to generate a hybrid nucleic acid
  • the methods contemplated herein comprise obtaining the sequences of the hybrid nucleic acid molecules from step e).
  • steps a) through e) are repeated at least about twice and a sequence alignment is performed using the hybrid nucleic acid molecule sequences obtained from the at least two e) steps.
  • At least two different multifunctional capture probe modules are used in the at least two a) steps, wherein the at least two a) steps employ one multifunctional capture probe module each.
  • At least one multifunctional capture probe module hybridizes downstream of the genomic target region and at least one multifunctional capture probe module hybridizes upstream of the genomic target region.
  • a method for determining copy number of a specific genomic target region comprising: (a) hybridizing a tagged genomic library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module comprises selectively hybridizes to a specific genomic target region in the genomic library; (b) isolating the tagged genomic library-multifunctional capture probe module complex from a); (c) performing 3 '-5' exonuclease enzymatic processing on the isolated tagged genomic library-multifunctional capture probe module complex from b) using an enzyme with 3 '-5' exonuclease activity to remove the single stranded 3' ends; (d) performing a PCR reaction on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is replicated in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the genomic target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence;
  • steps a) through e) are repeated at least about twice and the targeted genetic analysis of f) comprises performing a sequence alignment of the hybrid nucleic acid molecule sequences from the at least two e) steps.
  • At least two different multifunctional capture probe modules are used in the at least two a) steps, wherein the at least two a) steps employ one multifunctional capture probe module each.
  • At least one multifunctional capture probe module hybridizes downstream of the genomic target region and at least one multifunctional capture probe module hybridizes upstream of the genomic target region.
  • a method for targeted genetic analysis comprising: (a) hybridizing a tagged genomic library with a multifunctional capture probe hybrid module complex, wherein the multifunctional capture probe hybrid module selectively hybridizes to a specific genomic target region in the genomic library; (b) isolating the tagged genomic library-multifunctional capture probe hybrid module complex from a); (c) performing 5' to 3' DNA polymerase extension of the multifunctional capture probe on the complex from b) to replicate a region of the captured, tagged genomic target region that is 3 Of the multifunctional capture probe in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the multifunctional capture probe hybrid module and the complement of a region of the tagged genomic target region that is located in the 3' direction from the location where the multifunctional capture probe hybrid module hybridizes to the genomic target region; and (d) performing targeted genetic analysis on the hybrid nucleic acid molecule from c).
  • steps a) through c) are repeated at least about twice and the targeted genetic analysis of d) comprises a sequence alignment of the hybrid nucleic acid molecule sequences obtained from the at least two d) steps.
  • At least two different multifunctional capture probe modules are used in the at least two a) steps, wherein the at least two a) steps employ one multifunctional capture probe module each.
  • At least one multifunctional capture probe module hybridizes downstream of the genomic target region and at least one multifunctional capture probe module hybridizes upstream of the genomic target region.
  • a method for determining copy number of a specific genomic target region comprising: (a) hybridizing a tagged genomic library with a multifunctional capture probe hybrid module complex, wherein the
  • multifunctional capture probe hybrid module selectively hybridizes to a specific genomic target region in the genomic library; (b) isolating the tagged genomic library- multifunctional capture probe hybrid module complex from a); (c) performing 5' to 3' DNA polymerase extension of the multifunctional capture probe on the complex from b) to replicate a region of the captured tagged genomic target region that is 3 ' of the multifunctional capture probe in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the multifunctional capture probe hybrid module and the complement of a region of the tagged genomic target region that is located in the 3 'direction from the location where the multifunctional capture probe hybrid module hybridizes to the genomic target region; and (d) performing PCR amplification of the hybrid nucleic acid molecule in c); and (e) quantitating the PCR reaction in d), wherein the quantitation allows for a determination of copy number of the specific genomic target region.
  • the methods contemplated herein comprise obtaining the sequences of the hybrid nucleic acid molecules from step d).
  • steps a) through d) are repeated at least about twice and a sequence alignment of the hybrid nucleic acid molecules from the at least two d) steps.
  • At least two different multifunctional capture probe modules are used in the at least two a) steps, wherein the at least two a) steps employ one multifunctional capture probe module each.
  • At least one multifunctional capture probe module hybridizes downstream of the genomic target region and at least one multifunctional capture probe module hybridizes upstream of the genomic target region.
  • the targeted genetic analysis is sequence analysis.
  • the tagged genomic library is amplified by PCR to generate an amplified tagged genomic library.
  • the genomic DNA is from a biological sample selected from the group consisting of blood, skin, hair, hair follicles, saliva, oral mucous, vaginal mucous, sweat, tears, epithelial tissues, urine, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid), excreta, biopsy, ascites, cerebrospinal fluid, lymph, and tissue extract sample or biopsy sample.
  • a biological sample selected from the group consisting of blood, skin, hair, hair follicles, saliva, oral mucous, vaginal mucous, sweat, tears, epithelial tissues, urine, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid), excreta, biopsy, ascites, cerebrospinal fluid, lymph, and tissue extract sample or biopsy sample.
  • a tagged genomic library comprising tagged genomic sequences, wherein each tagged genomic sequence comprises:
  • fragmented end-repaired genomic DNA a random nucleotide tag sequence; a sample code sequence; and a PCR primer sequence.
  • a tagged cDNA library comprising tagged cDNA sequences, wherein each tagged cDNA sequence comprises: fragmented end-repaired cDNA; a random nucleotide tag sequence; a sample code sequence; and a PCR primer sequence.
  • a hybrid tagged genomic library comprising hybrid tagged genomic sequences for use in targeted genetic analysis, wherein each hybrid tagged genomic sequence comprises: fragmented end-repaired genomic DNA; a random nucleotide tag sequence; a sample code sequence; a PCR primer sequence; a genomic target region; and a multifunctional capture probe module tail sequence.
  • a hybrid tagged cDNA library is provided comprising hybrid tagged cDNA sequences for use in targeted genetic analysis, wherein each hybrid tagged cDNA sequence comprises: fragmented end-repaired cDNA; a random nucleotide tag sequence; a sample code sequence; a PCR primer sequence; a cDNA target region; and a multifunctional capture probe module tail sequence.
  • a multifunctional adaptor module comprising: a first region comprising a random nucleotide tag sequence; a second region comprising a sample code sequence; and a third region comprising a PCR primer sequence.
  • a multifunctional capture probe module comprising: a first region capable of hybridizing to a partner oligonucleotide; a second region capable of hybridizing to a specific genomic target region; and a third region comprising a tail sequence.
  • the first region is bound to a partner oligonucleotide.
  • a multifunctional adaptor probe hybrid module comprising: a first region capable of hybridizing to a partner oligonucleotide and capable of functioning as a PCR primer and a second region capable of hybridizing to a specific genomic target region.
  • the first region is bound to a partner oligonucleotide.
  • the partner oligonucleotide is chemically modified.
  • composition comprising a tagged genomic library, a multifunctional adaptor module and a multifunctional capture probe module is provided.
  • composition comprising a hybrid tagged genomic or c DNA library according to any of the preceding embodiments is provided.
  • reaction mixture for performing a method of any one of the preceding embodiments is provided.
  • a reaction mixture capable of generating a tagged genomic library comprising: fragmented genomic DNA; and DNA end- repair enzymes to generate fragmented end-repaired genomic DNA.
  • a reaction mixture capable of generating a tagged genomic library comprising: fragmented cDNA; and DNA end-repair enzymes to generate fragmented end-repaired cDNA.
  • a reaction mixture comprises a multifunctional adaptor module.
  • a reaction mixture comprises a multifunctional capture probe module.
  • a reaction mixture comprises an enzyme with 3 '-5' exonuclease activity and PCR amplification activity.
  • a method for DNA sequence analysis comprising: obtaining one or more clones, each clone comprising a first DNA sequence and a second DNA sequence, wherein the first DNA sequence comprises targeted genomic DNA sequence and the second DNA sequence comprises a capture probe sequence; performing a paired end sequencing reaction on the one or more clones and obtaining one or more sequencing reads; and ordering or clustering the sequencing reads of the one or more clones according to the probe sequence of the sequencing reads.
  • a method for DNA sequence analysis comprising: obtaining one or more clones, each clone comprising a first DNA sequence and a second DNA sequence, wherein the first DNA sequence comprises targeted genomic DNA sequence and the second DNA sequence comprises a capture probe sequence; performing a sequencing reaction on the one or more clones in which a single long sequencing read of greater than about 100 nucleotides is obtained, wherein the read is sufficient to identify both the first DNA sequence and the second DNA sequence; and ordering or clustering sequencing reads of the one or more clones according to the probe sequence of the sequencing reads.
  • sequences of the one or more clones are compared to one or more human reference DNA sequences.
  • sequences that do not match the one or more human reference DNA sequences are identified.
  • non-matching sequences are used to create a de novo assembly from the non-matching sequence data.
  • the de novo assemblies are used to identify novel sequence rearrangements associated with the capture probe.
  • a method for genomic copy number determination analysis comprising: obtaining one or more clones, each clone comprising a first DNA sequence and a second DNA sequence, wherein the first DNA sequence comprises a random nucleotide tag sequence and a targeted genomic DNA sequence and the second DNA sequence comprises a capture probe sequence; performing a paired end sequencing reaction on the one or more clones and obtaining one or more sequencing reads; and ordering or clustering the sequencing reads of the one or more clones according to the probe sequence of the sequencing reads.
  • a method for genomic copy number determination analysis comprising: obtaining one or more clones, each clone comprising a first DNA sequence and a second DNA sequence, wherein the first DNA sequence comprises a random nucleotide tag sequence and a targeted genomic DNA sequence and the second DNA sequence comprises a capture probe sequence; performing a sequencing reaction on the one or more clones in which a single long sequencing read of greater than about 100 nucleotides is obtained, wherein the read is sufficient to identify both the first DNA sequence and the second DNA sequence; and ordering or clustering sequencing reads of the one or more clones according to the probe sequence of the sequencing reads.
  • the random nucleotide tag sequence is about 2 to about 50 nucleotides in length.
  • the methods contemplated herein comprise analyzing all sequencing reads associated with a second read sequence by: determining the distributions of unique and redundant sequencing reads; counting the number of times a unique read is encountered; fitting a frequency distribution of the unique reads to a statistical distribution; inferring a total number of unique reads; and normalizing the total number of inferred unique read to an assumption that most human genetic loci are generally diploid.
  • an inferred copy number of one or more targeted loci are determined.
  • the one or more target loci that deviate from an expected copy number value are determined.
  • the one or more targeted loci of a gene are grouped together in a collection of loci and the copy number measurements from the collection of targeted loci are averaged and normalized.
  • the inferred copy number of a gene is represented by the normalized average of all the target loci representing that gene.
  • a method for generating a tagged R A expression library comprising: fragmenting a cDNA library; treating the fragmented cDNA library with end-repair enzymes to generate fragmented end-repaired cDNA; and ligating a multifunctional adapter molecule to the fragmented end-repaired c DNA to generate a tagged RNA expression library.
  • a method for generating a tagged RNA expression library comprising: preparing a cDNA library from the total RNA of one or more cells; fragmenting the cDNA library; treating the fragmented cDNA with end- repair enzymes to generate fragmented end-repaired cDNA; and ligating a
  • multifunctional adapter molecule to the fragmented end-repaired c DNA to generate a tagged RNA expression library.
  • the cDNA library is an oligo-dT primed cDNA library.
  • the cDNA library is primed by random
  • oligonucleotides comprising about 6 to about 20 random nucleotides.
  • the cDNA library is primed by random hexamers or random octamers.
  • the cDNA library is fragmented to a size of about 250 bp to about 750 bp.
  • the cDNA library is fragmented to a size of about 500 bp.
  • the multifunctional adaptor module comprises: a first region comprising a random nucleic acid tag sequence, and optionally; a second region comprising a sample code sequence, and optionally a third region comprising a PCR primer sequence.
  • the multifunctional adaptor module comprises a first region comprising a random nucleic acid tag sequence, a second region comprising a sample code sequence, and a third region comprising a PCR primer sequence.
  • the methods contemplated herein comprise hybridizing a tagged cDNA library with a multifunctional capture probe module to form a complex, wherein the multifunctional capture probe module hybridizes to a specific target region in the cDNA library.
  • the methods contemplated herein comprise isolating the tagged cDNA library-multifunctional capture probe module complex.
  • the methods contemplated herein comprise 3 '-5' exonuclease enzymatic processing of the isolated tagged cDNA library-multifunctional capture probe module complex to remove the single stranded 3' ends.
  • the enzyme for use in the 3 '-5' exonuclease enzymatic processing is T4 DNA polymerase.
  • the methods contemplated herein comprise performing PCR on the 3 '-5' exonuclease enzymatically processed complex, wherein a tail portion of the multifunctional capture probe molecule is copied in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the cDNA target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence.
  • a method for targeted gene expression analysis comprising: (a) hybridizing a tagged RNA expression library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module selectively hybridizes to a specific target region in the tagged RNA expression library; (b) isolating the tagged RNA expression library-multifunctional capture probe module complex from a); (c) performing 3 '-5' exonuclease enzymatic processing on the isolated tagged RNA expression library-multifunctional capture probe module complex from b) using an enzyme with 3 '-5' exonuclease activity to remove the single stranded 3' ends; (d) performing PCR on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is copied in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence; and (e) performing
  • a method for targeted gene expression analysis comprising: (a) hybridizing a tagged R A expression library with a multifunctional capture probe hybrid module complex, wherein the multifunctional capture probe hybrid module selectively hybridizes to a specific target region in the RNA expression library; (b) isolating the tagged RNA expression library- multifunctional capture probe hybrid module complex from a); (c) performing 5' to 3' DNA polymerase extension of the multifunctional capture probe on the complex from b) to replicate a region of the captured, tagged target region that is 3 ' of the
  • the hybrid nucleic acid molecule comprises the multifunctional capture probe hybrid module and the complement of the tagged target region that is located in the 3 'direction from the location where the multifunctional capture probe hybrid module hybridizes to the target region; and (d) performing targeted genetic analysis on the hybrid nucleic acid molecule from c).
  • a method for targeted gene expression analysis comprising: (a) hybridizing a tagged cDNA library with a multifunctional capture probe hybrid module complex, wherein the multifunctional capture probe hybrid module selectively hybridizes to a specific target region in the cDNA library; (b) isolating the tagged cDNA library-multifunctional capture probe hybrid module complex from a); (c) performing 5' to 3' DNA polymerase extension of the
  • At least two different multifunctional capture probe modules are used in the at least two (a) steps, wherein the at least two a) steps employ one multifunctional capture probe module each.
  • At least one multifunctional capture probe module hybridizes downstream of the target region and at least one multifunctional capture probe module hybridizes upstream of the target region.
  • a method for cDNA sequence analysis comprising: (a) obtaining one or more clones, each clone comprising a first cDNA sequence and a second cDNA sequence, wherein the first cDNA sequence comprises targeted genomic cDNA sequence and the second cDNA sequence comprises a capture probe sequence; (b) performing a paired end sequencing reaction on the one or more clones and obtaining one or more sequencing reads; and (c) ordering or clustering the sequencing reads of the one or more clones according to the probe sequence of the sequencing reads.
