WO2018009677A1 - Enrichissement rapide de cible par pcr relais multiplexée avec des amorces à bulles modifiées - Google Patents

Enrichissement rapide de cible par pcr relais multiplexée avec des amorces à bulles modifiées Download PDF

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WO2018009677A1
WO2018009677A1 PCT/US2017/040917 US2017040917W WO2018009677A1 WO 2018009677 A1 WO2018009677 A1 WO 2018009677A1 US 2017040917 W US2017040917 W US 2017040917W WO 2018009677 A1 WO2018009677 A1 WO 2018009677A1
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Prior art keywords
primer
stranded
universal
double
primers
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PCT/US2017/040917
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English (en)
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Yuan Jiang
Hongdong Tan
Radoje T. Drmanac
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Complete Genomics, Inc.
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Publication of WO2018009677A1 publication Critical patent/WO2018009677A1/fr

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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups

Definitions

  • a library of nucleic acids are provided as input into a high-throughput sequencing platform, where the individual nucleic acids of the library contain defined ends that flank a region to be sequenced and are compatible with the sequencing platform.
  • Such libraries are often produced using either a hybrid capture method or a multiplex amplification method such as multiplex PCR.
  • multiple sequence-specific PCR primer pairs are used to amplify different nucleic acid regions of interest from a sample.
  • the multiplex primers can contain universal primer binding sites that can then be used in a subsequent universal amplification step to provide sufficient quantities of nucleic acid for sequencing.
  • Such multiplex amplification strategies can provide a high degree of on-target amplification and a quick turn-around time as compared to hybrid capture methods.
  • the present invention provides a method comprising: a) forming a reaction mixture comprising a double-stranded target polynucleotide, a DNA polymerase, a forward degradable bubble (DB) primer, and a reverse DB primer, wherein: the forward DB primer comprises (i) at least two selectively degradable nucleotides (SDNs) and (ii) a first universal primer binding site or the complement thereof, wherein the forward DB primer hybridizes under hybridization conditions to a first site on one strand of a double-stranded target polynucleotide and the reverse DB primer comprises (i) at least two SDNs and (ii) a universal primer binding site or the complement thereof, wherein the reverse DB primer hybridizes under hybridization conditions to a second site on an opposite strand of the double-stranded target polynucleotide, wherein the first and second sites flank a region of interest of the double-stranded target polyn
  • the method further comprises: b) amplifying the region of interest with 3-15 cycles of denaturation and DNA polymerase-mediated DB primer extension to generate a DB primer amplicon having at least four SDNs, a forward universal primer binding site, and a reverse universal primer binding site, wherein the forward and reverse universal primer binding sites flank the region of interest.
  • the method further comprises: c) selectively degrading the SDNs of the DB primer amplicon; and d) selectively degrading the SDNs of DB primers that have not been extended, if present.
  • the reaction mixture further contains a forward and a reverse universal primer
  • the amplifying further comprises generating an amplicon that does not have an SDN and comprises a forward universal primer binding site and a reverse universal primer binding site, wherein the forward and reverse universal primer binding sites flank the region of interest.
  • the method further comprises d) amplifying the region of interest with 10-30 cycles of denaturation and DNA polymerase-mediated universal primer extension.
  • the reaction mixture comprises a forward universal primer and a reverse universal primer, wherein the forward and reverse universal primers do not comprise SDNs, and the step of amplifying produces an amplicon that (i) does not comprise an SDN (ii) comprises a forward universal primer binding site, and (iii) comprises a reverse universal primer binding site, wherein the forward and reverse universal primer binding sites flank the region of interest.
  • the present invention provides a degradable bubble (DB) primer, wherein the DB primer is an oligonucleotide comprising a loop region, a 3' arm that is 3' to the loop region, and a 5' arm that is 5' to the loop region, wherein: a) the 3' arm has a 3' end and a 5' end, wherein the 3' arm specifically hybridizes under hybridization conditions to a first portion of a first strand of a double-stranded target polynucleotide, and wherein the 3' end is a substrate for a polymerase extension reaction when hybridized to the first strand; b) the 5' arm has a 3' end and a 5' end, wherein the 5' arm specifically hybridizes under hybridization conditions to a second portion of the first strand; c) the loop region comprises at least two selectively degradable nucleotides (SDNs); and d) the loop region comprises a universal primer binding site or
  • SDNs selectively degrad
  • the 5' arm is from 12 to 50 nucleotides in length. In some embodiments, the 3' arm is from 6 to 24 nucleotides in length. In some embodiments, the 5' arm is from 12 to 50 nucleotides in length, the 3' arm is from 6 to 24 nucleotides in length, and the loop region is from 6 to 50 nucleotides in length. In some embodiments, the loop region comprises a first barcode. In some embodiments, the first barcode is from 3 to 20 nucleotides in length, preferably from 8 to 12 nucleotides in length. In some embodiments, the first barcode of the loop region is 3' to each of the at least two SDNs.
  • each of the at least two SDNs is independently selected from the group consisting of a deoxyuridine nucleotide, a deoxyinosine nucleotide, an oxidized pyrimidine nucleotide, an oxidized purine nucleotide, an alkylated purine, 5- hydroxyuracil, 5-hydroxymethyluracil, and 5-formyluracil.
  • the loop region comprises at least three SDNs. In some embodiments, the loop region comprises from 3 to 30 SDNs. In some embodiments, the universal primer binding site or complement thereof is at least 4 nucleotides in length, from 5 to 30 nucleotides in length, or from 6 to 25 nucleotides in length. In some embodiments, the universal primer binding site or complement thereof is from 7 to 20 or from 7 to 12 nucleotides in length. In some embodiments, the SDNs of the loop region include a 5'-most SDN and a 3'-most SDN, and there are at least 4, preferably from 4 to 30, intervening nucleotides positioned between the 5'-most SDN and 3'-most SDN.
  • the 5'-most and 3'-most SDNs are separated by from 7 to 18, preferably from 7 to 10, intervening nucleotides that are not SDNs.
  • the present invention provides a composition comprising at least 10 DB forward primers, and at least 10 DB reverse primers, wherein: each DB forward primer has the same first universal primer binding site or complement thereof; each DB reverse primer has the same second universal primer binding site or complement thereof; and the 3' arms of each of the individual DB forward primers and DB reverse primers are different from each other; the 5' arms of each of the individual DB forward primers and DB reverse primers are different from each other; orthe 3' arms and 5' arms of each of the individual DB forward primers and DB reverse primers are different from each other.
  • composition comprises from 10-1,000 DB forward primers and from 10-1,000 DB reverse primers. In some embodiments, the composition comprises from 10- 15,000 DB forward primers and from 10-15,000 DB reverse primers. In some embodiments, the composition comprises at least 1,000 DB forward primers and at least 1,000 DB reverse primers. In some embodiments, the composition comprises at least 10,000 DB forward primers and at least 10,000 DB reverse primers.
  • the present invention provides a composition comprising a double- stranded target polynucleotide, a forward DB primer, and a reverse DB primer, wherein: the forward DB primer hybridizes under hybridization conditions to a first site on one strand of a double- stranded target polynucleotide and the reverse DB primer hybridizes under hybridization conditions to a second site on an opposite strand of the double-stranded target polynucleotide, wherein the first and second sites flank a region of interest of the double-stranded target polynucleotide, wherein the forward DB primer and the reverse DB primer each independently comprises a structure according to any one of the foregoing DB primer aspects or embodiments.