  • a method for cDNA sequence analysis comprising: (a) obtaining one or more clones, each clone comprising a first cDNA sequence and a second cDNA sequence, wherein the first cDNA sequence comprises targeted genomic DNA sequence and the second cDNA sequence comprises a capture probe sequence; (b) performing a sequencing reaction on the one or more clones in which a single long sequencing read of greater than about 100 nucleotides is obtained, wherein the read is sufficient to identify both the first cDNA sequence and the second cDNA sequence; and (c) ordering or clustering sequencing reads of the one or more clones according to the probe sequence of the sequencing reads.
  • the methods contemplated herein comprise analyzing all sequencing reads associated with a second read sequence by: determining the distributions of unique and redundant sequencing reads; counting the number of times a unique read is encountered; fitting a frequency distribution of the unique reads to a statistical distribution; inferring a total number of unique reads; and converting unique read counts into transcript abundance using normalization to the total reads collected within each cDNA library sample.
  • a method for targeted genetic analysis comprising: (a) hybridizing a tagged DNA library with a multifunctional capture probe hybrid module complex, wherein the multifunctional capture probe hybrid module selectively hybridizes to a specific target region in the DNA library; (b) isolating the tagged DNA library-multifunctional capture probe hybrid module complex from a); (c) performing a concerted enzymatic processing of the tagged DNA library- multifunctional capture probe hybrid module complex from b) that includes 5' FLAP endonuclease activity, 5' to 3' DNA polymerase extension, and nick closure by a DNA ligase to join the complement of the multifunctional capture probe to the target region that is 5 Of the multifunctional capture probe binding site in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the complement of the multifunctional capture probe hybrid module and a region of the tagged target region that is located 5' of the location where the multifunctional capture probe hybrid module hybridizes to the genomic target region; and (
  • steps a) through c) are repeated at least about twice and the targeted genetic analysis of d) comprises a sequence alignment of the hybrid nucleic acid molecule sequences obtained from the at least two d) steps.
  • At least two different multifunctional capture probe modules are used in the at least two a) steps, wherein the at least two a) steps employ one multifunctional capture probe module each.
  • At least one multifunctional capture probe module hybridizes downstream of the target region and at least one multifunctional capture probe module hybridizes upstream of the target region.
  • a method for determining copy number of a specific target region comprising: (a) hybridizing a tagged DNA library with a multifunctional capture probe hybrid module complex, wherein the multifunctional capture probe hybrid module selectively hybridizes to a specific target region in the genomic library; (b) isolating the tagged DNA library-multifunctional capture probe hybrid module complex from a); (c) performing a concerted enzymatic processing of the tagged DNA library-multifunctional capture probe hybrid module complex from b) that includes 5' FLAP endonuclease activity, 5' to 3' DNA polymerase extension, and nick closure by a DNA ligase to join the complement of the multifunctional capture probe to the target region that is 5 Of the multifunctional capture probe binding site in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the complement of the multifunctional capture probe hybrid module and a region of the tagged target region that is located 5' of the location where the multifunctional capture probe hybrid module hybridizes to
  • the methods contemplated herein comprise obtaining the sequences of the hybrid nucleic acid molecules from step d).
  • steps a) through d) are repeated at least about twice and a sequence alignment of the hybrid nucleic acid molecules from the at least two d) steps.
  • At least two different multifunctional capture probe modules are used in the at least two a) steps, wherein the at least two a) steps employ one multifunctional capture probe module each.
  • At least one multifunctional capture probe module hybridizes downstream of the genomic target region and at least one multifunctional capture probe module hybridizes upstream of the genomic target region.
  • the targeted genetic analysis is sequence analysis.
  • the target region is a genomic target region and the DNA library is a genomic DNA library.
  • the target region is a cDNA target region and the DNA library is a cDNA library.
  • Figure 1 Construction of an am lifiable, sample coded, tagged genomic DNA library.
  • Purified genomic DNA was isolated from a source such as whole blood or a buccal cheek swab.
  • the DNA was fragmented (e.g. by mechanical, enzymatic or chemical means) and the ends of the DNA were repaired, in this example, to blunt ends.
  • the repaired DNA was ligated to a multifunctional adaptor module that contained a universal amplification sequence, a random nucleotide tag sequence and a sample code sequence.
  • a specific example of a typical adaptor duplex molecule is shown by way of example.
  • FIG. 2 Genomic capture probe design.
  • A Structure of a prototypical 114 nt probe. Region 1 is a 34 nt sequence that shares perfect complementarity to a highly modified, 34 nt partner oligonucleotide. The partner oligonucleotide was modified on its 5' end with a biotin - TEG chemical entity that enabled capture on streptavidin- coated magnetic beads. B stands for the "Bio-TEG" modification.
  • Region 2 is the 60 nt probe region designed to interact with genomic DNA targets.
  • Region 3 is a 20 nt tail that introduced PCR amplification sequences into the captured genomic fragments.
  • B An example of the highly modified partner strand that is complementary to region 1 of each individual probe.
  • FIG. 3 Sequence "spreading” in conventional, hybridization-based capture experiments.
  • A Flanking fragments can "hitch-hike” into capture libraries by way of legitimate hybridization triplexes involving probe-fragment- flanking fragment interactions.
  • B The net result of cross-fragment hybridization is sequence
  • “spreading”, meaning sequences that are within proximity to the target region (dashed lines) but fall outside the boundaries of the desired target.
  • Figure 4 Enzymatic processing of fragment: probe hybridized complexes.
  • A The purified complex of fragment (light gray) and probe (black) prior to processing.
  • B biotin affinity modification.
  • DNA polymerase e.g. T4 DNA polymerase
  • C Upon encountering the probe: fragment duplex region, the polymerase copies the probe tail segment onto the hybridized genomic fragment.
  • D The final modified fragment in which the tail segment of the multifunctional capture probe hybrid module has been copied onto the target genomic fragment.
  • Figure 5 Enzymatic processing of capture complexes "focuses" sequencing reads on the target region.
  • A Enzymatically processed fragment shown in relation to the capture probe. The orientation of the sequencing read is specified by the probe and is denoted by the thin black arrow.
  • B Hypothetical trace of focused reads where "spreading" is minimized.
  • Figure 6 The "focusing effect" of directional probes.
  • A Typical exons average 100 - 150 bp.
  • Directional capture probes are positioned in intronic region flanking the target segment.
  • B The sequence read distribution for each individual probe is shown by the dashed line.
  • the additive coverage is shown by the solid line. The directional nature of coverage can be used, as shown in this example, to obtain sharp focus on query regions.
  • Figure 8 Even distribution of average fragment size following Covaris fragmentation of gDNA.
  • Male (M) and female (F) human gDNA (received from Promega Corporation, Madison, WI, USA) was sheared via Covaris conditions, and 2 ⁇ 1 ( ⁇ 120ng) or 5 ⁇ 1 ( ⁇ 300ng) of the pre-fragmented (U) or the post-fragmented (C) samples were loaded on a 2% agarose gel.
  • the average fragment size was an even distribution centered around 200bp.
  • Figure 9 Amplification traces for proteolipid protein 1 (PLPl) qPCR assay 6 demonstrating expected copy number differences between male and female samples. Triplicate male or female genomic DNA templates were amplified by real-time PCR on the Illumina Eco instrument using PLPl qPCR assay 6. Amplification traces clearly demonstrated copy number differences between the female and male samples.
  • PLPl proteolipid protein 1
  • Figure 10 Demonstration of expected amplicon size and uniqueness following conventional PCR with PLPl qPCR assay primers.
  • Male or female genomic DNA templates were amplified by conventional PCR using qPCR assay primer sets 1-8 (Example 3) and the unpurified PCR reactions were loaded directly on 2% agarose gels. The upper bands of each doublet were consistent with the projected mobility of the assay PCR products. The lower "fuzzy" material was most likely unused PCR primers.
  • Figure 11 Analysis of PLPl qPCR assay performance using ABI 2x SYBR mix and conditions. DNA fragments from genomic library I (constructed in Example 4) were used as template to measure the performance of the PLPl qPCR assays when set up at room temperature using ABI 2x SYBR master mix in a 2 step PCR reaction. No template control traces (A) and +gDNA traces (B) are shown to provide a qualitative picture of assay performance.
  • FIG 12 Reduced insert size following post-capture processing with T4- DNA polymerase.
  • Four samples of adapter ligated gDNA fragments from genomic library I (constructed in Example 4) were captured as described (Example 6 - PLPl EXON 2). Two of these samples utilized universal binding oligo CI, while the other two samples were bound with oligo CIO. Samples were then either treated with T4- DNA polymerase (T4 processed) or processed similarly in a reaction solution lacking the T4 polymerase (untreated). Post-capture processing with T4-polymerase induced an overall reduction in the size distribution of the sample, suggesting a reduction in the average size of insert. Additionally, T4 processing resulted in the appearance of two faint bands (-250 bp and ⁇ 175bp).
  • Figure 13 Direct measurement of post-capture processing sensitivity.
  • PLPl exon 2 specific genomic DNA fragments were isolated by pull down/pull out from the female gDNA library (Example 1) using single PLPl capture probes in independent reactions. Captured material was quantified using an adjacent PLPl qPCR assay primer pair, as illustrated in (A). Following enzymatic processing the amount of processed complex was measured again by qPCR using one PLPl specific primer and one probe-specific primer, as show in (B). The ratio of the measurements in [B/A x 100%] will yield an estimate of processing efficiency.
  • the PCR products from realtime reactions were extracted and subjected to gel analysis to verify that amplicons of the expected length were produced (C). This was possible since both PCR reactions had discrete start and stop points. Processing efficiency was inferred from pull-outs that yielded interpretable data from A + B + C.
  • Figure 14 Gel analysis of qPCR products from real-time quantification of pre- and post-processed PLPl exon 2 captured DNA fragments.
  • Six independent capture reactions (two with probe #1, two with probe #4, one with probe #2, and one with probe #3) were processed as described in Figure 16.
  • the probes were from the B10 universal oligo set (Example 4) and comprised universal oligo and probe ultramer. Under these conditions, assay sets 3 (probe 4), 5 (probe 2) and 6 (probe 3) yielded PCR products consistent with the assay amplicon (top gel) or the post-processed PLPl to adaptor amplicons (bottom gel), while no detectable products were observed in the other assay sets.
  • Figure 15 Alternative enzymatic processing of fragment: probe hybridized complexes.
  • this alternative approach shifted from having the clone copy the probe to having the probe copy the clone.
  • This reversal in polarity means that the 5' end of the probe was used as both the pull-down sequence and the reverse PCR sequence. The 3' end of the probe was left unmodified thus it was able to copy the clone using DNA polymerase; 5 '-3' DNA polymerase extended the multifunctional capture probe utilizing the isolated tagged DNA library fragments as template.
  • Figure 16 Experimental design employed to test the alternative enzymatic processing concept.
  • Four well behaved qPCR assays, (10, 14, 15 and 16) were matched with probes that "pointed" at those assays.
  • the target sequences of the probe and qPCR assays were directed to regions within proximity to one another, they did not overlap. Thus the effect of the processing could be directly tested using these well-established assay sets.
  • Figure 17 Decrease in the average insert size of the library induced by the alternative processing method. DNA fragments were hybridized to capture
  • Figure 18 Enhanced focusing of target sequences by the alternative processing method.
  • the sequence reads obtained in Example 13 were displayed in the UCSC Genome Browser to assess the coverage and distribution of captured fragments within specific target sites.
  • the density of sequence reads corresponding to 'capture only' and 'processed' libraries are shown (black) for two target regions on the X chromosome, one corresponding to exons of the PLP1 gene (A and B) and the other an intronic segment of the ZNF630 gene (C and D).
  • Reads from libraries generated by the alternative processing method (B and D) are more highly concentrated in target sites than libraries constructed by capture alone (A and C). Capture probe binding sites are shown in red. Each track is scaled to the maximal read density values (y-axis) observed for a given stretch of genomic coordinates (x-axis).
  • Figure 19 Diagram of vertical alignment. All next generation sequence (NGS) analysis starts with alignment to a reference genome.
  • NGS next generation sequence
  • A Initial read alignment is based on configurable word string searches that can accommodate single nucleotide variants (SNVs) and, to a limited extent, insertions/deletions.
  • B The collective set of aligned reads is analyzed for SNVs. In the example shown, the candidate SNV was observed twice, but the read coordinates for these two reads was identical. The vertical alignment paradigm generates a large number of SNVs and/or insertions/deletion hypotheses that require orthogonal validation.
  • Figure 20 Diagram of data analysis scheme. Step one is to match reads to probes. Step two will be to analyze the sequence information connected "horizontally" to each probe.
  • Figure 21 Diagram of horizontal alignment "forces" reads associated with probe 1 and probe 2. Assembly will generate two contigs, one with a wild-type exon structure and one with an insertion structure. Two important principles emerge: 1) Overlapping reads from adjacent probes will support or refute the hypothesis of an indel-containing allele of the captured exon and 2) micro-CNV alleles outside of capture probes are readily detectable by horizontal methodologies.
  • Figure 22 Diagram of "low confidence" SNV calls.
  • Candidate nucleotide variants can be actual events harbored in the individual from whom the sample was collected and processed, but they can also be artifacts introduced during processing and sequencing of samples. The methods described here were designed to differentiate between actual, "high confidence” variant calls and artifactual "low confidence” variant calls.
  • Sequencing reads covering a target region are collected from multiple different clones in both possible sequencing orientations, and each read is annotated with tag information.
  • the tags allow reads derived from the same cloning event to be identified and grouped. SNVs and indels that arise within only one set of clones that are all derived from the same cloning event are low confidence calls that are discarded from further analysis.
  • FIG. 23 Diagram of "high confidence" SNV calls.
  • Candidate nucleotide variants can be actual events harbored in the individual from whom the sample was collected and processed, but they can also be artifacts introduced during processing and sequencing of samples.
  • the methods described here were designed to differentiate between actual, "high confidence” variant calls and artifactual "low confidence” variant calls.
  • Sequencing reads covering a target region are collected from multiple different clones in both possible sequencing orientations, and each read is annotated with tag information.
  • the tags allow reads derived from distinct cloning events to be identified. Examples shown are (A) reads with the same start point but different sequence labels,
  • (C) reads in opposite orientations.
  • the occurrence and detect of a variant in independent cloning events marks that variant with high confidence, and such variants are followed up with further, orthogonal validation methods.
  • Figure 24 Molecularly annotated sequencing read.
  • A Forward flow cell (Illumina chemistry) graft sequences and sequencing primer binding site.
  • B Reverse flow cell graft sequence and reverse sequencing primer annealing site.
  • Sample label. Sample label.
  • (4) Sequence of genomic fragment. Genomic index (probe sequence). The combination of (1) + (3) constitutes the unique read tag that is critical for both variant calling and copy number determination.
  • Figure 25 The most significant classes of DNA sequence variants (insertions, deletions, runs of point mutations and/or translocations) are also the most difficult to detect by alignment based methods.
  • Figure 26 Dual probe interrogation of target regions (e. g. exons).
  • A Typical exons average 100 - 150 bp. Capture probes are positioned in the intronic region flanking the target segment. These probes have opposite sequence polarity (one queries the "+" stand, the other the "-" strand.
  • B The sequence read distribution for each individual probe is indicated by the shaded areas and the read orientations are specified by arrows. Key aspects are that target regions are sequenced by multiple reads in both orientations. Moreover, each probe captures reads that sequence the adjacent probe binding sites. This arrangement is one element that increases the confidence of variant calls.
  • Sequence tags are comprised of a nucleotide code (ovals; in the case of Clearfork a collection of 16 possible three nucleotide sequences) and a ragged, arbitrary clone fragment end sequence.
  • a false-positive variant call is one in which a variant is identified among a collection of sibling sequences that all bear identical sequence tags.
  • B A high confidence variant call is found among a collection of sequences that have different sequence tags.
  • Figure 28 Copy determination using read observation statistics.
  • Figure 29 Molecularly annotated sequencing read.
  • A Forward flow cell (Illumina SBS chemistry) graft sequences and sequencing primer binding site.
  • B Reverse flow cell graft sequence and reverse sequencing primer annealing site.
  • the combination of (1) + (3) constitutes the unique read tag that is critical for copy number determination.
  • the combination of (5) + (6) constitutes a genomic index tag that can be used to monitor and quantify capture events.
  • the forward sequencing read 1 that determines the sequences of annotation elements 1, 2, 3, and 4, and paired-end reverse read 2 that determines the sequences of annotation elements 5 and 6 are indicated.
  • Probes e.g., multifunctional capture probes
  • Tail sequences that add additional functionality e.g., PCR primer binding site, binding site for partner oligo that enables biotin pull-out, etc
  • Sixty nucleotide targeting sequences are chosen with the following constraints and criterion: (1) The probe is positioned from -100 to +50 nt relative to the start of the target sequence.
  • the "start" of the target sequence is the intron:exon junction; (2) Probes are designed with redundancy, as illustrated, such that the resulting sequences from a pair of probes are overlapping in opposite orientations; (3) Probes are selected (where possible) to possess GC content not less than 33% (>20 G's or C's per 60 mer) and not more than 67% ( ⁇ 40 G's or C's per 60 mer); (4) Probes are selected to avoid repeats wherever possible.
  • Figure 31 Processing to create targeted genomic sequencing libraries.
  • the initial capture complex comprises a "standard” tagged genomic library fragment, a tailed capture probe that targets a genomic "target region” that is 5' to the probe, and a biotinylated partner Oligonucleotide that is common to all probes.
  • Processing of the complex into a sequence-ready clone comprises 3 steps: (1) the 5' FLAP
  • DNA polymerase holoenzyme e.g., full length Bst polymerase
  • the polymerase extends the partner oligo sequence by polymerization (can occur concurrently with step 1); and (3) Taq ligase repairs the nick between the partner oligo and the genomic fragment.
  • Figure 32 shows post-capture/processing of PCR products.
  • Lane 1 is ACA2 single primer amplified, unprocessed capture complex.
  • Lanes 2-4 were amplified with AF+CR dual PCR primers.
  • Figures 33-35 show a library free method for generating tagged genomic DNAs and related capture, processing, and analysis methods.
  • Figure 36 shows a suppressive PCR strategy for avoiding primer-dimer artifacts in library free methods for generating tagged genomic DNAs.
  • Figure 37 shows gel electrophoresis results of raw and sonicated gDNAs used in library free methods for generating tagged genomic DNAs.
  • Figure 38 shows a qPCR amplification plot of four gDNA samples prepared by the library free method.
  • Figure 39 shows the gel electrophoresis results from raw PCR products amplified from samples prepared by the library free methods.
  • Figure 40 shows the gel electrophoresis results from bead-cleaned PCR products amplified from samples prepared by the library free methods.
  • Figure 41 shows a qPCR amplification plot of samples prepared by the library free methods using different combinations of enzymes: T4 DNA polymerase (P), T4 DNA ligase (L), and T4 gene 32 protein (32), or no enzyme control.
  • P T4 DNA polymerase
  • L T4 DNA ligase
  • 32 T4 gene 32 protein
  • Figure 42 shows the gel electrophoresis results of PCR amplified (10 cycles or 16 cycles) samples prepared by the library free methods using different combinations of enzymes: T4 DNA polymerase (P), T4 DNA ligase (L), and T4 gene 32 protein (32), or no enzyme control.
  • P T4 DNA polymerase
  • L T4 DNA ligase
  • 32 T4 gene 32 protein
  • Figure 43 shows the gel electrophoresis results of individual samples prepared by the library free methods prior to pooling.
  • Figure 44 shows CNV for PLP1 in relation to the normalizing autosomal loci KRAS and MYC across samples with variable dosages of the X chromosome. Samples were prepared using library free methods.
  • Figure 45 shows the DNA sequence start points for chrX region 15 in the XXXX (4X dosage) sample relative to the capture probe sequence. Reads go from left to right and samples were prepared using library free methods.
  • Figure 46 shows the gel electrophoresis results of cDNA prepared from RNA samples using the RNA -seq methods contemplated herein, captured cDNAs, and cDNA preps sized with the Pippin automated DNA size selector.
  • Figure 47 shows the correlations of the gene expression of various transcripts in heart versus liver in libraries prepared using a total RNA versus targeted expression strategy for the library preparation.
  • Figure 48 shows the correlation of absolute expression levels of various transcript measured in total RNA-seq compared to targeted RNA-seq.
  • the present invention is based at least in part, on the discovery that the coordinated utilization of several key molecular modules can be employed in
  • the term "about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • the term "about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ⁇ 15%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%>, or ⁇ 1%) about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • isolated means material that is substantially or essentially free from components that normally accompany it in its native state.
  • obtained or “derived” is used synonymously with isolated.
  • DNA refers to deoxyribonucleic acid.
  • DNA refers to genomic DNA, recombinant DNA, synthetic DNA, or cDNA.
  • DNA refers to genomic DNA or cDNA.
  • the DNA comprises a "target region.”
  • DNA libraries contemplated herein include genomic DNA libraries and cDNA libraries constructed from RNA, e.g., an RNA expression library.
  • the DNA libraries comprise one or more additional DNA sequences and/or tags.
  • target region refers to a region of interest within a DNA sequence.
  • targeted genetic analyses are performed on the target region.
  • the target region is sequenced or the copy number of the target region is determined.
  • the present invention contemplates, in part, a method for generating a tagged genomic library.
  • the method comprises treating fragmented DNA, e.g., genomic DNA or cDNA, with end-repair enzymes to generate fragmented end-repaired DNA followed by ligating a random nucleic acid tag sequence to generate the tagged genomic library.
  • a sample code sequence and/or a PCR primer sequence are optionally ligated to the fragmented end-repaired DNA.
  • the present invention contemplates, in part, a method for generating a tagged DNA library.
  • the method comprises treating fragmented DNA with end-repair enzymes to generate fragmented end-repaired DNA followed by ligating a random nucleic acid tag sequence to generate the tagged DNA library.
  • a sample code sequence and/or a PCR primer sequence are optionally ligated to the fragmented end-repaired DNA.
  • Illustrative methods for fragmenting DNA include, but are not limited to:
  • any method known in the art for fragmenting DNA can be employed with the present invention.
  • the fragmented DNA is processed by end-repair enzymes to generate end-repaired DNA.
  • the end-repair enzymes can yield for example blunt ends, 5 '-overhangs, and 3 '-overhangs.
  • the end-repaired DNA contains blunt ends.
  • the end-repaired DNA is processed to contain blunt ends.
  • the blunt ends of the end-repaired DNA are further modified to contain a single base pair overhang.
  • end-repaired DNA containing blunt ends can be further processed to contain adenine (A)/thymine (T) overhang.
  • end-repaired DNA containing blunt ends can be further processed to contain adenine (A)/thymine (T) overhang as the single base pair overhang.
  • the end-repaired DNA has non-templated 3' overhangs.
  • the end-repaired DNA is processed to contain 3 '-overhangs.
  • the end-repaired DNA is processed with terminal transferase (TdT) to contain 3 '-overhangs.
  • TdT terminal transferase
  • a G-tail can be added by TdT.
  • the end-repaired DNA is processed to contain overhang ends using partial digestion with any known restriction enzymes (e.g., with the enzyme Sau3A, and the like.
  • DNA fragments are tagged using one or more "random nucleotide tags” or “random nucleic acid tags.”
  • random nucleotide tag or “random nucleic acid tag” refer to a polynucleotide of discrete length wherein the nucleotide sequence has been randomly generated or selected.
  • the length of the random nucleic acid tag is from about 2 to about 100 nucleotides, from about 2 to about 75 nucleotides, about 2 to about 50 nucleotides, about 2 to about 25 nucleotides, about 2 to about 20 nucleotides, about 2 to about 15 nucleotides, about 2 to about 10 nucleotides, about 2 to about 8 nucleotides, or about 2 to about 6 nucleotides.
  • the length of the random nucleotide tag is from about 2 to about 6 nucleotides (see, e.g., Figure 1).
  • the random nucleotide tag sequence is about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides.
  • tags can be employed.
  • Tagmentation is a Nextera
  • transposon protein complexes with the random nucleotide tags and/or the multifunctional adaptor modules of the invention.
  • the loaded transposon complexes can then be used in creation of the tagged genomic library according to described methods.
  • DNA for use in the present methods can come from any source known to those of skill in the art.
  • DNA can be collected from any source, synthesized from RNA as copy DNA (cDNA), and processed into pure or substantially pure DNA for use in the present methods.
  • the size of the fragmented DNA is in the range of about 2 to about 500 base pairs, about 2 to about 400 base pairs, about 2 to about 300 base pairs, about 2 to about 250 base pairs, about 2 to about 200 base pairs, about 2 to about 100 base pairs or about 2 to about 50 base pairs.
  • the combination of the introduced “random nucleic acid tag” or “random nucleic acid tags” with the DNA fragment end sequence constitutes a combination of two elements that will hereafter be referred to as “the genomic tag” or the “the cDNA tag.”
  • the uniqueness of "the genomic tag” or the “the cDNA tag” can be determined by the combinatorial product of the diversity within the attached random nucleotide tag pool multiplied by the diversity of the DNA fragment end sequence pool.
  • the present invention also contemplates, in part, a multifunctional adaptor module.
  • the term "multifunctional adaptor module” refers to a polynucleotide comprising: (i) a first region comprising a random nucleotide tag sequence; optionally (ii) a second region comprising a sample code sequence; and optionally (iii) a third region comprising a PCR primer sequence.
  • a multifunctional adaptor module comprises a PCR primer sequence, a random nucleotide tag, and a sample code sequence.
  • a multifunctional adaptor module comprises a PCR primer sequence and a random nucleotide tag or a sample code sequence.
  • the second region comprising the sample code is optional.
  • the multifunctional adaptor module does not include a second region, but instead only a first and third region.
  • the multifunctional adaptor module of the present invention can include blunt or complementary ends appropriate for the ligation method employed, including those disclosed elsewhere herein, as well as any others known to those of skill in the art for ligating the multifunctional adaptor module to the fragmented DNA.
  • a first region comprises a random nucleotide tag sequence.
  • the first region comprises a random nucleotide tag sequence that is from about 2 to about 100 nucleotides, from about 2 to about 75 nucleotides, about 2 to about 50 nucleotides, about 2 to about 25 nucleotides, about 2 to about 20 nucleotides, about 2 to about 15 nucleotides, about 2 to about 10 nucleotides, about 2 to about 8 nucleotides, or about 2 to about 6 nucleotides, or any intervening number of nucleotides.
  • a second region when optionally present, comprises a sample code sequence.
  • sample code sequence refers to a polynucleotide that is used to identify the sample.
  • the second region comprises a sample code sequence that is from about 1 to about 100 nucleotides, from about 2 to about 75 nucleotides, about 2 to about 50 nucleotides, about 2 to about 25 nucleotides, about 2 to about 20 nucleotides, about 2 to about 15 nucleotides, about 2 to about 10 nucleotides, about 2 to about 8 nucleotides, or about 2 to about 6 nucleotides, or any intervening number of nucleotides.
  • a third region when optionally present, comprises a PCR primer sequence.
  • the third region comprises a PCR primer sequence that is from about 5 to about 200 nucleotides, from about 5 to about 150 nucleotides, from about 10 to about 100 nucleotides from about 10 to about 75 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 20 to about 40 nucleotides, or about 20 to about 30 nucleotides, or any intervening number of nucleotides.
  • a ligation step comprises ligating a multifunctional adaptor module to the fragmented end-repaired DNA.
  • This ligation reaction can be used to generate the tagged DNA library, which comprises end-repaired DNA ligated to a multifunctional adaptor molecule and/or random nucleotide tag.
  • a single multifunctional adaptor module is employed. In some embodiments, more than one multifunctional adaptor module is employed. In some embodiments, a single multifunctional adaptor module of identical sequence is ligated to each end of the fragmented end-repaired DNA.
  • the present invention also provides a multifunctional capture probe module.
  • multifunctional capture probe module refers to a
  • a multifunctional capture probe module comprises a region capable of hybridizing to a partner oligonucleotide, a region capable of hybridizing to a DNA target sequence, and a tail sequence.
  • a multifunctional capture probe module comprises a region capable of hybridizing to a partner oligonucleotide and a region capable of hybridizing to a genomic target sequence.
  • the multifunctional capture probe module optionally comprises a random nucleotide tag sequence.
  • a first region comprises a region capable of hybridizing to a partner oligonucleotide.
  • partner oligonucleotide refers to an oligonucleotide that is complementary to a nucleotide sequence of the
  • the first region capable of hybridizing to a partner oligonucleotide is a sequence that is from about 20 to about 200 nucleotides, from about 20 to about 150 nucleotides, about 30 to about 100 nucleotides, about 30 to about 75 nucleotides, about 20 to about 50 nucleotides, about 30 to about 45 nucleotides, or about 35 to about 45 nucleotides.
  • the first region capable of hybridizing to a partner oligonucleotide is a sequence that is from about 20 to about 200 nucleotides, from about 20 to about 150 nucleotides, about 30 to about 100 nucleotides, about 30 to about 75 nucleotides, about 20 to about 50 nucleotides, about 30 to about 45 nucleotides, or about 35 to about 45 nucleotides.
  • the region is about 30 to about 50 nucleotides, about 30 to about 40 nucleotides, about 30 to about 35 nucleotides or about 34 nucleotides, or any intervening number of nucleotides.
  • a second region when optionally present, comprises a region capable of hybridizing to a specific DNA target region.
  • DNA target region refers to a region of the genome or cDNA selected for analyses using the compositions and methods contemplated herein.
  • the second region comprises a region capable of hybridizing to a specific target region is a sequence from about 20 to about 200 nucleotides, from about 30 to about 150 nucleotides, about 50 to about 150 nucleotides, about 30 to about 100 nucleotides, about 50 to about 100 nucleotides, about 50 to about 90 nucleotides, about 50 to about 80 nucleotides, about 50 to about 70 nucleotides or about 50 to about 60 nucleotides.
  • the second region is about 60 nucleotides, or any intervening number of nucleotides.
  • a third region when optionally present, comprises a tail sequence.
  • the term "tail sequence" refers to a polynucleotide at the 5' end of the multifunctional capture probe module, which in particular embodiments can serve as a PCR primer binding site.
  • the third region comprises a tail sequence that is from 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.
  • the third region is from about 10 to about 50 nucleotides, about 15 to about 40 nucleotides, about 20 to about 30 nucleotides or about 20 nucleotides, or any intervening number of nucleotides.
  • a multifunctional capture probe module comprises a region capable of hybridizing to a partner oligonucleotide and a region capable of hybridizing to a genomic target sequence.