  • the composition comprises: (i) at least 2 different double-stranded target polynucleotides, each comprising one or more regions of interest; (ii) at least 10 different forward DB primers; (iii) and at least 10 different reverse DB primers;, wherein the reverse DB primers and the forward DB primers comprise at least 10 different DB primer pairs; and (iv) optionally one or more (e.g., tens (e.g., at least 10), hundreds (e.g., at least 100), thousands (e.g., at least 10 3 ), or millions (e.g., at least 10 s ), or more) double-stranded target polynucleotides that do not comprise a region of interest, wherein each DB primer pair comprises a forward DB primer and a reverse DB primer that hybridize to opposite strands of a double-stranded target polynucleotide under hybridization conditions and flank a region of interest, wherein the at least 2 different double-stranded
  • the composition comprises at least 10 different double-stranded target polynucleotides, at least 1,000 different forward DB primers, and at least 1,000 different reverse DB primers, wherein the reverse DB primers and the forward DB primers comprise at least 1,000 different DB primer pairs, wherein individual different primer pairs comprise forward DB primers and reverse DB primers that hybridize to opposite strands of a double-stranded target polynucleotide under hybridization conditions and flank a region of interest, wherein the at least 1,000 different DB primer pairs flank at least 1,000 different regions of interest, wherein each forward DB primer and each reverse DB primer independently comprises a structure according to any one of the foregoing DB primer aspects or embodiments.
  • the composition comprises at least 100 different double-stranded target polynucleotides, at least 10,000 different forward DB primers, and at least 10,000 different reverse DB primers, wherein the at least 10,000 different reverse DB primers and at the least 10,000 different forward DB primers comprise at least 10,000 different DB primer pairs, wherein individual different primer pairs of the at least 10,000 different DB primer pairs comprise forward DB primers and reverse DB primers that hybridize to opposite strands of a double-stranded target
  • the polynucleotide under hybridization conditions and flank a region of interest wherein the at least 10,000 different DB primer pairs thereby flank at least 10,000 different regions of interest
  • the at least 10,000 forward DB primers comprise a structure according to any one of the foregoing DB primer aspects or embodiments
  • the at least 10,000 reverse DB primers comprises a structure according to any one of the foregoing DB primer aspects or embodiments.
  • the composition comprises 10-15,000 different forward DB primers, and 10-15,000 different reverse DB primers, wherein the 10-15,000 different reverse DB primers and 10-15,000 different forward DB primers comprise 10-15,000 different DB primer pairs, wherein individual different primer pairs of the 10-15,000 different DB primer pairs comprise forward DB primers and reverse DB primers that hybridize to opposite strands of a double-stranded target polynucleotide under hybridization conditions and flank a region of interest, wherein the 10-15,000 different DB primer pairs thereby flank 10-15,000 different regions of interest, wherein the 10- 15,000 forward DB primers comprise a structure according to any one of the foregoing DB primer aspects or embodiments, and wherein the 10-15,000 reverse DB primers comprises a structure according to any one of the foregoing DB primer aspects or embodiments.
  • the present invention provides a method comprising: a) amplifying at least 10 different regions of interest using one of the foregoing compositions comprising at least 10 DB forward primers and at least 10 DB reverse primers; b) amplifying at least 1,000 different regions of interest using one of the foregoing compositions comprising at least 1,000 DB forward primers and at least 1,000 DB reverse primers; c) amplifying at least 10,000 different regions of interest using one of the foregoing compositions comprising at least 10,000 DB forward primers and at least 10,000 DB reverse primers; or d) amplifying 10-15,000 regions of interest using a using one of the foregoing compositions comprising at least 10-15,000 DB forward primers and at least 10-15,000 DB reverse primers.
  • the present invention provides a double-stranded amplicon, wherein a first strand of the double-stranded amplicon comprises a DNA polymerase extension product of a first DB primer having a structure according to any one of the foregoing DB primers, and a second strand of the double-stranded amplicon comprises a DNA polymerase extension product of a second DB primer having a structure according to any one of the foregoing DB primers, wherein the first and second DB primer extension products are reverse complements of each other, wherein the double- stranded amplicon comprises: i) a first end region comprising at least two SDNs on the first strand; and ii) a second end region comprising at least two SDNs on the second strand, and wherein the double stranded amplicon comprises a region of interest flanked by the first end region and second end region, wherein the region of interest does not contain an SDN and/or consists of nu
  • the region of interest is at least 50 base pairs in length, preferably from 150 to 250 base pairs in length.
  • the at least two SDNs of the first end region comprise a 5'-most SDN and a 3'-most SDN and there are from 4 to 30 intervening nucleotides positioned between the 5'-most and 3'-most SDNs; and ii) the at least two SDNs of the second end region comprise a 5'-most SDN and a 3'-most SDN and there are from 4 to 30 intervening nucleotides positioned between the 5'-most and 3'-most SDNs.
  • the present invention provides a double-stranded amplicon having a first strand hybridized to a second strand, wherein the amplicon comprises a first end region comprising at least two SDNs on the first strand, and a second end region comprising at least two SDNs on the second strand, wherein the at least two SDNs of each strand comprise a 5'-most and a 3'-most SDN, and the 5'-most and 3'-most SDNs of each strand independently have from 4 to 30 intervening nucleotides positioned between the 5'-most and 3'-most SDNs, and wherein the double stranded amplicon comprises a region of interest flanked by the first and second ends that does not contain an SDN, and/or consists of nucleotides selected from A, C, G, and T.
  • two of the at least two SDNs of the first strand are less than 200 nucleotides from the 5' -terminal nucleotide of the first strand; and ii) two of the at least two SDNs of the second strand are less than 200 nucleotides from the 5'-terminal nucleotide of the second strand.
  • the region of interest is at least 50 base pairs in length, preferably from 150 to 250 base pairs in length.
  • the present invention provides a selectively-degraded double-stranded amplicon having a first single-stranded universal primer binding site of at least 4-30 nucleotides in a first end region near the 3' end of a first strand and a second single-stranded universal primer binding site of at least 4-30 nucleotides in a second end region near the 3' end and of an opposite strand of the degraded double-stranded amplicon, and wherein: i) the first end region comprises a gap opposite the first single-stranded universal primer binding site of the amplicon, wherein the gap is flanked by a nucleotide comprising a 5'-phosphate and a nucleotide comprising a 3' blocking group, wherein the nucleotide comprising the 5'-phosphate is 3' to the gap, and the nucleotide comprising the 3' blocking group is 5' to the gap; and ii) the second end region comprises a
  • nucleotide comprising the 3' blocking group of the first end region is covalently linked to a polynucleotide of at least 6 to 12 nucleotides in length that is hybridized to the first strand; and ii) the nucleotide comprising the 3' blocking group of the second end region is covalently linked to a polynucleotide of at least 6 to 12 nucleotides in length that is hybridized to the opposite strand.
  • the present invention provides a construct comprising: a first nucleic acid (1) and second nucleic acid (2) hybridized to a third nucleic acid (3) at positions that result in a gap (4) between a 3' nucleotide of the first nucleic acid (5) and a 5' nucleotide of the second nucleic acid (6), wherein the 3' nucleotide comprises a 3' blocking group and the 5' nucleotide comprises a 5' phosphate, and wherein the third nucleic acid comprises a first single-stranded universal primer binding site (7); and a fourth nucleic acid (8) hybridized to the second nucleic acid at positions that result in a gap (9) between a 3' nucleotide of the fourth nucleic acid (10) and a 5' nucleotide of the third nucleic acid (11), wherein the 3' nucleotide of the fourth nucleic acid comprises a 3' blocking group and the 5' nucleotide
  • the present invention provides a reaction mixture comprising any one of the foregoing selectively degraded double-stranded amplicons or constructs, wherein the reaction mixture further comprises: i) a first universal primer hybridized to the first single-stranded universal primer binding site, wherein a 3'-most nucleotide of the first universal primer is adjacent to the 5' phosphate of the second nucleic acid; and ii) a second universal primer hybridized to the second single-stranded universal primer binding site, wherein a 3'-most nucleotide of the second universal primer is adjacent to the 5' phosphate of the third nucleic acid.