  • the partner oligo may also function as a tail sequence or primer binding site.
  • a multifunctional capture probe module comprises a tail region and a region capable of hybridizing to a genomic target sequence.
  • the multifunctional capture probe comprises a specific member of a binding pair to enable isolation and/or purification of one or more captured fragments of a tagged DNA library that hybridizes to the multifunctional capture probe.
  • the multifunctional capture probe is conjugate to biotin or another suitable hapten, e.g., dinitrophenol, digoxigenin.
  • the present invention further contemplates, in part, hybridizing a tagged DNA library with a multifunctional capture probe module to form a complex.
  • the multifunctional capture probe module substantially hybridizes to a specific genomic target region in the DNA library.
  • Hybridization or hybridizing conditions can include any reaction conditions where two nucleotide sequences form a stable complex; for example, the tagged DNA library and multifunctional capture probe module forming a stable tagged DNA library— multifunctional capture probe module complex.
  • reaction conditions are well known in the art and those of skill in the art will appreciated that such conditions can be modified as appropriate and within the scope of the present invention.
  • Substantial hybridization can occur when the second region of the multifunctional 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 first region of the multifunctional capture probe module does not substantially hybridize to the region of the tagged DNA library to which the second region does substantially hybridize.
  • the third region of the multifunctional capture probe module does not substantially hybridize to the region of the tagged DNA library to which the second region of the multifunctional capture probe module does substantially hybridize.
  • the first and third regions of the multifunctional capture probe module do not substantially hybridize to the region of the tagged DNA library to which the second region of the
  • multifunctional capture probe module does substantially hybridize.
  • the methods contemplated herein comprise isolating a tagged DNA library— multifunctional capture probe module complex.
  • methods for isolating DNA complexes are well known to those skilled in the art and any methods deemed appropriate by one of skill in the art can be employed with the methods of the present invention (Ausubel et al. , Current Protocols in
  • the complexes are isolated using biotin— streptavidin isolation techniques.
  • the partner oligonucleotide capable of hybridizing to the first region of the multifunctional capture probe module is modified to contain a biotin at the 5'- end or 3 '-end which is capable of interacting with streptavidin linked to a column, bead or other substrate for use in DNA complex isolation methods.
  • a first region of a multifunctional capture probe module is bound to a partner oligonucleotide.
  • the partner oligonucleotide In some embodiments, the
  • multifunctional capture probe module is bound to the partner oligonucleotide prior to formation of a tagged DNA library— multifunctional capture probe module complex. In some embodiments, the multifunctional capture probe module is bound to the partner oligonucleotide after the formation of a tagged DNA library— multifunctional capture probe module complex. In some embodiments, the multifunctional capture probe module is bound to the partner oligonucleotide simultaneously with the formation of a tagged DNA library— multifunctional capture probe module complex. In some embodiments, the partner oligonucleotide is chemically modified.
  • removal of the single stranded 3 '-ends from the isolated tagged DNA library-multifunctional capture probe module complex is contemplated.
  • the methods comprise 3 '-5' exonuclease enzymatic processing of the isolated tagged DNA library-multifunctional capture probe module complex to remove the single stranded 3' ends.
  • the methods comprise performing 5 '-3' DNA polymerase extension of multifunctional capture probe utilizing the isolated tagged DNA library fragments as template.
  • the methods comprise creating a hybrid multifunctional capture probe-isolated tagged DNA target molecule through the concerted action of a 5 ' FLAP endonuclease, DNA polymerization and nick closure by a DNA ligase.
  • a variety of enzymes can be employed for the 3 '-5' exonuclease enzymatic processing of the isolated tagged DNA library-multifunctional capture probe module complex.
  • suitable enzymes which exhibit 3 '-5' exonuclease enzymatic activity, that can be employed in particular embodiments include, but are not limited to: T4 or Exonucleases I, III, V (see also, Shevelev IV, Hubscher U., "The 3' 5' exonucleases,” Nat Rev Mol Cell Biol. 3(5):364-76 (2002)).
  • the enzyme comprising 3 '-5' exonuclease activity is T4 polymerase.
  • an enzyme which exhibits 3 '-5' exonuclease enzymatic activity and is capable of primer template extension can be employed, including for example T4 or Exonucleases I, III, V. Id. 3 '5'
  • the methods contemplated herein comprise performing PCR on the 3 '-5' exonuclease enzymatically processed complex discussed supra and elsewhere herein.
  • a tail portion of a multifunctional capture probe molecule is copied in order to generate a hybrid nucleic acid molecule.
  • the hybrid nucleic acid molecule generated comprises the target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence.
  • a method for targeted genetic analysis comprises: a) hybridizing a tagged DNA library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module selectively hybridizes to a specific target region in the genomic library; b) isolating the tagged DNA library-multifunctional capture probe module complex from a); c) performing 3'- 5' exonuclease enzymatic processing on the isolated tagged DNA library- multifunctional capture probe module complex from b) using an enzyme with 3 '-5' exonuclease activity to remove the single stranded 3' ends; d) performing PCR on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is copied in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence; and
  • a method for determining copy number of a specific target region comprises: a) hybridizing a tagged DNA library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module comprises selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library-multifunctional capture probe module complex from a); c) performing 3 '-5' exonuclease enzymatic processing on the isolated tagged DNA library-multifunctional capture probe module complex from b) using an enzyme with 3 '-5' exonuclease activity to remove the single stranded 3' ends; d) performing a PCR reaction on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is replicated in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the target region capable of hybridizing to the multifunctional capture
  • a method for targeted genetic analysis comprises: a) hybridizing a tagged DNA library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module selectively hybridizes to a specific target region in the genomic library; b) isolating the tagged DNA library-multifunctional capture probe module complex from a); c) performing 5'- 3' DNA polymerase extension of multifunctional capture probe utilizing the isolated tagged DNA library fragments as template; d) performing PCR on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is copied in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence; and e) performing targeted genetic analysis on the hybrid nucleic acid molecule from d).
  • a method for determining copy number of a specific target region comprises: a) hybridizing a tagged DNA library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module comprises selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library-multifunctional capture probe module complex from a); c) performing 5 '-3' DNA polymerase extension of multifunctional capture probe utilizing the isolated tagged DNA library fragments as template; d) performing a PCR reaction on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is replicated in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence; e) performing PCR amplification of the hybrid nucleic acid in d); and
  • a method for targeted genetic analysis comprises: a) hybridizing a tagged DNA library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module selectively hybridizes to a specific target region in the genomic library; b) isolating the tagged DNA library-multifunctional capture probe module complex from a); c) creating a hybrid multifunctional capture probe-isolated tagged DNA target molecule through the concerted action of a 5 ' FLAP endonuclease, DNA polymerization and nick closure by a DNA ligase; d) performing PCR on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is copied in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the target region capable of hybridizing to the multifunctional capture probe module and the complement of the multifunctional capture probe module tail sequence; and e) performing targeted genetic analysis on the
  • a method for determining copy number of a specific target region comprises: a) hybridizing a tagged DNA library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module comprises selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library-multifunctional capture probe module complex from a); c) creating a hybrid multifunctional capture probe-isolated tagged DNA target molecule through the concerted action of a 5 ' FLAP endonuclease, DNA polymerization and nick closure by a DNA ligase; d) performing a PCR reaction on the enzymatically processed complex from c) wherein the tail portion of the multifunctional capture probe molecule is replicated in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the target region capable of hybridizing to the multifunctional capture probe module and the complement of the multi
  • PCR can be performed using any standard PCR reaction conditions well known to those of skill in the art.
  • the PCR reaction in e) employs two PCR primers.
  • the PCR reaction in e) employs a first PCR primer that hybridizes to the target region.
  • the PCR reaction in e) employs a second PCR primer hybridizes to the hybrid molecule at the target region/tail junction.
  • the PCR reaction in e) employs a first PCR primer that hybridizes to the target region and a second PCR primer hybridizes to the hybrid molecule at the target genomic region/tail junction.
  • the second primer hybridizes to the target region/tail junction such that at least one or more nucleotides of the primer hybridize to the target region and at least one or more nucleotides of the primer hybridize to the tail sequence.
  • the hybrid nucleic acid molecules obtained from step e) are sequenced and the sequences aligned horizontally, i.e., aligned to one another but not aligned to a reference sequence.
  • steps a) through e) are repeated one or more times with one or more multifunctional capture probe module complexes.
  • the multifunctional capture probe complexes can be the same or different and designed to target either DNA strand of the target sequence.
  • the multifunctional capture probe complexes when they hybridize near the same target region within the tagged DNA library. In one embodiment, one or more multifunctional capture probe hybridize within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more bp of the target region in a tagged DNA library, included all intervening distances from the target region
  • the method can be performed using two multifunctional capture probe modules per target region, wherein one hybridizes to the "Watson" strand (non-coding or template strand) upstream of the target region and one hybridizes to the "Crick" strand (coding or non-template strand) downstream of the target region.
  • the methods contemplated herein can further be performed multiple times with any number of multifunctional probe modules, for example 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more multifunctional capture probe modules per target region any number of which hybridize to the Watson or Crick strand in any combination.
  • the sequences obtained can be aligned to one another in order to identify any of a number of differences.
  • a plurality of target regions are interrogated, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 10000, 50000, 100000, 500000 or more in a single reaction, using one or more multifunctional probe modules.
  • Copy number can provide useful information regarding unique reads and duplicative reads, as well as assisting with searching for variants of known reads.
  • the terms "read,” “read sequence,” or “sequencing read” are used synonymously and refer to a polynucleotide sequence obtained by sequencing a polynucleotide.
  • DNA tags e.g., a random nucleotide tag, can be used to determine copy number of a nucleic acid sequence being analyzed.
  • a multifunctional capture probe hybrid module comprises: (i) a first region capable of hybridizing to a partner oligonucleotide and capable of functioning as a PCR primer and (ii) a second region capable of hybridizing to a specific genomic target region.
  • a first region of a multifunctional capture probe hybrid module comprises a PCR primer sequence.
  • this first region comprises a PCR primer sequence that is from about 5 to about 200 nucleotides, from about 5 to about 150 nucleotides, from about 10 to about 100 nucleotides from about 10 to about 75 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 20 to about 40 nucleotides, or about 20 to about 30 nucleotides, including any intervening number of nucleotides.
  • a first region of a multifunctional capture probe hybrid module is bound to a partner oligonucleotide.
  • the multifunctional capture hybrid probe module is bound to the partner oligonucleotide prior to formation of a tagged DNA library— multifunctional capture probe hybrid module complex.
  • the multifunctional capture probe hybrid module is bound to the partner oligonucleotide after the formation of a tagged DNA library— multifunctional capture probe hybrid module complex.
  • the multifunctional capture probe hybrid module is bound to the partner oligonucleotide simultaneously with the formation of a tagged DNA library— multifunctional capture hybrid probe module complex.
  • the partner oligonucleotide is chemically modified.
  • the methods contemplated herein comprise performing PCR on the tagged DNA library— multifunctional capture probe hybrid module complex so as to copy the captured tagged DNA library sequence to generate a hybrid nucleic acid molecule comprising the multifunctional capture probe hybrid module complex and a sequence complementary to a region of the captured tagged DNA library sequence located 3 Or 5 Of the multifunctional capture probe sequence relative to where the hybrid module hybridizes to the genomic target.
  • the copied target region is anywhere from 1 to 5000 nt from the, 3' or 5 '-end of sequence where the multifunctional capture probe hybrid module hybridizes to the genomic target.
  • the complementary sequence of the region that is 3 ' to the location where the multifunctional capture probe hybrid module hybridizes is copied in order to generate a hybrid nucleic acid molecule.
  • the hybrid nucleic acid molecule generated comprises the multifunctional capture probe hybrid module and the complement of a region of captured tagged DNA library sequence that is located 3 Or 5' from the location where the multifunctional capture probe hybrid module hybridizes to the target region.
  • the methods contemplated herein comprise processing a tagged DNA library-multifunctional capture probe module complex to generate a hybrid nucleic acid molecule ⁇ i.e., a hybrid multifunctional capture probe-isolated tagged DNA target molecule).
  • a hybrid nucleic acid molecule comprises the multifunctional capture probe hybrid module and the complement of a region of the tagged DNA library sequence that is located 3 ' relative to the location where the multifunctional capture probe hybrid module hybridizes to the target region.
  • the hybrid nucleic acid molecule is generated by 3 '-5' exonuclease enzymatic processing that removes single stranded 3'- ends from an isolated tagged DNA library-multifunctional capture probe module complex and/or 5 '-3' DNA polymerase extension of the multifunctional capture probe.
  • a hybrid nucleic acid molecule comprises the multifunctional capture probe hybrid module and the complement of a region of the tagged DNA library sequence that is located 5 ' relative to the location where the multifunctional capture probe hybrid module hybridizes to the target region.
  • the hybrid nucleic acid molecule is generated by the concerted action of a 5 ' FLAP endonuclease, DNA polymerization and nick closure by a DNA ligase.
  • a method for targeted genetic analysis comprises: a) hybridizing a tagged DNA library with a multifunctional capture probe hybrid module complex, wherein the multifunctional capture probe hybrid module selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library- multifunctional capture probe hybrid module complex from a); c) performing PCR on the complex from b) to form a hybrid nucleic acid molecule and d) performing targeted genetic analysis on the hybrid nucleic acid molecule from c).
  • a method for targeted genetic analysis comprises: a) hybridizing a tagged DNA library with a multifunctional capture probe hybrid module complex, wherein the multifunctional capture probe hybrid module selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library- multifunctional capture probe hybrid module complex from a); c) performing PCR on the complex from b) to form a hybrid nucleic acid molecule and d) performing targeted genetic analysis on the hybrid nucleic acid molecule from c).
  • the hybrid nucleic acid molecules obtained from step c) are sequenced and the sequences aligned horizontally, i.e., aligned to one another but not aligned to a reference sequence.
  • steps a) through c) are repeated one or more times with one or more multifunctional capture probe module.
  • the multifunctional capture probe modules can be the same or different and designed to hybridize against either strand of the genome. In some embodiments, when the multifunctional capture probe modules are different, they hybridize anywhere from 1 to 5000 nt of the same target region in the tagged DNA library.
  • the method can be performed twice, using two multifunctional capture probe modules, wherein one hybridizes upstream of the genomic target region ⁇ i.e., at the 5'-end; i.e., a forward multifunctional capture probe module or complex) and one hybridizes downstream of the genomic target region on the opposite genomic strand (i.e., at the 3'-end; i.e., a reverse multifunctional capture probe module or complex).
  • one or more multifunctional capture probe hybridize within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more bp of the target region in a tagged DNA library, included all intervening distances from the target region.
  • the method can further be performed multiple times with any number of multifunctional probe modules, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more multifunctional capture probe modules per target region, any number of which hybridize to the Watson or Crick strand in any combination.
  • a plurality of target regions are interrogated, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 10000, 50000, 100000, 500000 or more in a single reaction, using one or more multifunctional probe modules.
  • sequences obtained by the method can be aligned to one another in order to identify mutations and without being aligned to a reference sequence.
  • sequences obtained in can optionally be aligned to a reference sequence.