  • the present invention provides a reaction mixture comprising a selectively- degraded double-stranded amplicon having a first single-stranded universal primer binding site of at least 4-30 nucleotides at a first end region near a 3' end of a first strand and a second single- stranded universal primer binding site of at least 4-30 nucleotides at a second end region near a 3' end and on an opposite strand of the degraded double-stranded amplicon, and wherein: i) the first strand comprises a 5'-phosphate at the 5' end, wherein a 3' end of the opposite strand extends further than the 5' end of the first strand; and ii) the opposite strand comprises a 5'-phosphate at the 5' end, wherein a 3' end of the first strand extends further than the 5' end of the opposite strand, wherein the reaction mixture further comprises: i) a first universal primer hybridized to the
  • the present invention provides a method comprising: i) providing any one of the foregoing reaction mixtures; ii) ligating the first universal primer to the adjacent 5'- phosphate; and iii) ligating the second universal primer to the adjacent 5'-phosphate.
  • the present invention provides a method for enriching a region of interest of a target polynucleotide from a sample, the method comprising: a) amplifying the target polynucleotide from the sample with a first and a second DB primer, wherein the first DB primer comprises a structure according to any one of the foregoing DB primers and the second DB primer comprises a structure according to any one of foregoing DB primers, thereby producing a selectively degradable double-stranded amplicon comprising the SDNs of the DB primers; b) degrading the SDNs of the degradable double-stranded amplicon to produce a degraded double-stranded amplicon comprising a first single-stranded universal primer binding site near a 3' end of a first strand of the degraded double-stranded amplicon and a second single-stranded universal primer binding site near a 3' end of
  • the universal primer pair comprises the first and second universal primers. In some embodiments, the universal primer pair comprises a third universal primer and a fourth universal primer. In some embodiments, the method comprises: i) combining all reagents for a)-e) together at the same time in a single reaction mixture and performing the amplifying of a), the degrading of b), the hybridizing of c), the ligating of d) and the amplifying of e) in the single reaction mixture; ii) combining the reagents for a) into a DB primer amplification reaction mixture and performing the amplifying of a) in the DB primer amplification reaction mixture, and then combining all the reagents for b)-e) into a single reaction mixture and performing the degrading of b), the hybridizing of c), the ligating of d) and the amplifying of e) in the single reaction mixture; or iii) combining the reagents for a
  • the method comprises: i) combining all reagents for a)-e) together at the same time in a single reaction mixture and performing the amplifying of a), the degrading of b), the hybridizing of c), the ligating of d) and the amplifying of e) in the single reaction mixture; ii) combining the reagents for a) into a DB primer amplification reaction mixture and performing the amplifying of a) in the DB primer amplification reaction mixture, and then combining all the reagents for b)-e) into a single reaction mixture and performing the degrading of b), the hybridizing of c), the ligating of d) and the amplifying of e) in the single reaction mixture; or iii) combining the reagents for a)-d) into a first reaction mixture and performing the amplifying of a), the degrading of b), the hybridizing of c), and the ligating
  • the present invention provides a method for enriching a region of interest of a target polynucleotide from a sample, the method comprising: a) providing a reaction mixture comprising the sample, wherein the target polynucleotide in the sample is double-stranded, and a DB amplification primer pair comprising a first and a second DB primer, wherein the first and second DB primers hybridize to opposite strands of the double-stranded target polynucleotide and flank the region of interest under hybridization conditions, and wherein the first DB primer comprises a structure according to any one of the foregoing DB primers, and the second DB primer comprises a structure according to any one of the foregoing DB primers, wherein the loop region of the first or second DB primer optionally comprises a molecular barcode; b) heating the reaction mixture to melt the double-stranded target polynucleotide and then cooling the reaction mixture to thereby hybridize
  • the 5' arm of the degraded first and second DB primer extension products are blocked from extension by a polymerase.
  • the block comprises a 3' terminal phosphate of the 5' arm of the degraded DB primer extension products.
  • the first or second universal primers comprise a 5' region having a second barcode, and the ligating of g) incorporates the second barcode into the enriched target polynucleotide.
  • the 5' arm of the degraded first and second DB primer extension products do not hybridize to the target polynucleotide under the hybridization conditions of f).
  • the selectively degrading of e) comprises contacting the selectively degradable double-stranded polynucleotide with an enzyme selected from the group consisting of APE I, Endo III, Endo IV, Endo V, DNA glycosylase-lyase Endonuclease VIII, Fpg, hOGGI, hNEILl, T7 Endo I, T4 PDG, Uracil DNA glycosylase (UDG), Afu, and a mixture of UDG or Afu and the DNA glycosylase-lyase Endonuclease VIII, or a combination thereof.
  • the selectively degrading of e) the introducing and hybridizing of f), and the ligating of g) are performed simultaneously in the same reaction mixture.
  • the present invention provides a method for enriching a plurality of different regions of interest from a plurality of different target polynucleotides from a sample, the method comprising performing any one of the foregoing methods with a plurality of different DB amplification primer pairs and the sample comprising the plurality of different target polynucleotides, thereby enriching regions of interest from the sample.
  • the method comprises enriching at least 1,000 different regions of interest from the sample with at least 1,000 different DB amplification primer pairs.
  • the method comprises enriching at least 10,000 different regions of interest from the sample with at least 10,000 different DB amplification primer pairs.
  • the present invention provides a method for enriching a target polynucleotide from a sample, the method comprising: a) amplifying the target polynucleotide from the sample with: i) a DB forward primer comprising at least two SDNs and a DB reverse primer comprising at least two SDNs, wherein the DB forward primer comprises a structure according to any one of the foregoing DB primers, and the DB reverse primer comprises a structure according to any one of the foregoing DB primers; and ii), a forward universal primer and a reverse universal primer, wherein the forward universal primer hybridizes under hybridizing conditions to a forward universal primer binding site positioned within the loop region of the DB forward primer and the reverse universal primer hybridizes under hybridizing conditions to a reverse universal primer binding site positioned within the loop region of the DB reverse primer; thereby producing a selectively degradable double-stranded amplicon comprising the SDNs of the DB primer
  • the present invention provides a method for enriching a target polynucleotide from a sample, the method comprising: a) providing a reaction mixture comprising: i) the sample, wherein the target polynucleotide in the sample is double stranded; ii) a DB
  • amplification primer pair comprising a first and a second DB primer, wherein the first and second DB primer hybridize to opposite strands of the double-stranded target polynucleotide and flank a region of interest, and wherein each of the first and second DB primer comprise a structure according to any one of the foregoing DB primers, wherein the loop region of the first or second DB primer optionally comprises a molecular barcode; and iii) a first and second universal primer, wherein the first and second universal primers hybridize under hybridizing conditions to a first and second universal primer binding site positioned within the loop region of the first and second DB primer respectively; b) heating the reaction mixture to melt the double-stranded target polynucleotide and then cooling the reaction mixture to thereby hybridize the first and second DB primer to the opposite strands of the target polynucleotide; c) extending the hybridized first and second DB primers with a polymerase; d) repeating the melting and
  • the present invention provides a method for creating a sequencing library from multiple samples, the methods comprising: a) performing any one of the foregoing methods on a plurality of different samples, wherein the method is performed in a separate sample reaction mixture for each sample, wherein each sample reaction mixture comprises a sample barcode and each sample barcode comprises a different sequence for each different sample reaction mixture, and the method further comprises pooling different sample reaction mixtures after amplification with DB primer pairs, after universal primer ligation, or after universal amplification.
  • each sample reaction mixture comprises a universal primer comprising the sample barcode
  • the method comprises pooling different sample reaction mixtures after universal primer ligation, or after universal amplification.
  • each sample reaction mixture comprises a DB primer comprising the sample barcode
  • the method comprises pooling different sample reaction mixtures after amplification with DB primer pairs.
  • each sample reaction mixture comprises a DB primer comprising the sample barcode, and the method comprises pooling different sample reaction mixtures after amplification with DB primer pairs and before universal amplification.
  • FIG. 1 illustrates a method for fast target enrichment using a pair of degradable bubble primers (a DB primer pair), each having target-specific 3' arms and 5' arms (green/blue) and a loop region (red/purple) between the arms.
  • the loop regions contain one or more selectively degradable nucleotides (herein deoxyuracil or "U” but more generally "SDN”).
  • the loop regions can also contain a barcode ("NNN").
  • a pair of DB primers are incubated in a reaction mixture containing target polynucleotide and a uracil-tolerating DNA polymerase such as PFUCX, and subjected to DB primer PCR amplification conditions to generate DB primer amplicons in a first bubble primer amplification phase (Phase I: BP).
  • the DB primer amplicons are subject to selective degradation, in which single-stranded universal primer binding sites are exposed in the selectively degraded amplicons (USER to degrade U_BP), and universal primer hybridization and ligation (UP Lig).
  • the selective degradation and universal primer hybridization and ligation reagents can be present in the reaction mixture at the same time as depicted here, or introduced separately.
  • the selective degradation can and universal primer hybridization or hybridization and ligation can be performed simultaneously or sequentially by, e.g., controlling the reaction mixture temperature.
  • One or more universal primers can contain a sample barcode.
  • universal amplification is performed with a molar excess of universal amplification primers.
  • FIG. 2 illustrates an alternative embodiment in which the first phase of DB primer PCR amplification is performed in the presence of universal primers (Phase I: U_BP/UP amplification), and selectively degradable nucleotides are then removed, thereby removing DB primers from the reaction mixture (USER to degrade U_BP). Following selective degradation, amplicons are amplified by universal amplification.
  • Phase I U_BP/UP amplification
  • FIG. 3 illustrates additional detail regarding use of a pair of DB primers to amplify a target polynucleotide region of interest and thereby produce a DB primer amplicon (bottom figure in bracketed region).
  • the DB primer amplicon is degraded to produce a degraded DB primer amplicon having exposed single-stranded universal primer binding sites at each end.
  • FIG. 4 illustrates annealing of universal primers to the exposed single-stranded universal primer binding sites at each end of the degraded DB primer amplicon and ligation of the universal primers to generate a ligated DB primer amplicon. Amplification of the ligated DB primer amplicon with a molar excess of forward and reverse universal primers can result in the amplicon depicted at bottom. [0040] FIG.
  • FIG. 5 illustrates a target polynucleotide (top), a DB primer amplicon (2 nd from top), a degraded DB primer amplicon (3 rd from top), a ligated DB primer amplicon (4 th from top), a DB primer (5 th from top), and a degraded DB primer (bottom).
  • FIG. 6 illustrates product efficiency of different fast target enrichment strategies.
  • primers were not degraded prior to, during, or after universal amplification.
  • DB primers and universal primers were present in the reaction mixture at the same time (see method illustrated in Fig. 2). Competition was reduced relative to the method used in lane 1 due to the selective degradation.
  • selective degradation of DB primer amplicons was performed after an initial amplification with DB primers, thereby exposing single-stranded universal primer binding sites. Universal primers then hybridized to the exposed universal primer binding sites and were ligated. Universal amplification was then performed (see method illustrated in Fig. 1).
  • T4 Ligase (3) T4 Ligase (3)
  • Taq Ligase (4) Ampligase (5)
  • 9N Ligase (6) T4 Ligase and Taq Ligase demonstrated the highest product efficiency.
  • a color coded system was used to correspond to the approximate location of the universal amplification product for each gene (arrow head) and identify the combination of oligos and size of expected gene (Table)
  • FIG. 7 illustrates the effect of different reaction conditions on amplification product yield for methods of the present invention that employ a universal primer ligation step.
  • sample 4 is a reaction mixture containing 0.2 pmol of bubble primer at 65°C annealing temperature for 10 min. At 65°C, there is an increase in yield that is comparable to 1 pmol bubble primer at 5 min (sample 5). Uniformity between gene sets is also observed when compared to 56°C (sample 2) and 60°C (sample 3). Sample 2 demonstrates that at 0.2 pmol bubble primer at 56°C for 10 has higher efficiency compared to 5 min incubations (sample 1). The significant target enrichment at lower bubble primer concentrations (e.g., sample 4) indicates that more primer sets can be accommodated.
  • FIG. 8 illustrates an embodiment of a construct produced by selectively degrading a DB primer amplicon.
  • the construct contains a first nucleic acid (1) and a second nucleic acid (2) hybridized to a third nucleic acid (3) at positions that result in a gap (4) between the 3'-most nucleotide of the first nucleic acid (5) and the 5'-most nucleotide of the second nucleic acid (6), wherein the 3'-most nucleotide of the first nucleic acid contains a 3' blocking group and the 5'-most nucleotide of the second nucleic acid contains a 5' phosphate.
  • the gap produces a first single- stranded universal primer binding site (7) on the third nucleic acid.
  • the construct can further contain a fourth nucleic acid (8) hybridized to the second nucleic acid at a positions that results in a gap (9) between the 3'-most nucleotide of the fourth nucleic acid (10) and a 5'-most nucleotide of the third nucleic acid (11).
  • the 3'-most nucleotide of the fourth nucleic acid can contain a 3' blocking group and the 5'-most nucleotide of the third nucleic acid can contain a 5' phosphate.
  • the gap produces a second single-stranded universal primer binding site (12) on the second nucleic acid.
  • DB primers degradable bubble primers
  • methods of their use and products of polymerase-mediated extension of DB primers.
  • DB primers are oligonucleotide primers that contain one or more, preferably two or more, selectively degradable nucleotides (SDNs).
  • SDNs selectively degradable nucleotides
  • a DB primer can, e.g., be hybridized to a target polynucleotide template and extended by a polymerase, thereby generating a DB primer extension product that contains one or more SDNs.
  • a pair of DB primers can be used in a polymerase chain reaction (PCR) to produce a double-stranded amplification product ("a DB primer amplicon") that contains one or more SDNs from each DB primer of the pair.
  • a DB primer amplicon a double-stranded amplification product
  • the one or more SDNs in the DB primers, DB primer extension products, or DB primer amplicons can be selectively degraded by chemical or enzymatic methods as further described below.
  • DB primers, DB primer extension products, DB primer amplicons, and degradation products thereof can be used, e.g., for high-throughput sequencing library preparation.
  • the DB primers are used in an initial multiplex amplification reaction and then degraded to (i) reduce or eliminate interference with downstream processing and/or (ii) generate single-stranded universal primer binding sites in amplicons resulting from the multiplex DB primer amplification.