  • a method for determining copy number of a specific target region comprises: a) hybridizing a tagged DNA library with a multifunctional capture probe hybrid module complex, wherein the
  • multifunctional capture probe hybrid module comprises selectively hybridizes to a specific target region in the DNA library; b) isolating the tagged DNA library- multifunctional capture probe hybrid module complex from a); c) performing PCR on the complex from b) to form a hybrid nucleic acid molecule; d) performing PCR amplification of the hybrid nucleic acid in c); and e) quantitating the PCR reaction in d), wherein the quantitation allows for a determination of copy number of the specific target region.
  • PCR can be performed using any standard PCR reaction conditions well known to those of skill in the art.
  • the PCR reaction in d) employs two PCR primers.
  • the PCR reaction in d) employs two PCR primers each of which hybridize to a region downstream to the location where the multifunctional capture probe hybrid module hybridizes to the tagged DNA library.
  • the region where the PCR primers hybridize is located in the region amplified in step c).
  • the hybrid nucleic acid molecules obtained from step c) are sequenced and the sequences aligned horizontally, i.e., aligned to one another but not aligned to a reference sequence.
  • steps a) through c) are repeated one or more times with one or more multifunctional capture probe module.
  • the multifunctional capture probe modules can be the same or different and designed to hybridize to either strand of the genome.
  • one or more multifunctional capture probe hybridize within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more bp of the target region in a tagged DNA library, included all intervening distances from the target region.
  • the method can further be performed multiple times with any number of multifunctional probe modules, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more multifunctional capture probe modules per target region, any number of which hybridize to the Watson or Crick strand in any combination.
  • a plurality of target regions are interrogated, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 10000, 50000, 100000, 500000 or more in a single reaction, using one or more multifunctional probe modules.
  • a tagged DNA library is amplified, e.g., by PCR, to generate an amplified tagged DNA library.
  • All genomic target regions will have a 5 '-end and a 3 '-end.
  • the methods described herein can be performed with two multifunctional capture probe complexes which provide for amplification of a targeted genomic region from both the 5' and 3' directions, respectively.
  • one or more multifunctional capture probe hybridize within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more bp of the target region in a tagged DNA library, included all intervening distances from the target region.
  • the method can further be performed multiple times with any number of multifunctional probe modules, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more multifunctional capture probe modules per target region, any number of which hybridize to the Watson or Crick strand in any combination.
  • a plurality of target regions are interrogated, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 10000, 50000, 100000, or more in a single reaction, using one or more multifunctional probe modules.
  • the targeted genetic analysis is a sequence analysis.
  • sequence analysis comprises any analysis wherein one sequence is distinguished from a second sequence.
  • sequence analysis excludes any purely mental sequence analysis performed in the absence of a composition or method for sequencing.
  • sequence analysis includes, but is not limited to: sequencing, single nucleotide polymorphism (SNP) analysis, gene copy number analysis, haplotype analysis, mutation analysis, methylation status analysis (as determined by example, but not limited to, bisulfite conversion of unmethylated cytosine residues), targeted resequencing of DNA sequences obtained in a chromatin-immunoprecipitation experiment (CHIP-seq), paternity testing in sequence captured fetal DNA collected from pregnant maternal plasma DNA, microbial presence and population assessment in samples captured with microbe-specific capture probes, and fetal genetic sequence analysis (for example, using fetal cells or extracellular fetal DNA in maternal samples).
  • SNP single nucleotide polymorphism
  • haplotype analysis haplotype analysis
  • mutation analysis methylation status analysis (as determined by example, but not limited to, bisulfite conversion of unmethylated cytosine residues)
  • methylation status analysis as determined by example, but not limited to, bisul
  • Copy number analyses include, but are not limited to analyses that examine the number of copies of a particular gene or mutation that occurs in a given genomic DNA sample and can further include quantitative determination of the number of copies of a given gene or sequence differences in a given sample.
  • sequence alignment analysis methods for sequence alignment analysis that can be performed without the need for alignment to a reference sequence, referred to herein as horizontal sequence analysis (exemplified in, for example, Figure 20). Such analysis can be performed on any sequences generated by the methods contemplated herein or any other methods.
  • sequence analysis comprises performing sequence alignments on the hybrid nucleic acid molecules obtained by the methods contemplated herein.
  • one or more multifunctional capture probe hybridize within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more bp of the target region in a tagged DNA library, included all intervening distances from the target region.
  • the method can further be performed multiple times with any number of multifunctional probe modules, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more multifunctional capture probe modules per target region, any number of which hybridize to the Watson or Crick strand in any combination.
  • a plurality of target regions are interrogated, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 10000, 50000, 100000, or more in a single reaction, using one or more multifunctional probe modules.
  • DNA can be isolated from any biological source.
  • Illustrative sources for DNA include, but are not limited to: blood, skin, hair, hair follicles, saliva, oral mucous, vaginal mucous, sweat, tears, epithelial tissues, urine, semen, seminal fluid, seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid), excreta, biopsy, ascites, cerebrospinal fluid, lymph, or tissue extract sample or biopsy sample.
  • tagged DNA libraries for use with the methods
  • a tagged DNA library comprises tagged genomic sequences.
  • each tagged DNA sequence includes: i) fragmented end-repaired DNA; ii) one or more random nucleotide tag sequences; iii) one or more sample code sequences; and iv) one or more PCR primer sequences.
  • a hybrid tagged DNA library comprises hybrid tagged DNA sequences.
  • each hybrid tagged DNA sequence includes: i) fragmented end-repaired DNA comprising a target region; ii) one or more random nucleotide tag sequences; iii) one or more sample code sequences; iv) one or more PCR primer sequences; and v) a multifunctional capture probe module tail sequence.
  • kits and compositions of reagents used in the methods contemplated herein include a tagged DNA library, a multifunctional adaptor module and a multifunctional capture probe module.
  • the composition includes a tagged genomic library.
  • the composition includes a hybrid tagged genomic library.
  • reaction mixtures for carrying out the methods contemplated herein are provided.
  • the reaction mixture is a reaction mixture for performing any of the methods contemplated herein.
  • the reaction mixture is capable generating a tagged DNA library.
  • the reaction mixture capable of generating a tagged DNA library includes: a) fragmented DNA and b) DNA end-repair enzymes to generate fragmented end-repaired DNA.
  • the reaction mixture further comprises a multifunctional adaptor module.
  • the reaction mixture further comprises a multifunctional capture probe module.
  • the reaction mixture further comprises an enzyme with 3 '-5' exonuclease activity and PCR amplification activity.
  • the method comprises obtaining one or more or a plurality of tagged DNA library clones, each clone comprising a first DNA sequence and a second DNA sequence, wherein the first DNA sequence comprises targeted DNA sequence and the second DNA sequence comprises a capture probe sequence; performing a paired end sequencing reaction on the one or more clones and obtaining one or more sequencing reads or performing a sequencing reaction on the one or more clones in which a single long sequencing read of greater than about 100, 200, 300, 400, 500 or more nucleotides is obtained, wherein the read is sufficient to identify both the first DNA sequence and the second DNA sequence; and ordering or clustering the sequencing reads of the one or more clones according to the probe sequences of the sequencing reads.
  • sequence reads can be compared to one or more human reference DNA sequences. Sequence reads that do not match the reference sequence may be identified and used to create a de novo assembly from the non-matching sequence data. In particular embodiments, the de novo assemblies are used to identify novel sequence rearrangements associated with the capture probe.
  • a method for copy number determination analysis comprising obtaining one or more or a plurality of clones, each clone comprising a first DNA sequence and a second DNA sequence, wherein the first DNA sequence comprises a random nucleotide tag sequence and a targeted DNA sequence and the second DNA sequence comprises a capture probe sequence.
  • a paired end sequencing reaction on the one or more clones is performed and one or more sequencing reads are obtained.
  • a sequencing reaction on the one or more clones is performed in which a single long sequencing read of greater than about 100 nucleotides is obtained, wherein the read is sufficient to identify both the first DNA sequence and the second DNA sequence.
  • the sequencing reads of the one or more clones can be ordered or clustered according to the probe sequence of the sequencing reads.
  • a method for determining the copy number comprises obtaining one or more or a plurality of clones, each clone comprising a first DNA sequence and a second DNA sequence, wherein the first DNA sequence comprises a random nucleotide tag sequence and a targeted DNA sequence and the second DNA sequence comprises a capture probe sequence; ordering or clustering sequencing reads of the one or more clones according to the probe sequence of the sequencing reads.
  • the random nucleotide tag is about 2 to about 50 nucleotides in length.
  • the methods may further comprise analyzing all sequencing reads associated with a second read sequence by determining the distributions of unique and redundant sequencing reads; counting the number of times a unique read is encountered; fitting a frequency distribution of the unique reads to a statistical distribution; inferring a total number of unique reads; and normalizing the total number of inferred unique read to an assumption that humans are generally diploid.
  • the methods contemplated herein can be used to calculate an inferred copy number of one or more targeted loci and the deviation of that calculation, if any, from an expected copy number value.
  • one or more targeted loci of a gene are grouped together in a collection of loci and the copy number measurements from the collection of targeted loci are averaged and normalized.
  • the inferred copy number of a gene can be represented by the normalized average of all the target loci representing that gene.
  • compositions and methods contemplated herein are also applicable to generating and analyzing RNA expression.
  • any of the methods and compositions that are used to make tagged gDNA libraries may also be used to create tagged cDNA libraries, and capture and process target regions corresponding to RNA sequences embodied in the cDNA for subsequent RNA expression analysis, including, without limitation, sequence analysis.
  • a method for generating a tagged RNA expression library comprises first obtaining or preparing a cDNA library. Methods of cDNA library synthesis are known in the art and may be applicable to various embodiments.
  • the cDNA library may be prepared from one or a plurality of the same or different cell types depending on the application.
  • the method comprises fragmenting a cDNA library; treating the fragmented cDNA library with end-repair enzymes to generate fragmented end-repaired cDNA; and ligating a multifunctional adapter molecule to the fragmented end-repaired cDNA to generate a tagged RNA expression library.
  • a tagged RNA expression library (cDNA library) is prepared by obtaining or preparing a cDNA library from the total RNA of one or more cells; fragmenting the cDNA library; treating the fragmented cDNA with end-repair enzymes to generate fragmented end-repaired cDNA; and ligating a multifunctional adapter molecule to the fragmented end-repaired cDNA to generate a tagged RNA expression library.
  • the cDNA library is an oligo-dT primed cDNA library. In certain embodiments, the cDNA library is primed by random
  • oligonucleotides comprising about 6 to about 20 random nucleotides.
  • the cDNA library is primed by random hexamers or random octamers.
  • the cDNA libraries may be sheared or fragmented using known methods in order to achieve a desired average library fragment size.
  • the cDNA library is fragmented to an average size of about 250 bp to about 750 bp. In a certain embodiment, the cDNA library is fragmented to an average size of about 500 bp.
  • RNA expression libraries contemplated herein may be captured, processed, amplified, and sequenced, etc., using any of the methods contemplated herein for capturing, processing, and sequencing tagged genomic DNA libraries, without or without minor variations.
  • a method for targeted gene expression analysis comprising: hybridizing a tagged RNA expression library with a multifunctional capture probe module complex, wherein the multifunctional capture probe module selectively hybridizes to a specific target region in the tagged RNA expression library; isolating the tagged RNA expression library-multifunctional capture probe module complex; performing 3 '-5' exonuclease enzymatic processing and/or 5 '-3' DNA polymerase extension on the isolated tagged RNA expression library-multifunctional capture probe module complex; performing PCR on the enzymatically processed complex, wherein a tail portion ⁇ e.g., a PCR primer binding site) of the multifunctional capture probe molecule is copied in order to generate a hybrid nucleic acid molecule, wherein the hybrid nucleic acid molecule comprises the complement of the target region, the specific multifunctional capture probe sequence and the capture module tail sequence; and performing targeted gene expression analysis on the hybrid nucleic acid molecule.
  • a method for targeted gene expression analysis comprises: hybridizing a tagged RNA expression library with a multifunctional capture probe hybrid module complex, wherein the multifunctional capture probe hybrid module selectively hybridizes to a specific target region in the RNA expression library;
  • At least two different multifunctional capture probe modules are used in the at least two hybridization steps, wherein the at least two hybridization steps employ one multifunctional capture probe module each.
  • at least one multifunctional capture probe module hybridizes 5' of the target region and at least one multifunctional capture probe module hybridizes 3' of the target region.
  • one or more multifunctional capture probe hybridize within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more bp of the target region in a tagged RNA expression or cDNA library, included all intervening distances from the target region.
  • the method can further be performed multiple times with any number of multifunctional probe modules, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more multifunctional capture probe modules per target region, any number of which hybridize to the Watson or Crick strand in any combination.
  • a plurality of target regions are interrogated, e.g., 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 10000, 50000, 100000, or more in a single reaction, using one or more multifunctional probe modules.
  • any of the sequencing methods contemplated herein may be adapted to sequence the cDNA libraries with little or no deviation from their application to sequencing tagged genomic clones.
  • the statistical distribution of tagged cDNA sequencing reads of a target region of a cDNA in the RNA expression analyses contemplated herein correlates to the level of gene expression of the target region in a cell from which the cDNA library was prepared or obtained.
  • the methods contemplated herein comprise the coordinated utilization of several key molecular modules.
  • each module is described separately.
  • the interconnection of modules is described.
  • Genomic DNA from an individual can be collected, processed into pure DNA, fragmented and random nucleotide sequences of one nucleotide or more, in some embodiments in the range of 2 - 100 nucleotides, or in the range of 2 - 6 nucleotides are attached to the random ends of genomic DNA fragments ( Figure 1).
  • the combination of the introduced random nucleotide tag sequence together with the genomic fragment end sequence constitutes a unique combination of two elements that will hereafter be referred to as a the first region of the multifunctional adaptor module.
  • the uniqueness of first region of the multifunctional adaptor module is determined by the combinatorial product of the diversity within the attached first region of the multifunctional adaptor module pool times the diversity of the genomic fragment end sequences.
  • Multifunctional adapter molecules can further comprise sample-specific codes (referred to herein as the second region of the multifunctional adaptor module) and universal amplification sequences (referred to herein as the PCR primer sequence or the third region of the multifunctional adaptor module).
  • sample-specific codes referred to herein as the second region of the multifunctional adaptor module
  • universal amplification sequences referred to herein as the PCR primer sequence or the third region of the multifunctional adaptor module.
  • each segment that is attached to fragmented genomic DNA may include an additional set of nucleotides that is common to each sample but different between samples such that the DNA sequence of this region can be used to uniquely identify a given samples sequences in a set of sequences where multiple samples have been combined together (in other words, sample barcoding).
  • the attached nucleotide sequence may contain a universal sequence that can be used to amplify ⁇ e.g., by PCR) the
  • the combined elements of the random nucleotide tag sequence, the sample code, and the universal amplification sequence constitute an "adaptor" (also refer to as a multifunctional adaptor module) that is most commonly attached to the fragmented genomic DNA by means of nucleotide ligation.
  • FIG. 1 An illustrative example of a multifunctional adaptor module ligated to fragmented gDNA are illustrated in Figure 1 and (while not wishing to be limited by example) an exemplary set of such sequences is shown in Table 1.
  • Table 1 the sets of adaptor sequences are clustered into four sets of adaptor sequences. Within each column, all adaptors share the same two base code and all 16 possible random tags are represented. The 16 possible adaptors are mixed prior to ligation to fragment. Only the top, "ligation strand" of each adaptor is shown; this is the strand that becomes covalently attached to end-repaired DNA fragments. The bottom, partner strand that is eventually lost, while shown in Figure 1 , is not included in Table 1.
  • GACCAGAGAATTCGAATACAGGCT 43 GACCAGAGAATTCGAATACAGGTG 59
  • GACCAGAGAATTCGAATACATGCT 47 GACCAGAGAATTCGAATACATGTG 63
  • a single group of adaptors i.e., a universal amplification sequence, a sample-specific code, and a set of random tags; also referred to as a multifunctional adaptor module
  • a single amplification sequence on both ends of the genomic fragment
  • the two strands of any given fragment are eventually separated from one another and will behave in the present invention as independent molecules.
  • present inventors contemplate that this may be because the rare adaptor dimer species that are formed rapidly form tight hairpin structures during the steps of denaturation and annealing that are necessary for PCR amplification and it is further contemplated that these hairpin structures are completely resistant to further primer-directed amplification.
  • the ability to make adaptor-dimer free libraries is a significant technical feature in extremely low input applications like single-to-few cell genomic analysis, circulating DNA analysis (as in fetal diagnostics, tissue transplantation rejection surveillance, or cancer screening applications) or single cell transcriptome analysis. As such, the present methods provide significant utility in such applications.
  • an additional aspect of the present methods for genomic analysis strategy is that the "coverage depth" is known, that is, the average number of genomic copies present in a library are known or can be determined.
  • the cover depth is measured using purified ligation reactions prior to the bulk amplification of the library that is necessary for subsequent steps.
  • a portion of the adaptor ligated genomic fragment library equivalent to 20 - 100 fold coverage depth will be amplified using standard PCR techniques with a single, universal primer sequence driving amplification.
  • Region 1 comprises a 34 nt region common to all probes that hybridizes to a modified complementary oligonucleotide (also referred to as a partner oligonucleotide).
  • This modified oligonucleotide further comprises on the 5' end a biotin - TEG modification that is a biotin capable of binding tightly to streptavidin protein, a long hydrophilic spacer arm that alleviates steric hindrance of biotin binding.
  • the oligonucleotide terminates with a dideoxy-cytosine residue that renders this partner oligonucleotide inert to primer extension.
  • This element of probe design allows for adapting an unlimited number of probes with biotin capture functionality without having to directly modify said probes.
  • Region 2 comprises custom 60 nt regions that are target-specific and that interact with gDNA fragment molecules. This region is designed by computational methods that account for uniqueness of sequence within the genome, the presence of common SNPs that may compromise binding efficiency, and secondary structure considerations. Region 3 comprises a 20 nt segment that will serve as a PCR primer binding site in subsequent fragment amplification. This feature is described in further detail in the next paragraph.
  • a multiplicity of probes can be used to capture genomic regions of interest (multiplexing of probes). At least two probes may be employed to thoroughly query a typical coding exon of 100 - 150 bp in length. By way of example, this indicates that 20 probes will be used to capture a typical 10 exon gene and a total of 2000 probes will be used to interrogate a 100 gene panel.
  • the hybridization of genomic library fragments to probes can be performed by thermal denaturation followed by reannealing. In one embodiment, the steps comprise:
  • probe sequences refers to the combination of individual probes together with an equal molar quantity of the highly modified partner oligonucleotide
  • a specific target to probe ratio ranging anywhere from 1 part target to 1 part probe to 1 part target to 1,000,000 parts probe. In one embodiment, the optimal ratio is about 1 part fragment to 10,000 parts probe.
  • the wash step is performed at least twice.
  • hybridization-based sequence capture methods generally result in a suboptimal enrichment of target sequences. From literature and commercial publications it can be estimated that, at best, about 5% - 10% of reads map to their intended target sequences. The remaining reads often map near the intended target and commercial vendors have resorted to redefining "on-target” as reads that land anywhere within -1000 bases of the intended locus. The reasons for this "spreading" effect are incompletely understood, but they are likely a result of legitimate sequence hybridization events (see, e.g., Figure 3).
  • a DNA polymerase that also possesses a 3' to 5' exonuclease activity is employed.
  • An illustrative example of such an enzyme is T4 DNA polymerase. This enzyme will "chew off dangling tail sequences down to the duplex region formed between the probe and target sequence. It will then copy the tail segment on the probe. See, e.g., Figure 4. Benefits provided by this step include, but are not limited to:
  • the final sequencing library is a chimeric (hybrid) set of molecules derived from both the fragment and the probe.
  • the probe is strand-specific and therefore the captured target has an inherent directionality to it (illustrated in Figure 5). This means that only one of two strands generated from a single fragment will interact with probe, and processing will "focus” reads to a region 5' of the probe sequence. At this point, the complementary strand of the fragment becomes a completely independent species.
  • a target region e.g. an exon
  • Target molecules that are legitimately cross-hybridized with target fragments do not acquire essential probe sequence and are therefore lost in the subsequent amplification step.
  • fragment libraries contemplated herein have a single species on the end; called "PI".
  • the enzymatic processing step achieves two things. First, it "erases” (by 3' to 5' exonuclease activity) one of these PI ends. Second, it “adds” (by DNA polymerase copying of the probe tail sequence) the basis of a P2 end that is dissimilar from PI .
  • Enzymatically-modified target molecules with legitimate P1-P2 ends can be selectively enriched in the PCR amplification step that follows processing. This is achieved by the use of long PCR primers. In particular long primers are necessary to add the full functionality required for next-generation sequencing, and they also confer selectivity to amplification. Residual PI -PI library fragments that are "contaminants" from the first round of amplification fail to amplify with longer PI primers. This is a significant advantage of the present method. The initial PI -PI library amplifies effectively with a single, 25 nt PCR primer.
  • Example 3 Construction of single-adaptor genomic library.
  • the preferential amplification of Pl- insert-P2 processed DNA fragments is shown in Example 3 (Construction of single- adaptor genomic library).
  • Example 3 further demonstrates the substantial improvement in target specificity that accompanies processing.
  • the "sensitivity" of processing meaning the percent of initial complexes that are processed, is demonstrated to be on the order of 10% of all captured complexes in Example 9 (Direct measurement of post-capture processing).
  • the core adaptor and primer sequences applied to initial proof-of-concept experiments are shown in Table 2.
  • Enzymatically processed complex from step 6 is added directly to a PCR amplification reaction that contains full-length forward and reverse PCR primers.
  • the library can be purified, quantified, loaded on a high-throughput, next-generation sequencer (in this embodiment, the libraries are configured for the Illumina reversible terminator-based platforms), and the sequence of -millions of fragments is determined.
  • single reads of >36 nt, preferably 72 or 100+ nt in length can be observed.
  • CAC3 full length reverse primer (FLRP).
  • FLRP full length reverse primer
  • the first is the identification of sequence variants - single nucleotide variants, micro-insertions and/or micro-deletions relative to an established set of reference sequences. While complex, these methods are well documented in the field and one of skill in the art would understand such methods.
  • the second is the determination of copy number variation from the targeted sequencing data.
  • Copy number determination finds a variety of uses in the field of DNA sequencing.
  • massive parallel DNA sequencing technologies provide at least two opportunities to interrogate and analyze biological samples.
  • One well-established aspect is determination of DNA sequences, meaning the de novo sequences present in the sample ⁇ e.g., the sequencing of a newly isolated microbe) or the resequencing of known regions for variants ⁇ e.g., the search for variants within known genes).
  • a second aspect of massive parallel sequencing is quantitative biology and the ability to count the number of times a particular sequence is encountered. This is a fundamental aspect of technologies like “RNA-seq” and “CHIP- seq", where counting is used to infer gene expression or the association of a particular protein with genomic DNA, respectively. This example relates to the quantitative, counting-based aspects of DNA sequencing.
  • DNA fragments are most often counted as constellations of sequences that share high degrees of similarity (i.e., they align to specific regions of a known genomic sequence). Often the sequences within these clusters are identical. Note that DNA sequences with either a) different starting and ending DNA sequence reads or b) with high quality sequence differences from other reads within the set are most often considered "unique reads". Thus, different starting sequence location and sequence variation are one form of "tagging" that is used to differentiate unique events from clones. In the present example, random nucleotide tags (e.g., random six nucleotide sequences) are also introduced onto genomic fragments during the course of library construction.
  • random nucleotide tags e.g., random six nucleotide sequences
  • the combination of 1) the random nucleotide tag sequence with 2) the start point of the DNA sequencing read and 3) the actual sequence of the read collectively make up a tag.
  • This tag enables one to differentiate between convergent events where the same fragment was cloned twice (such fragments will have different random nucleotide tag sequences that were introduced during library construction) and fragments of the same origin that were replicated during library amplification (these "clones” will have the same random nucleotide segment and the same clone start points).
  • This type of tagging further enables quantitative analysis of genomic DNA specifically, and populations of DNA molecules (e.g., RNA-seq libraries) more generally.
  • tagging may be applied to RNA-seq, where cDNA molecules produced from mRNA of a sample are cloned by methods that create tags. Such approaches may substantially increase the fidelity of sequence-based gene expression analysis.
  • tagging can increase the resolution of chromatin immunoprecipitation (CHIP-seq) experiments. In various embodiments, tagging will enhance the quantitative aspects of sequence counting used to determine the presence and abundance of microbes in microbiome compartments and environmental samples.
  • the goal of this example was to create a genomic DNA library from
  • the adaptor-alone ligation reaction had no detectable adaptor dimer species present. This was very significant in the context of extremely low input sequencing library preparation technology, as with current methods, the limits of input are invariably determined by the background levels of adaptor dimer. Highly specialized technologies have been applied in attempts to keep adaptor dimer contamination in check. These include size exclusion methods such as columns or gel purification, expensive custom oligonucleotide modifications designed to minimize adaptor self-ligation events, and adaptor sequence modifications that allow for restriction digestion destruction of adaptor dimers following library construction.
  • the simple, single adaptor, single primer concept contemplated herein addresses the adaptor dimer issue with a simple solution that evokes the basic principles of DNA structural principles.
  • This extremely low input technology will be useful for the construction of genomic libraries for genomic analysis, for transcriptome analysis of cloned double-stranded cDNA as, for example, in RNA-seq applications of one or a few specific cells, and in rescuing the few intact fragments that may be present in a highly modified, poorly preserved formalin-fixed, paraffin-embedded (FFPE) nucleic acid sample.
  • FFPE paraffin-embedded
  • Another essential feature of the adaptor design of the present invention is the ability to "turn on” and "turn off PCR amplification of the target amplicon library by using different PCR primer lengths.
  • the optimal primer length for library amplification was 25 nt primer species with a projected Tm (under standard ionic strength conditions) > 55°C.
  • Tm under standard ionic strength conditions
  • a shorter, lower Tm primer displayed less efficient amplification of and appeared to favor amplicons with smaller average insert sizes.
  • primers in this size class work well when paired with opposite primers of dissimilar sequence.
  • Hydrated primers that were received from IDT to 100 ⁇ in TEzero (10 mM Tris pH 8.0, 0.1 mM EDTA).
  • ACA2 primers it may be useful to titrate in the amount of the ACA2 primer and monitor yield.
  • Normal high-yield PCR primers possess 1 ⁇ of both the forward and reverse primers for a total of 2 ⁇ in primer (per 100 ⁇ PCR reaction). Therefore, addition of ACA2 to 2 ⁇ (since it is both the forward and reverse primer) may increase yield.
  • human gDNA from male and female was needed.
  • the present example employs human female and male gDNA from Promega. Based on quantities shown on the tubes, these were diluted to 1000 ⁇ of 100 ng/ ⁇ DNA and subjected them to Covaris conditions that were intended to generate fragments in the range of 200 bp.
  • the proteo lipid protein 1 (PLPl) gene on chromosome X was examined for initial proof-of-concept capture studies. This gene was chosen because it is relevant to cancer and resides on the X chromosome, meaning it has a natural copy variation between males and females.
  • NM000533.3 of PLPl was used as the target region.
  • the ability to monitor regions in and around PLPl exon2 by qPCR was needed. This example provides description of the design and validation of eight such assays.
  • the example shows that all eight assays produce a PCR fragment that is consistent with the predicted sizes of the amplicons.
  • the example showed that the PLPl assays, which were situated on chromosome X, appropriately had higher specific activities per ng of input gDNA in females than in males. These data validated the use of these assays in further experiments to monitor gDNA capture.
  • a 400 bp region centered around the PLPl exon 2 was submitted to PRIMER3 for generation of amplicons that were 80-100 bp in length, for primers that were 24 nt in length, on average, and that had Tms of 60°-65°C.
  • the search region was manipulated to obtain primer pairs (qPCR amplicons) that "walk" from the 5' intron- exon boundary of exon 2 through the CDS and into the 3' exon-intron boundary.
  • proximity capture assays were also designed that were distal to exon 2, toward exon 3, and positioned -200 nt and -1000 nt away from exon 2. These would be used to monitor "hitchhiker" genomic fragments that are captured in secondary hybridization events. Finally, one assay was created on chr9 to monitor bulk genomic DNA levels during experiments. The primer sequences of these assays are shown below and the details are appended to the end of this example.
  • Reverse qPCR primer that is 1000 bp downstream of PLP1 exon2 (actually in
  • PCR reaction set up For each female or male PCR reaction, a 250 ⁇ master mix was made on ice containing:
  • the conventional PCR reaction was cycled for 30 cycles of 94°C - 30sec, 60°C - 30 sec and 72°C - 30 sec; 72°C rest for 2 min, 10°C hold.
  • a total of 5 ⁇ of product was loaded directly on a 2% agarose gel without purification and is shown in Figure 10.
  • the upper bands of each doublet were consistent with the projected mobility of the assay PCR products.
  • the lower "fuzzy" material was most likely unused PCR primers.
  • PLP1 gene Transcript ID NM 000533.3; Exon 2 - 187 nt; CDS2 From UCSC browser CDS in BOLD UPPERCASE UNDERLINED; primer sequences are shaded.
  • the Clearfork Bioscience vl .O DNA capture strategy entails the use of multifunctional probes targeted at specific genomic target regions.
  • the goal was to validate the approach using ultramersTM (Integrated DNA Technologies (IDT), Coralville, IA; ultramers is the trade name given to specialty synthesis synthetic oligonucleotides ranging in length from 45 - 200 nt) targeted to PLPl exon 2.
  • ultramersTM Integrated DNA Technologies (IDT), Coralville, IA
  • ultramers is the trade name given to specialty synthesis synthetic oligonucleotides ranging in length from 45 - 200 nt) targeted to PLPl exon 2.
  • Region 1 was the binding site for a 34 nt universal, 5' biotin-TEG and 3' dideoxycytosine-modified universal "pull-down" oligo. Two of these universal regions were designed to validate/verify equivalent (hopefully) performance.
  • Sequence 1 is 47% GC and has a 76°C melting temp in 1M NaCl.
  • Sequence 2 has a 57% GC content and an 86°C melting temp in high salt.
  • the sequences selected, 1 and 10 are the actual "universal" 5' Biotin TEG ⁇ ddC
  • Region 2 encompassed the portion of the probe that was designed to contact genomic sequences in sample genomic libraries.
  • the target region was exon 2 of PLP1.
  • the DNA sequence of PLP1 exon 2 is shown below.
  • the CDS exon 2 is highlighted in BOLD UPPERCASE UNDERLINED type. The evenly spaced-capture probe sequences are shaded.