  • SDN selectively degradable nucleotide
  • dA deoxyadenosine
  • dC deoxycytidine
  • dG deoxyguanosine
  • dT deoxythymidine
  • the SDNs must be capable of functioning as a template nucleotide in a template-directed and polymerase-mediated primer extension reaction (e.g., a PCR amplification reaction).
  • exemplary SDNs include, but are not limited to, deoxyuridine, deoxyinosine, an oxidized pyrimidine nucleotide, an oxidized purine nucleotide, an alkylated purine, 5-hydroxyuracil, 5-hydroxymethyluracil, and 5- formyluracil.
  • a DB primer can contain any combination of at least one or at least two of the foregoing SDNs. In an exemplary embodiment, the DB primer contains at least two deoxyuridine SDNs. Table 1 lists, for illustration and not limitation, exemplary SDNs and exemplary corresponding degradation conditions. TABLE 1
  • methyltartronylurea oxidized purine nucleotide Fpg, hOGGl, hNEILl, or a
  • an AP site such as:
  • a DB primer contains (from 5' to 3'), a 5' arm, a loop region, and a 3' arm.
  • the loop region can contain a universal primer binding site or complement thereof, and at the least one or two SDNs.
  • the loop region contains a universal primer binding site and at least two SDNs, where the universal primer binding site includes at least one of the at least two SDNs.
  • the 5' and 3' arms may be designed to hybridize to complementary sequences in a target polynucleotide of known sequence in a sample.
  • the loop sequence may be designed to not hybridize to the target polynucleotide (or more specifically, designed to not hybridize to a region of the target polynucleotide that lies between the above-noted complementary sequences).
  • a pair of DB primers may hybridize to opposite strands of a double-stranded target polynucleotide.
  • each strand may be referred to as a "template strand" in relation to the DB primer annealed to that strand.
  • reference to a pair of DB primers hybridizing to a double-stranded target polynucleotide may refer to a first DB primer hybridizing to one strand of the target polynucleotide (first template strand) and to a second DB primer hybridizing to a second strand of the target polynucleotide (second template strand).
  • a double-stranded DB primer amplicon can be viewed as containing two "template strands," each of which comprises a sequence of one DB primer, and the compliment of another DB primer.
  • Removal of an SDN from a single-stranded DB primer breaks the contiguous chain of nucleotides to produce at least two oligonucleotide fragment products.
  • the length of the 5' and 3' arms, and the relative position(s) of the one or more SDNs are selected such that, after degradation of the SDNs, one or more, or all, of the oligonucleotide fragment products do not efficiently hybridize to the target polynucleotide under primer extension conditions (e.g., about 60- 80 °C).
  • the selective degradation is performed by chemical or enzymatic methods that produce one or more "blocked" ends in oligonucleotide fragment products.
  • blocked ends include, e.g, a 3' phosphate, a 3' ring-opened sugar such as a 3 ' -phospho-a, ⁇ -unsaturated aldehyde (PA), or a 3' phosphate ester.
  • blocked ends cannot be extended by a polymerase even when hybridized to a template polynucleotide. Thus, selective degradation can be used to remove excess DB primers from a reaction mixture and reduce interference with downstream processes.
  • selective degradation of SDNs in a double-stranded amplicon produced by PCR amplification of a target polynucleotide with a pair of DB primers can be used to remove of one or more SDNs from the amplicon. Such selective degradation can produce a single-stranded site at the position of the removed SDN.
  • multiple SDNs can be selectively removed from a DB primer amplicon to produce single-stranded region(s) that include the positions of the removed SDNs and the position of intervening polynucleotide fragments that do not contain SDNs, provided that such intervening fragments have an annealing temperature that is below the temperature of the reaction mixture.
  • selective degradation of a pair of SDNs in a double-stranded amplicon can result in loss of one or more single-stranded oligonucleotide(s) lying between SDN sites.
  • selective degradation of a DB primer amplicon can be used to expose a single-stranded region of a template strand that contains a universal primer binding site.
  • a universal primer binding site, or complement thereof can be incorporated into a loop region of a DB primer or a pair of DB primers.
  • PC amplification with a pair of target-specific DB primers, each containing such a universal primer binding site or its complement can then incorporate this feature into the resulting double-stranded amplicon.
  • the single-stranded universal primer binding sites are exposed, as illustrated in FIG. 1 and FIG. 3.
  • the universal primer binding site can, in turn, be used for universal amplification of a DB primer amplicons by, e.g., PCR using a pair of universal PCR primers that hybridize to the exposed universal primer binding sites, as illustrated in FIG. 1, 2, and 4.
  • the use of DB primers to generate amplicons having universal primer binding sites provides certain advantages over other methods such the use of primers having 5' universal primer binding site tails.
  • the DB primers can be selectively degraded into fragments that are too short to support PCR amplification.
  • DB primers can be used to support target-specific nested universal amplification of target polynucleotides, and non-extended primers may be degraded to reduce or eliminate interference with downstream processes.
  • combinations of DB primers can be used for multiplex amplification of a large number of target polynucleotide regions in a single reaction tube.
  • DB primers can be used to amplify from 1 to 10; 1 to 10,000; 10 to 15,000; 10 to 50,000; 10 to 100,000; 1,000 to 10,000; 1,000 to 15,000; 1,000 to 50,000; 1,000 to 100,000; or more target polynucleotide regions in a single reaction tube.
  • the amplification of a large number of target polynucleotide regions of interest from a sample and preparation of sequencing libraries containing such regions of interest can be useful for, e.g., genome-wide, exome-wide, or transcriptome-wide nucleic acid sequence analysis, analysis of target organism populations, or analysis of environmental samples.
  • DB primer amplification can be useful in any sequencing library preparation method in which it is desirable to reduce sample input requirements, reduce library preparation time, increase the specificity of the library preparation [e.g., as indicated by the percent of on-target reads produced in a subsequent sequencing step), increase the sensitivity of the sequencing analysis (e.g., as indicated by the lower limit of detection for specific sequences), or a combination thereof.
  • DB primer amplification is compatible with high-throughput sequencing platforms including, but not limited to sequencing by ligation (e.g., combinatorial probe anchor ligation (cPAL)), or sequencing by synthesis, methods known in the art. It will be recognized that sequencing libraries having essentially any desired adaptor sequences may be prepared using the DB primer system.
  • the DB primers can be used to amplify one or more target polynucleotides from a sample.
  • the target polynucleotides in the sample can be double or single-stranded, or may contain portions of both double-stranded and single-stranded regions.
  • target polynucleotides in the sample can be single- or double-stranded genomic DNA, single- or double-stranded cDNA, m NA, or a DNA/RNA hybrid (e.g., mRNA hybridized to first strand cDNA).
  • the target polynucleotides are genomic DNA.
  • Sample containing target polynucleotides can be obtained from any suitable source.
  • the sample can be obtained or provided from any organism of interest.
  • organisms include, for example, plants; animals (e.g., mammals, including humans and non-human primates); or pathogens, such as bacteria and viruses.
  • the sample can be, or can be obtained from, cells, tissue, or polynucleotides of a population of such organisms of interest.
  • the sample can be an environmental sample, such as a sample of water, air, or soil.
  • Samples from an organism of interest, or a population of such organisms of interest can include, but are not limited to, samples of bodily fluids (including, but not limited to, blood, urine, serum, lymph, saliva, anal and vaginal secretions, perspiration and semen); cells; tissue; biological warfare agent samples; research samples (e.g., products of nucleic acid amplification reactions, such as PCR amplification reactions); purified samples, such as purified genomic DNA; RNA preparations; and raw samples (bacteria, virus, genomic DNA, etc.).