  • Region 3 was complementary to a validated PCR primer called CAC3.
  • the sequence of the CAC3 PCR primer is: CACGGGAGTTGATCCTGGTTTTCAC (SEQ ID NO: 72).
  • Solution 1 - binding probes hydrated universal binding partners and PLP1 probes to 100 ⁇ .
  • two separate tubes combined 92 ⁇ TEz + 0.05% Tween-20 buffer, 4 ⁇ universal oligo and 1 ⁇ each of the four cognate (with the universal oligo) probes. This generated two of 1 ⁇ stock probe solution. Diluted each of these 4 ⁇ into 1000 ⁇ of TEz+Tween to provide a 4 nM working solution of probes.
  • 4X Binding buffer 4M NaCl, 40 mM Tris pH 8.0, 0.4 mM EDTA, and 0.4% Tween 20. Made 50 mis by combining 40 mis of 5M NaCl, 2 mis of 1M Tris pH 8.0, 2 mis of 10% Tween20, 40 ⁇ of 0.5 M EDTA and 6 mis of water.
  • Wash buffer 25% formamide, 10 mM Tris pH 8.0, 0.1 mM EDTA and 0.05% Tween 20. Made 50 mis by combining 37 mis of water, 12.5 mis of formamide, 500 ⁇ of 1M Tris pH8.0, 10 ⁇ of 0.5M EDTA, and 250 ⁇ of 10% Tween 20.
  • Beads Combined 250 ⁇ of 4X bind and 750 ⁇ of water to make IX binding buffer. Added 10 ⁇ of beads to 90 ⁇ of IX bind, pulled aside with magnet, washed the beads 2X with 100 ⁇ of IX bind, and resuspended washed beads in 100 ⁇ IX bind. Ten microliters of washed beads is equivalent to 1 ⁇ of beads as they come out of the manufacturer's tube.
  • a set of four tubes was treated with T4 DNA polymerase.
  • a cocktail was made by combining 10 ⁇ of New England Biolab 10X Quick blunting buffer, 10 ⁇ of 1 mM dNTPs from the same kit, 10 ⁇ of water and 1 ⁇ of T4 DNA polymerase. 20 ⁇ was added to a set of four tubes and the reaction was incubated at 20°C for 15 min.
  • the non-T4 treated samples were amplified with ACA2-25 (TGCAGGACCAGAGAATTCGAATACA; SEQ ID NO: 67) in single primer reactions.
  • the T4-treated samples were amplified with ACA2FL and CAC3FL primers
  • the core reaction mixes contained, per 400 ⁇ reaction, 120 ⁇ of water, 40 ⁇ of 10X STD Taq buffer (NEB), 40 ⁇ of 25 mM MgC12, 80 ⁇ of 10 ⁇ single primer or 40 ⁇ + 40 ⁇ of F and R primer, 20 ⁇ of DMSO, 20 ⁇ of 10 mM dNTPs and 4 ⁇ of Taq polymerase. 80 ⁇ aliquots were added to beads that had been resuspended in 20 ⁇ of TEz (binding only) or 20 ⁇ of T4 mix. The final volumes were 100 ⁇ .
  • T4 treated samples were not assayed because gel analysis showed that only aberrant material was treated by PCR amplification.
  • the data were meant to be more of a qualitative overview than a rigorous quantitative measurement.
  • the data are shown in the tables below.
  • the top table is raw Cq values.
  • the next table is Cq values converted to absolute values based on the assumption that all samples and assays conform to the
  • the bottom table shows the quotient of captured sample divided by gDNA library. This provides a sense of fold-enrichment following capture.
  • the PLP1 qPCR assays are preferably not used to produce fragments, but only to measure locus enrichment. In this type of situation, qPCR reaction set up at room temperature and fast cycling are advantages. In this experiment, the eight PLP1 assays in ABI 2X SYBR mix were tested. These are the same primer assays as described in the Example 5 (Validation of PLP1 qPCR assays).
  • PLP1 assay performance was measured against the female gDNA library (Example 3: hgDNA library from Promega female).
  • Example 3 hgDNA library from Promega female.
  • Per 10 ⁇ well 5 ⁇ ABI 2X SYBR master mix, 0.2 ⁇ of 10 ⁇ stock F+R primer, 1 ⁇ of gDNA library (20 ng/ ⁇ ) and 3.8 ⁇ water (larger volume master mixes were made and aliquoted).
  • the called Cq values for each well are shown in Table 12 below.
  • the NTC is very clean; the gDNA Cq's are variable, likely due to pipetting.
  • the general theme is that Assay 1 and 7 were poor performers while the remaining assays worked reasonably well in SYBR space.
  • the NTC traces (A) and +gDNA traces (B) were copied to provide a qualitative picture of assay performance.
  • Example 6 Capture of PLP1 exon 2 the successful capture was described, however the post-capture processing step where T4 polymerase was not removed prior to PCR yielded an artifactual library.
  • T4 was heat inactivated at 95°C for 1 min prior to PCR.
  • each sample contained 50 ⁇ of 20 ng/ ⁇ genomic DNA, 20 ⁇ of 4X binding buffer, 1 ⁇ of binding probes and either 9 ⁇ of water for a final volume of 80 ⁇ . Samples were melted at 95°C for one minute and annealed by cooling the temperature in 1°C, 2min increments to 60°C (35 cycles of AutoX on our AB 12720 thermal cycler) followed by cooling to RT. A total of 10 ⁇ of washed beads
  • T4 processing of two of the samples we prepared 50 ⁇ of enzymatic processing mix that contained 40 ⁇ of water, 5 ⁇ of 10X quick blunt buffer (New England Biolabs), 5 ⁇ of 1 mM dNTPs and 0.5 ⁇ of T4 DNA polymerase. Two aliquots of complex were suspended in 20 ⁇ (each) of T4 mix and incubated at 20°C for 15 min, 95°C for 1 min, and cooled to RT. The "untreated" controls were suspended in 20 ⁇ of the same buffer (40 ⁇ of water, 5 ⁇ of 10X quick blunt buffer (New England Biolabs), 5 ⁇ of 1 mM dNTPs) that lacked T4 polymerase.
  • both the capture alone and capture + processing samples were amplified by 30 cycles of PCR.
  • the DNA was then quantified and PLP1 assay signals measured in a specific and known amount of amplified DNA.
  • two amplification reactions were set-up.
  • amplification was performed with ACA2-25 (TGCAGGACCAGAGAATTCGAATACA; SEQ ID NO: 67) since these libraries are only amplifiable with this single primer.
  • amplification was performed with ACA2FL and CAC3FL primers
  • PCR mix contained 10 ⁇ of 10X STD Taq buffer (all reagents NEB unless otherwise specified), 10 ⁇ of 25 mM MgC12, 20 ⁇ of 10 ⁇ single primer or 10 ⁇ + 10 ⁇ of 10 ⁇ dual primers, 20 ⁇ of template (untreated control or T4 processed, beads and all), 5 ⁇ of DMSO, 5 ⁇ of 10 mM dNTPs and 1 ⁇ of Taq DNA polymerase (all set up one ice prior to amplification). Samples were amplified for 30 cycles of PCR in a three step, 95°C-30sec, 60°C-30sec, 72°C-60sec, protocol followed by 72°C for 2 min and rested at 10°C.
  • the untreated CIO capture amplicons were diluted 1000-fold and the processed CIO amplicons 15,000 fold prior to qPCR; this was done to bring Cq values into a measurable range.
  • the starting gDNA library was then examined and these diluted samples in quadruplicate wells of the qPCR plate across two on-target assays (Assays 2 and 5) and two off-target assays (Assays 6 and 7). The Cq values of the quadruplicate wells were averaged and these values are shown in Table 15 below.
  • Example 6 it was observed that untreated capture enrichment of 82,000X for CI and 28,000X for CIO (as in all these experiments, the gDNA
  • sensitivity i. e. the percentage of the initially captured complexes that are recovered in the final sequencing library.
  • enzymatic processing is effective for >10% of the initially captured sequences.
  • FIG. 4 a schematic illustration of post-capture processing is shown in Figure 4.
  • the sensitivity of processing was measured in a three-step procedure, which is illustrated on the lower right of Figure 13.
  • single PLPl capture probes were used in independent reactions to pull down/pull out PLPl exon 2 specific genomic DNA fragments from the female gDNA library (Example 3 : hgDNA library from Promega female). As there were four probes, four pull-downs were performed. The amount of captured material was measured using an adjacent PLPl qPCR assay primer pair, as illustrated in Figure 13(A).
  • reactions were distributed in quads and cycled for 40 cycles of 94°C-30sec, 55°C-30sec, 72°C-60sec. Following PCR, reaction mix was pooled from each of the four wells of the quad and 5 ⁇ was analyzed on 2% agarose gels. RESULTS
  • Step 1 gDNA. Repaired gDNA was prepared.
  • Step 2 All 16 possible adaptor codes were generated. These codes are four base structures in which the base positions at -4 and -3 (relative to the insert) are random bases and the base positions at -2 and -1 are sample codes. There are four "clusters" of sample codes. These are:
  • Cluster 1 AC, GA, CT, TG
  • Cluster 2 AA, GC, CG, TT
  • Cluster 3 AG, GT, CA, TC
  • Cluster 4 AT, GG, CC, TA
  • Clusters 2 - 4 were ordered as 100 ⁇ oligos in plates. One set of plates had ligation strand and one set of plates had partner strands. The plate array was Al-Hl, A2-H2, etc.
  • Step 3 It is easiest to create 16 ligation for female DNA and 16 ligations for male DNA in which both types received the same set of 16 unique adaptor types. This will allow us to decide later which combinations of samples we want to create with maximum flexibility. To do this, End-repaired gDNA from the experiment was used. I will perform the requisite 32 ligation in 20 ⁇ /rxn as follows:
  • the cocktails were mixed and aliquoted into 16 tubes with 18 ⁇ each. 2 ⁇ of adaptors and 0.5 ⁇ HC T4 ligase were added and the resulting reactions were incubated at 22°C for 60 min, 65°C for 10 min, and cooled to RT. 80 ⁇ TEz, then 120 ⁇ Ampure beads were also added to the reactions, mixed and incubated 10 min at RT. The reactions were wash twice with 200 ⁇ of 70% EtOH/water (v/v), air dried, and resuspended in 100 ⁇ TEz.
  • Step 4 qPCR. Make qPCR master mix containing:
  • the second plate had the layout shown in Table 18 below.
  • Table 19 shows the Cq values of STDs and samples (the average of duplicate measurements except (i) the experiment repeated on plate 2 and (ii) Ml, M2 and M3 were measured in three sets of duplicates -average of the three measurements taken).
  • the goal of this experiment was to create ligation mixes containing gDNA library and to quantify genome equivalents per ⁇ of ligation mix so that measured numbers of genomes can be amplified into microgram quantities of library material.
  • Table 20 above shows the genomes per ⁇ for each library that was made.
  • the goal of Table 21, shown below, was to convert designated samples (picked by random drawing) into 10 copy, 20 copy, 40 copy, 80 copy, etc. libraries for downstream capture tests.
  • the table transposes the genomes per ⁇ into ⁇ per PCR reaction to achieve the indicated depth of coverage.
  • the table assumes a 200 ⁇ PCR per sample and 40 ⁇ of template input.
  • Eight segments for assay design that represent a sampling of the 49 probe target regions were chosen. To design assays, the DNA segment that is within 200 bp of the 5' end of the probe was identified. The eight regions, as shown in Table 22 below, were chosen to be a more-or-less random selection of target regions. The 200 bp segments were submitted to PRIMER3 PCR primer picking in which we specified amplicons of 50-100 bp, primer Tm's of 65°C (optimal) and primer lengths of 24 nt (optimal). Table 22 below shows the region and unique genomic attributes, the forward (F) and reverse (R) primer sequences, the expected amplicon length, and the actual amplicon within the context of the genomic sequence.
  • each primer pair was investigated by performing 100 ⁇ PCR reactions that contained 200 ng (2 ng/ ⁇ ) of female genomic DNA.
  • the reaction mixes contained, per 100 ⁇ , 50 ⁇ water, 10 ⁇ 10X STD Taq buffer, 10 ⁇ 25 mM MgC12, 10 ⁇ of an F+R primer blend in which each primer was present at 10 ⁇ , 10 ⁇ of 20 ng/ ⁇ gDNA, 5 ⁇ of DMSO, 5 ⁇ of 10 mM dNTPs, and 1 ⁇ of Taq polymerase. Reactions were set up on ice.
  • Amplifications were performed for 30 cycles of 94°C-30sec, 60°C-30 sec, and 72°C-30sec, followed by a 2 min incubation at 72°C and hold at 10°C. Five ⁇ of PCR product was examined on a 2% agarose gel.
  • PCR products were purified on Qiagen PCR purification columns by combining the remaining 95 ⁇ of PCR product with 500 ⁇ PB. The material was spun through the column at 6 KRPM for 30 sec and washed with 750 ⁇ PE, spun at 13.2 KRPM. The products were eluted from the columns with 50 ⁇ of EB and quantified by Qubit.
  • chrX-154376051 region (assays 10 & 11) was examined in more detail.
  • the purified PCR products were diluted to 100 fg/ ⁇ , 10 fg/ ⁇ and 1 fg/ ⁇ .
  • Genomic DNA was diluted to 10 ng/ ⁇ .
  • Two microliters of either standards or gDNA was combined with 8 ⁇ of PCR master mix per well of the 48-well Eco qPCR plate.
  • Master mix contained, per 500 ⁇ of final reaction volume (that accounts for the addition of template) 175 ⁇ of water, 50 ⁇ of 10X STD Taq buffer, 50 ⁇ of 25 mM MgCl 2 , 50 ⁇ of F+R 10 ⁇ primer blend, 25 ⁇ of DMSO, 25 ⁇ of 10 mM dNTPs, 12.5 ⁇ EvaGreen, 10 ⁇ of ROX and 5 ⁇ of Taq polymerase. Thirty two ⁇ of mix was distributed to 16 wells and 8 ⁇ of templates were added. These were then distributed in quads to the qPCR plate. The plate layout is shown in Table 23 below.
  • Table 23 Plate layout for qPCR interrogation of chrX-154376051 region
  • the product yields were: Assay 9 - 18.4 ng/ ⁇ ; Assay 10 - 26.1 ng/ ⁇ ; Assay 11 - 13.9 ng/ ⁇ ; Assay 12 - 26.6 ng/ ⁇ ; Assay 13 - 7.9 ng/ ⁇ ; Assay 14 - 19.2 ng/ ⁇ ; Assay 15 - 23.1 ng/ ⁇ ; and Assay 16 - 20.4 ng/ ⁇ .
  • Quantitative analysis was performed with assays 10 & 11 that correspond to a cryptic segmental duplication on chromosome X such that females have four copies and males have two copies.
  • this step can be done in concert with PCR. Moreover, this step can be done at 72°C with a thermostable polymerase enzyme, meaning potential secondary structures of single strand clones are less of an issue. Finally, the implication was that probes were shortened from 114 nt to 95 nt; and this provided a cost saving advantage.
  • Example 11 Validation of eight new capture qPCR assays
  • assays 10, 14, 15 and 16 with probes that "point" at those assays were matched. While the probe and qPCR assays were within proximity to one another, their DNA sequences did not overlap with one another (See Figure 16). The sequences of the probes and corresponding assays are shown in Tables 26 and 27 below.