  • target polynucleotides e.g., genomic DNA
  • target polynucleotides comprise genomic DNA.
  • target nucleic acids comprise a subset of a genome (e.g., a subset of interest for a particular application, e.g., selected regions of the genome that may harbor mutations in a particular subset of a population such as individuals predisposed to cancer).
  • target nucleic acids comprise exome DNA.
  • target nucleic acids comprise all or part of a transcriptome.
  • target nucleic acids comprise all or part of a methylome, i.e., the population of methylated sites and the pattern of methylation in a genome or in a particular cell.
  • target polynucleotides are processed by fragmentation to produce fragments of one or more specific sizes or to produce a population of fragments having a narrow distribution of fragment lengths. Any method of fragmentation can be used.
  • the target nucleic acids are fragmented by mechanical means (e.g., ultrasonic cleavage, acoustic shearing, needle shearing, nebulization, or sonication); by chemical methods; or by enzymatic methods (e.g., using endonucleases). Methods of fragmentation are known in the art; see e.g., US 2012/0004126.
  • fragmentation is accomplished by ultrasound (e.g., Covaris or Sonicman 96-well format instruments).
  • target polynucleotides or fragmented target polynucleotides are subjected to a size selection step to obtain nucleic acid fragments having a certain size or distribution of sizes.
  • a size selection step to obtain nucleic acid fragments having a certain size or distribution of sizes. Any methods of size selection can be used.
  • fragmented target polynucleotides are separated by gel electrophoresis and the band or region corresponding to a fragment size or range of sizes of interest is extracted from the gel.
  • a spin column can be used to select for fragments having a certain minimum size.
  • paramagnetic beads can be used to selectively bind DNA fragments having a desired range of sizes.
  • solid-phase reversible immobilization (SPRI) methods can be used to enrich a sample for fragments having a certain size or distribution of sizes.
  • a combination of size selection methods can be used.
  • target polynucleotides e.g., genomic DNA
  • the methods, compositions, and kits described herein can be used with very large target polynucleotides, at least in part because multiple different primer pairs can target different regions of a single target polynucleotide.
  • target polynucleotides need not be subject to an active fragmentation step.
  • target polynucleotides are not fragmented, not nebulized, not sheared (e.g., hydrodynamically sheared, chemically sheared, or acoustically sheared), not sonicated, not fragmented with a non-specific nuclease (e.g., DNase I) or a restriction nuclease (e.g., a 4-cutter), or not fragmented with a transposase (e.g., tagmentase).
  • a non-specific nuclease e.g., DNase I
  • a restriction nuclease e.g., a 4-cutter
  • transposase e.g., tagmentase
  • target polynucleotides or fragmented target polynucleotides are about 50 to about 2000 bases in length, e.g., from about 50 to about 600 bases in length, from about 300 to about 1000 bases in length, from about 300 to about 600 bases in length, or from about 200 to about 2000 bases in length.
  • the target polynucleotides or fragmented target polynucleotides are 10-100, 50-100, 50-200, 50-300, 100-200, 200-300, 50-400, 100-400, 200- 400, 400-500, 400-600, 500-600, 50-1000, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 600- 1000, 700-1000, 700-900, 700-800, 800-1000, 900-1000, 1500-2000, or 1750-2000 bases in length.
  • At least 25%, 50%, 75%, or 90% of target polynucleotides or fragmented target polynucleotides in a sample are about 50 to about 2000 bases in length, e.g., from about 50 to about 600 bases in length, from about 300 to about 1000 bases in length, from about 300 to about 600 bases in length, or from about 200 to about 2000 bases in length.
  • At least 25%, 50%, 75%, or 90% of target polynucleotides or fragmented target polynucleotides in a sample are 10-100, 50-100, 50-200, 50-300, 100-200, 200- 300, 50-400, 100-400, 200-400, 400-500, 400-600, 500-600, 50-1000, 100-1000, 200-1000, 300-1000, 400-1000, 500-1000, 600-1000, 700-1000, 700-900, 700-800, 800-1000, 900-1000, 1500-2000, or 1750-2000 bases in length.
  • the target polynucleotides or fragmented target polynucleotides [e.g., genomic DNA) have a mean length of about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, or about 2000 bases in length.
  • genomic DNA e.g., genomic DNA
  • a DB primer comprises a 5' arm that can hybridize to a target polynucleotide in a sequence-specific manner and a 3' arm that can hybridize to the target polynucleotide in a sequence-specific manner, such that the 3' end of the 3' arm can be extended in a template- dependent and polymerase-mediated primer extension reaction.
  • the sequences of the 5' arm and 3' arm are selected to function additively or synergistically for target-specific hybridization to a target polynucleotide sequence of interest when linked together via the loop region.
  • the sequence of the 3' arm can be selected to hybridize to a first region of a target polynucleotide of interest and the sequence of the 5' arm can be selected to hybridize to a second region of a target polynucleotide of interest, wherein the first region of the target polynucleotide is 5' to the second region of the target polynucleotide.
  • sequences of the 5' arm and 3' arm are selected such that the first and second regions of the target polynucleotide are contiguous [i.e., the 5'-most nucleotide of the DB primer 3' arm and the 3'-most nucleotide of the DB primer 5' arm are positioned adjacent to each other when hybridized to the target polynucleotide).
  • sequences of the 5' arm and 3' arm are selected such that the first and second regions of the target polynucleotide are separated by a gap.
  • the length of the gap can be as long or short as desired, provided that a target-specific DB primer having a sufficient length and sequence between the 5' arm and 3' arm to bridge the gap can be synthesized and provided in practical quantity and purity.
  • the length of the gap is constrained by the length of a loop region positioned between the 5' arm and 3' arm of the DB primer.
  • the gap is no longer than or is shorter than the length of the loop region.
  • the loop region of a DB primer can bridge a loop of the target polynucleotide.
  • the gap length in nucleotides in the target polynucleotide can be longer than the length in nucleotides of the loop region.
  • sequences of the 5' arm and 3' arm are selected such that the first and second regions of the target polynucleotide are separated by a gap of from 0 (i.e., no gap) to no more than 100 contiguous nucleotides in length.
  • the gap is from 0 to no more than 70 contiguous nucleotides in length, from 0 to no more than 50 contiguous nucleotides in length, from 0 to no more than 30 contiguous nucleotides in length, from 0 to no more than 20 contiguous nucleotides in length, from 0 to no more than 15 contiguous nucleotides in length, or from 0 to no more than 10 contiguous nucleotides in length.
  • the gap is from 1 to no more than 70 contiguous nucleotides in length, from 1 to no more than 50 contiguous nucleotides in length, from 1 to no more than 30 contiguous nucleotides in length, from 1 to no more than 20 contiguous nucleotides in length, from 1 to no more than 15 contiguous nucleotides in length, or from 1 to no more than 10 contiguous nucleotides in length.
  • the gap is, is about, is at least, or is at least about, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, or 50 contiguous nucleotides in length.
  • the 5' arm and 3' arm of the DB primer can selected to have any suitable total combined length and sequence for target-specific hybridization to a target polynucleotide in a reaction mixture containing both target and non-target polynucleotides.
  • the combined length of the 5' arm and 3' arm together can be from 15 to 100 nucleotides or more, from 18 to 100 nucleotides, from 20 to 100 nucleotides, from 25 to 100 nucleotides, from 30 to 100 nucleotides, from 35 to 100 nucleotides, from 40 to 100 nucleotides, from 50 to 100 nucleotides, from 60 to 100 nucleotides, from 15 to 75 nucleotides, from 18 to 75 nucleotides, from 20 to 75 nucleotides, from 25 to 75 nucleotides, from 30 to 75 nucleotides, from 35 to 75 nucleotides, from 40 to 75 nucleotides, from 50 to 75 nucleotides, from 60 to 75 nucleotides, from 18 to 50 nucleotides, from 20 to 50 nucleotides, from 25 to 50 nucleotides, from 30 to 50 nucleotides, from 35 to 50 nucleotides, from 40 to 50 nucleo
  • the 5' arm can be any suitable length from about 5 to about 50 nucleotides, from about 6 to about 50 nucleotides, from about 7 to about 50 nucleotides, from about 8 to about 50 nucleotides, from about 9 to about 50 nucleotides, from about 10 to about 50 nucleotides, from about 11 to about 50 nucleotides, from about 12 to about 50 nucleotides, from about 13 to about 50 nucleotides, from about 14 to about 50 nucleotides, from about 15 to about 50 nucleotides, from about 16 to about 50 nucleotides, from about 17 to about 50 nucleotides, from about 18 to about 50 nucleotides, from about 19 to about 50 nucleotides, or from about 20 to about 50 nucleotides.
  • the 5' arm is, is about, is at least, or is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • the length and sequence of the 5' arm is selected such that, after selectively degrading the one or more SDNs of a DB primer, the 5' arm, or in the case of a 5' arm that contains SDNs, the oligonucleotide fragments of the 5' arm resulting from degradation of the 5' arm, has an annealing temperature that is too low for efficient hybridization and polymerase-mediated extension under universal PC amplification conditions.
  • the 5' arm, or fragments thereof can be less than about 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
  • the 5' arm, or fragments thereof can have a melting temperature with respect to a perfectly complementary target polynucleotide of less than about 45, 50, 55, 60, 65, 70, or 72 °C.
  • the selective degradation can be performed by chemical or enzymatic methods, or the combination thereof, that result in a 3' end of the 5' arm that is blocked from polymerase-mediated extension.
  • the 5' arm does not contain a selectively degradable nucleotide (SDN).
  • SDN selectively degradable nucleotide
  • the 5' arm can consist essentially of nucleotides selected from the group consisting of, A, C, G, and T.
  • the 5' arm can contain various derivatives of A, C, G, and T, or non- natural nucleotides such as locked nucleic acids, that are resistant to the selective degradation method used in a particular DB primer amplification method.
  • the 5' arm can contain one or more SDNs.
  • the SDN is capable of forming a specific Watson-Crick or
  • a 5' arm can contain a deoxyuracil nucleotide that forms a specific base pair with A.
  • the 5' arm can contain a so-called "universal base” SDN such as deoxyinosine.
  • the one or more SDNs of the 5' arm are different from the SDNs of the loop region.
  • the SDNs of the 5' arm can be uracil, while the SDNs of the loop region can be deoxyinosine, or vice versa.
  • the SDNs of the 5' arm are selected to be selectively degraded by a different method as compared to the SDNs of the loop region.
  • the 3' arm can be any suitable length from about 5 to about 50 nucleotides, from about 6 to about 50 nucleotides, from about 7 to about 50 nucleotides, from about 8 to about 50 nucleotides, from about 9 to about 50 nucleotides, from about 10 to about 50 nucleotides, from about 11 to about 50 nucleotides, from about 12 to about 50 nucleotides, from about 13 to about 50 nucleotides, from about 14 to about 50 nucleotides, from about 15 to about 50 nucleotides, from about 16 to about 50 nucleotides, from about 17 to about 50 nucleotides, from about 18 to about 50 nucleotides, from about 19 to about 50 nucleotides, or from about 20 to about 50 nucleotides.
  • the 5' arm is, is about, is at least, or is at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • the length and sequence of the 3' arm is selected such that, after selectively degrading the one or more SDNs of a DB primer, the 3' arm has an annealing temperature that is too low for efficient hybridization and polymerase-mediated extension under universal PC amplification conditions.
  • the 3' arm can be less than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
  • the 3' arm can have a melting temperature with respect to a perfectly complementary target polynucleotide of less than about 45, 50, 55, 60, 65, 70, or 72 °C.
  • the 3' arm does not contain a selectively degradable nucleotide (SDN).
  • SDN selectively degradable nucleotide
  • the 3' arm can consist essentially of nucleotides selected from the group consisting of, A, C, G, and T.
  • the 3' arm can contain various derivatives of A, C, G, and T, or non- natural nucleotides such as locked nucleic acids, that are resistant to, or not removed by, the selective degradation method used to remove one or more SDNs from a DB primer, DB primer extension product, or DB primer amplicon.
  • the 5' arm is longer than the 3' arm.
  • a longer 5' arm can, e.g., be advantageous for use in methods where the 5' arm is blocked from polymerase-mediated extension during selective degradation of one or more SDNs because such methods typically would not also block the 3' arm from polymerase-mediated extension.
  • a target-specific DB primer that has not been extended or substantially extended in a polymerase-mediated reaction can have a relatively short 3' arm, and a 5' arm such that the total length of 3' and 5' arms confers sufficient target-specificity.
  • Such a DB primer can be selectively degraded to produce a fragment that contains the 3' arm and exhibits reduced or eliminated interference with downstream sample processing (e.g., universal amplification) at least in part due to its short length.
  • downstream sample processing e.g., universal amplification
  • the 5' arm of a degraded DB primer or DB primer extension product can exhibit reduced or eliminated interference due to its short length or the presence of a blocked 3' end, or a combination thereof.
  • the 5' arm can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides longer than the length of the 3' arm.
  • the 5' arm can be the same length as the 3' arm, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides shorter than the length of the 3' arm.
  • the DB primer further contains a loop region that is positioned between the 5' arm and the 3' arm and does not hybridize to the target polynucleotide under hybridization, template- dependent primer extension, or DB-primer mediated PC amplification conditions, or a combination thereof.
  • the sequence of the loop region can be selected such that it is not exactly complementary to any expected sequence in a sample containing target polynucleotides. For example, where a sample is a sample of human genomic DNA, the sequence of the loop region can be selected such that it not exactly complementary to more than one region in the human genome.
  • the sequence of the loop region can be selected such that no portion of the loop region is exactly complementary to more than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 contiguous nucleotides of the human genome.
  • the sequence of the loop region can be selected such that it differs in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 positions from any contiguous sequence of human genomic DNA (e.g., any contiguous sequence having the same length as the loop region).
  • the loop region of a DB primer contains at least one SDN, typically at least two SDNs, and often at least 3 SDNs. In some cases, the loop region contains from 1 to 30 SDNs, from 1 to 25 SDNs, from 1 to 20 SDNs, from 1 to 15 SDNs, from 1 to 10 SDNs, from 1 to 5 SDNs, from 1 to 3 SDNs, from 2 to 30 SDNs, from 2 to 25 SDNs, from 2 to 20 SDNs, from 2 to 15 SDNs, from 2 to 10 SDNs, from 2 to 5 SDNs, from 2 to 3 SDNs, from 3 to 30 SDNs, from 3 to 25 SDNs, from 3 to 20 SDNs, from 3 to 15 SDNs, from 3 to 10 SDNs, or from 3 to 5 SDNs.
  • the loop region contains, contains about, contains at least, or contains at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 SDNs.
  • longer loop regions contain more SDNs such that fragments produced after degradation are too short to act as primers or otherwise interfere with downstream processes such as universal amplification.
  • a loop region of 30 nucleotides in length can preferably contain at least three SDNs spaced such that no loop region fragment is greater than about 10-12 nucleotides in length, or at least four SDNs spaced such that no loop region fragment is greater than 8-10 nucleotides in length.