  • a gDNA library was remade from samples F13-F16 (Example 10) by combining 20 ⁇ of each ligation mix into 80 ⁇ total and amplifying in 800 ⁇ total. Beads were cleaned to 400 ⁇ and pool concentration at 32 ng/ ⁇ was measured by Qubit.
  • Oligos from IDT listed below, were resuspended to 100 ⁇ . Ultramers come as 4 nmol, so these were suspended in 40 ⁇ TEzero. Four 2 ⁇ aliquots of the four test probes were combined with 8 ⁇ of 100 ⁇ universal tail sequence (derived from the first 35 bases of the full length reverse primer #9) to give a 50 ⁇ tube of duplex. This duplex was diluted 10 ⁇ into 990 ⁇ of TEzero + Tween to give 500 nM, and 10 into 990 again to get 5 nM.
  • Combined 40 ⁇ of gDNA was combined with 15 ⁇ of 4X bind and 5 ⁇ of capture duplex.
  • the reaction mix was annealed and captured on 2 of washed MyOne strep coated beads.
  • the reaction was washed four times with wash buffer and aspirated wash buffer from bead pellet.
  • To measure capture alone one bead pellet was resuspended in 100 ⁇ PCR mix that contained single PCR primer ACA2.
  • capture + processing another bead pellet was resuspended in 100 ⁇ of PCR mix that contained the full-length ACA2 forward primer (oligo #8) and full-length CAC3 reverse primer (oligo #9). The latter sample was incubated for 72°C for 2 min.
  • Both samples were amplified for 25 cycles of 94°C-30sec, 60°C - 30sec, and 72°C-60 sec. After a hold at 72°C for 2min and cooling to RT, the PCR amplicons were purified on bead and resuspended in 50 ⁇ of TEzero.
  • samples were assayed with assays 9 - 16 (assays 10, 14, 15, and 16 are targets), using EvaGreen as the reporter dye, ROX as the STD dye, and 3 step PCR of 94°C-30sec, 60°C-30sec and 72°C-60sec for 40 cycles.
  • the original gDNA library was present at 2 ng/ ⁇ final concentration.
  • the captured sample and capture+processed samples were present at 2 pg/ ⁇ final concentration (diluted in TEzero+0.05%
  • PCR yields of the capture only was 27.8 ng/ ⁇ and of the capture + processing was 40.4 ng/ ⁇ . These robust yields indicated that amplification went to completion.
  • the 2% agarose gel image shows the starting input library, the captured library and the capture + processing library ( Figure 17). If processing worked, the average insert size of the library should decrease, which it did. The fact that the bottom end of the library is more or less "a band" indicates that there may be some priming off of probe.
  • the purpose of this experiment was to assess the enrichment and coverage of target regions in sequencing libraries.
  • Two enrichment library pools were constructed from an equal mix of male and female human genomic DNA using a set of 49 capture probes that target sites within specific genes (KRAS, MYC, PLPl, CYP2D6 and AMYl) and duplicated regions on the X chromosome. Probe sequences are shown in Table 29 below
  • the first library pool was generated as described for the 'capture plus processing' library in Example 12.
  • the second pool was generated as described for the 'capture only' library in Example 12 except for the following modification.
  • a second round of PCR was performed to convert single primer, ACA2- amplified libraries into dual primer, heterologous end libraries suitable for Illumina sequencing. To do this, libraries were diluted and re-amplified with the following primers: primer #55 AATGATACGGCGACCACCGAGATCTACACGTCATGCAGGACCAGAG (SEQ ID NO: 199) and primer #56
  • the 100 ⁇ reaction mix contained 40 ng of library, 10 ⁇ of 10X STD Taq buffer, 10 ⁇ of 25 mM MgCl 2 , 10 ⁇ of 55 and 10 ⁇ of 56 primer that were both at 10 uM, 5 ⁇ of DMSO, 5 ⁇ of dNTP and 1 ⁇ of Taq DNA polymerase.
  • the samples were amplified for 2 cycles of 94°C-30sec, 50°C-30 sec, 52.5°C-30sec, 55°C-30sec, 57.5°C- 30sec, 60°C-30sec, 72°C-lmin. They were then amplified for 8 cycles of 94C-30sec, 60C-30 sec and 72C-60 sec followed by 72°C for 2 min.
  • PCR mixes were purified with beads and resuspended in 50 ⁇ each.
  • NGS Next Generation Sequencing
  • the paired-end read data obtained by the methods contemplated herein will have DNA tagged sequence information in read 1 and probe ID information in read 2.
  • the first step in data analysis is to match reads to probes.
  • Step two is analyzing the sequence information connected "horizontally" to each probe. See, e.g., Figure 20.
  • CNV copy number variation
  • SNV detection Homozygous variant alleles involving a single base are fairly straightforward to identify, but these changes are rare. More commonly, SNVs are heterozygous, and variants may occur at several contiguous or closely spaced positions (error prone repair tends to lay down tracks where several bases are non-consensus).
  • the heterozygous SNV hypotheses run the spectrum from true, high coverage detection where (strictly as an hypothetical example) 49 reads possess a SNV and 47 reads possess the wild-type, reference base.
  • a probe set is a unique and known collection of sequences, which can include one or two probes or even tens of thousands of probes. This means that read_2 can be used to identify any and all of the probes within an experiment. This of course assumes that read 2 is of sufficient length and that probes are designed such that the region interrogated by read_2 constitutes a unique identifier. Table 31 describes a collection of 192 probes and the 10 nt read_2 sequence that serves as a unique identifier for each probe.
  • read l and read_2 are derived from the same DNA clone. This implies that the read l genomic sequence (parts (3) and (4) in Figure 24) is present because it was associated with a particular probe (part (5) in Figure 24). Taken in total, this data indicates that each DNA sequence present in a collection of next-generation sequences can be associated with the probe sequence that targeted it. All DNA sequences associated with a particular probe can be retrieved.
  • PARSAR probe-associated-read-scaffold-assembly
  • Probe-based read grouping is used in conjunction with other aspects of molecular design to identify variants with a high-degree of first-pass confidence.
  • probes are generally designed to bracket target regions.
  • the overlapping aspect of reads allows potential variant sites to be queried by independent reads in both orientations.
  • this dual-probe design ensures that adjacent probe binding sites themselves are sequenced. This is an important feature where probe capture performance may be in question.
  • variant alleles where single nucleotide variants underlie one of the capture probe sequences are identified by this molecular design and can be accounted for in downstream informatics analysis.
  • sequence "tags” defined as the combination of a three base sequence label and ragged sequence start sites ((l)+(3) in Figure 24).
  • Sequence tags define the uniqueness of each sequencing clone. As illustrated in Figure 27, variants that occur within a collection of sibling clones that share identical sequence tags are likely to be false positives. In contrast, variants are shared among sequences with different tags - even if they occur at low frequency - have a higher probability of being true positive variants. This system of tagging sequences and using tags to assign confidence predictions to variant calling has the prospect of substantially decreasing the burden of downstream variant validation (which can be costly and time consuming). Molecular annotation is described in more detail in the document Example 16, which describes the molecular technology sequencing platform.
  • one of the distinguishing features of the technology platforms contemplated herein is the fact that all "annealing probe” events are copied into DNA clones that also possess additional molecular annotation. Sequences are segregated by probe and by sample labels into a collection belonging to a specific target region of a specific input sample. Combinations of alignment and de novo assembly can then be used for variant detection. Finally, redundancy in the appearance of candidate variants can be used to assign confidence in variant calling. In addition to variant analysis, methods for copy number determination were also provided. These two elements are tightly coupled, specifically because copy number determination is dependent on high confidence sequencing reads. The overall schema for determining copy number from sequence information is shown in Figure 28. EXAMPLE 16
  • the genomic sequencing platforms contemplated herein provide methods to: (1) Address genomic samples from multiple individuals in a single sequencing run; (2) Detect single (and/or multiple) nucleotide variants (SNVs) and single (and/or multiple) nucleotide insertions and deletions (SNIDs) with high confidence; (3) Detect large and small scale copy number variations (CNVs) in all queried gene environments; (4) Detect micro-scale translocations, inversions and insertion/deletion events in queried gene environments; (5) Develop a technological system that is scalable from > exome- scale investigation (> 1-2% of the overall human genome sequence) to ⁇ single gene- scale validation; (6) Achieve high specificity (low false-negative rate) and high sensitivity (low false-positive rate) in genomic variation tests; (7) Create a molecular and bioinformatics technology that is simple, portable and scalable in its execution; and (8) Provide molecular methods that are readily amenable to quality control
  • sequence label is a set of (contiguous*) nucleotides (i.e. a unique set of 3 mers) that is used in conjunction with the read start position (3) to establish the uniqueness of each sequencing read.
  • the combination of this label and the read start point were referred to as the "unique sequence tag.”
  • sequence label is the first set of bases encountered and in sequencing-by- synthesis (SBS) chemistry all four DNA bases must be equally represented at each read position, the constraints on the sequencing label are not only uniqueness, but also that the collection of bases used in the collective set of sequence labels must have all four bases present at all positions sequenced.
  • SBS sequencing-by- synthesis
  • sample label is a set of (contiguous*) nucleotide codes that uniquely identify a particular sample within a set of multiplexed samples.
  • the collection of sample labels must also contain all four bases to satisfy the requirements of SBS sequence base calling.
  • the sample codes are intentionally positioned next to genomic DNA fragments.
  • the driver for this design is ligation bias, meaning that there are base preferences for DNA ligation efficiency in the ⁇ 2 bases upstream and the 1-2 bases downstream of the ligation junction. By placing the sample code at the ligation junction, all fragments within a specific sample experience the ligation influences/biases.
  • sequence labels and sample labels could be created as inter-digitated nucleotide sequences.
  • the "read start point" within genomic fragments is one of two key elements that define a "unique sequence read.”
  • the unique identifying "tag” for each read is comprised of the sequencing label AND the read start point.
  • the collection of unique [(l)+(3)] sequence tags is essential for determination of large scale CNV.
  • large scale CNV is defined as any CNV that involves the entirety of at least one probe binding region plus some adjacent sequence. Large scale CNV can be as large as gain or loss of entire chromosomes.
  • sequence label and sample label are embedded in adaptor sequences that are ligated to end-repaired genomic fragments in the initial stage of library construction process where a whole genome library is created.
  • the sequencing read The sequence information from genomic fragments is, of course, a central focus of the genomic assays. Each read is considered in the context of multiple, overlapping reads produced within the same assay.
  • the probe level - "genomic indexing.”
  • the overall genomics assay strategy is to combine multiple sequence labels into a composite "molecular annotation" that places each sequencing read into a larger framework of genomic analysis.
  • Read 1 reveals elements (1-4) of each annotated clone.
  • Read 2 reveals the probe sequence that retrieved each clone by hybridization based capture and subsequent enzymatic processing.
  • the probe sequence information is central to the genomics strategy because all reads are initially clustered according to the probe that captured them. This clustering of information on a probe-by-probe basis is termed "genomic indexing" because each read is indexed to a genomic probe prior to analysis.
  • the constellation of all probe sequences within a capture reaction is well-defined (we know which probes went into the capture reaction). This implies that Read 2 does not necessarily need to cover the entire 60 nt probe sequence. Rather, Read 2 only needs to be of sufficient length to enable unambiguous identification of all probes within a specific reaction.
  • the probe set discussed in Example 15 consists of 192 probes that can be differentiated based on only 7 nt of 5' probe sequence (two of the probes with identical 7 nt 5' termini were tagged with dinucleotide codes so they could be informatically differentiated).
  • the capture label The composition of the libraries is determined by the intimate molecular interaction between probes and target sequences.
  • the performance of each unique probe sequence can be monitored using the capture label, which can be as simple as a string of several (4 - 6) random bases.
  • the diversity and statistical distribution of capture labels detected in sequencing is a direct measure of probe performance.
  • sequence under-representation may also be a consequence of sequences that do not ligate well to adaptors and or sequences that do not amplify well with the particular PCR regimen that is used.
  • capture labels allows differentiation of these failure modes. With poor probe performance, the very few capture events that do occur will manifest as very few capture labels that show up multiple times. In contrast, poor representation for reasons upstream of the actual capture reaction (ligation, PCR, end-repair, etc.) will result in a large constellation of capture labels that will be, by and large, uniquely represented. In particular
  • Probe sequence selection and the methods to use them have, necessarily, been developed in concert. This example describes probe selection criterion in Section I and the laboratory methods that make them most effective in Section II. See, e.g., Figure 30.
  • target enrichment probes are 60 nt in length. Probes are generally directional, meaning they capture sequences on one side (generally the 3' side) of their position. Tail sequences that add additional functionality (e.g., PCR primer binding site, binding site for complementary oligo that enables biotin pull-out, etc) are added in addition to the core targeting 60-mer. Sixty nucleotide targeting sequences are chosen with the following constraints and criterion: (1) The probe is positioned from -100 to +50 nt relative to the start of the target sequence.
  • the "start" of the target sequence is the intron:exon junction; (2) Probes are designed with redundancy, as illustrated, such that the resulting sequences from a pair of probes are overlapping in opposite orientations; (3) Probes are selected (where possible) to possess GC content not less than 33% (>20 G's or C's per 60 mer) and not more than 67% ( ⁇ 40 G's or C's per 60 mer); (4) Probes are selected to avoid repeats wherever possible.
  • the input to target enrichment is probes, a gDNA library, and buffers, which have been described elsewhere herein.
  • the first step in targeted enrichment is melting of the gDNA library, which begins in a form as double-stranded PCR fragments. This is achieved by denaturation of the gDNA, preferably at a concentration of 100 ng/ ⁇ in a total volume of 10 ⁇ , at 98°C for 2 min followed by immediate transfer to ice.
  • the gDNA library is suspended in a low salt buffer that contains 10 mM Tris pH 8.0 and 0.1 mM EDTA.
  • the second step is to add 5 ⁇ of concentrated binding buffer (4M NaCl, 40 mM Tris pH 8.0, 0.4 mM EDTA and 0.4% Tween20).
  • the overarching concept is that the concentration of salt must be increased to 2N osmolarity to achieve rapid kinetic association of complementary DNA strands. Five microliters of probe is also added such that the final concentration of probe is 250 pM in each probe. The mixture of gDNA library, buffer and probe is heated to 98°C for 2 min and cooled in 1°C increments for four min each down to 68°C. In the third step, probe: gDNA complexes (the probe has a biotin associated with it) are bound to magnetic beads that are coated with streptavidin.
  • stringent washing is used to remove unwanted associations between probe and non-target sequences that may occur because of, for example, short matches of nucleotide sequence between probe and gDNA.
  • Stringency is achieved by using low-salt, high- formamide wash buffer as, for example, a buffer containing 30%-35% formamide (v/v), 10 mM Tris pH 8.0, 0.1 mM EDTA and 0.5% Tween 20).
  • Several washes of the beads are used to achieve the desired purity of target sequence (e.g., four).
  • the washed beads possess target sequences bound to probe that are processed, amplified, and sequenced.
  • low salt melting of the gDNA library, high salt probe annealing, and high formamide washes are used in concert with probe designs to achieve high levels of target sequence enrichment.
  • Table 32 Exemplary Genome tags, sample codes and library information are shown in Tables 32-34 below.
  • Table 32 Exemplary Genome tags

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