  • a loop region of 4 nucleotides can contain at least two SDNs.
  • the 3'-most nucleotide of the loop region is an SDN.
  • the 5'-most nucleotide of the loop region is an SDN.
  • the length between SDNs is selected such that the products of selective degradation are sufficiently short to reduce or eliminate interference with downstream processes such as universal amplification.
  • two or more SDNs of the loop region may positioned about, or less than about, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides apart.
  • the SDNs of the loop regions are spaced no more than 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides apart.
  • the length of the intervening region between the 5'-most and 3'-most SDN of the loop region is selected to encompass a suitable length for a universal primer to hybridize to the intervening region or its complement, e.g., under T4 DNA ligase reaction conditions
  • thermostable ligase reaction conditions or universal primer amplification conditions, as further described below.
  • suitable lengths between a 5'-most and 3'-most SDNs of the loop region include, but are not limited to, from 2 to 50 nucleotides, from 2 to 40 nucleotides, from 2 to 35 nucleotides, from 2 to 30 nucleotides, from 2 to 25 nucleotides, from 2 to 20 nucleotides, from 2 to 18 nucleotides, from 2 to 15 nucleotides, from 2 to 12 nucleotides, from 2 to 10 nucleotides from 2 to 8 nucleotides, from 2 to 6 nucleotides, from 2 to 4 nucleotides, from 3 to 50 nucleotides, from 3 to 40 nucleotides, from 3 to 35 nucleotides, from 3 to 30 nucleotides, from 3 to 25 nucleotides, from 3 to 20 nucleotides, from 3 to 18 nucleotides, from
  • the 5'-most and 3'-most SDNs of the loop region are separated by 7 to 30, 7 to 25 nucleotides, 7 to 20 nucleotides, 7 to 18 nucleotides, 7 to 15 nucleotides, 7 to 12 nucleotides, or 7 to 10 nucleotides.
  • the nucleotides positioned between the 5'-most and 3'-most SDNs of the loop region are not SDNs.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides positioned between the 5'-most and 3'-most SDNs of the loop region are also SDNs.
  • At least 1 nucleotide positioned between the 5'-most and 3'-most SDNs of the loop region is also an SDN. In some cases, at least 2, or at least 3 nucleotides positioned between the 5'-most and 3'-most SDNs of the loop region are also SDNs. In some cases, at least 1 nucleotide positioned between the 5'-most and 3'-most SDNs of the loop region is not an SDN. In some cases, at least 2, or at least 3 nucleotides positioned between the 5'- most and 3'-most SDNs of the loop region is not an SDN.
  • the length of the loop region can be any suitable length. Typically, the loop region is selected such that the total length of the DB primer is, is less than, or is less than about, 200, 175, 125, 100, 75, 70, 60, 55, 50, 45, 40, 35, 30, or 25 nucleotides.
  • the loop region can have a length of from about 4 to 50 nucleotides, from 4 to 40 nucleotides, from 4 to 35 nucleotides, from 4 to 30 nucleotides, from 4 to 25 nucleotides, from 4 to 20 nucleotides, from 4 to 18 nucleotides, from 4 to 15 nucleotides, from 4 to 12 nucleotides, from 4 to 10 nucleotides from 4 to 8 nucleotides, or from 4 to 6 nucleotides.
  • the loop region has a length of 7 to 30, 7 to 25 nucleotides, 7 to 20 nucleotides, 7 to 18 nucleotides, 7 to 15 nucleotides, 7 to 12 nucleotides, or 7 to 10 nucleotides.
  • the loop region is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.
  • the loop region can contain a universal primer binding site or the complement of a universal primer binding site.
  • "universal primer binding site” has it usual meaning in the art, and refers to a nucleic acid region having a sequence shared amongst all, or a substantial fraction of all, polynucleotides in a reaction mixture that can be hybridized to a universal primer under primer hybridization conditions (e.g., PC primer annealing conditions or ligation conditions).
  • primer hybridization conditions e.g., PC primer annealing conditions or ligation conditions.
  • the universal primer binding site sequence of the loop region (or its complement) is the same in half (or about half) of the DB primers in a reaction mixture and the other half of the DB primers have a different universal primer binding site sequence or its complement.
  • DB primer pairs can together comprise a forward universal primer binding site or its complement and a reverse universal primer binding site or its complement.
  • a single forward universal primer and a single reverse universal primer can be used to amplify any region of interest that is positioned between the forward and reverse universal primer binding sites of different target polynucleotides, independent of the sequence of the region of interest.
  • multiple different sets of universal primers can be utilized to amplify DB primer amplification products containing multiple different sets of universal primer binding sites. Where two different forward and two different reverse universal primers are used, about 1 ⁇ 4 of the DB primer loop regions in a reaction mixture can share the same universal primer binding site sequence.
  • Such universal primer binding sites can be useful for providing a DB primer extension product or DB primer amplicon that can be further amplified by universal PC .
  • DB primers can be used for target-specific selection and/or amplification, e.g., from a complex mixture, and the selected target(s) can be universally amplified in a subsequent or simultaneous step.
  • the universal primer binding site (or its complement) can be the entire loop region sequence, or a portion thereof.
  • the universal primer binding site (or its complement) can include the 3'-most nucleotide of the loop region, the 5'-most nucleotide of the loop region, or the 3'-most and 5'-most nucleotide of the loop region.
  • the 3' most nucleotide of the universal primer binding site (or its complement) is an SDN.
  • the 5'-most nucleotide of the universal primer binding site (or its complement) is an SDN.
  • the 3' end and 5' end of the universal primer binding site (or its complement) are SDNs.
  • the universal primer binding (or its complement) site contains 1, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of the SDNs of the loop region.
  • the universal primer binding site does (or its complement) does not contain an SDN.
  • the loop region contains a universal primer binding site (or its complement) that is positioned between SDNs.
  • the universal primer binding site (or its complement) is selected to have a length sufficient to allow target-specific hybridization of a universal primer.
  • the universal primer binding site can have a length of from 4 to 25 nucleotides, from 4 to 20 nucleotides, from 4 to 18 nucleotides, from 4 to 15 nucleotides, from 4 to 12 nucleotides, from 4 to 10 nucleotides from 4 to 8 nucleotides, or from 4 to 6 nucleotides.
  • the universal primer binding site has a length of 7 to 30, 7 to 25 nucleotides, 7 to 20 nucleotides, 7 to 18 nucleotides, 7 to 15 nucleotides, 7 to 12 nucleotides, or 7 to 10 nucleotides.
  • the universal primer binding site is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, nucleotides in length.
  • the universal primer binding site (or its complement) is selected to have a sequence that is different from the sequence of one or more, or all, target polynucleotide sequences of the same length at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more positions.
  • the loop region can contain a barcode region.
  • the barcode identifies a set of target sequences with a common feature.
  • the barcodes may identify the chromosomal location of the target sequence (e.g., Y-chromosome target sequences can have a common barcode, and X-chromosome target sequences can have a different common barcode).
  • the loop region can contain a molecular barcode that is different for every DB primer molecule.
  • the loop region can contain a cellular barcode that is the same for every DB primer in a single-cell DB primer amplification reaction (e.g., a reaction in which the target polynucleotides in the sample are all from a single cell) but different for DB primers in amplification reactions directed to target polynucleotides from a different cell.
  • a cellular barcode that is the same for every DB primer in a single-cell DB primer amplification reaction (e.g., a reaction in which the target polynucleotides in the sample are all from a single cell) but different for DB primers in amplification reactions directed to target polynucleotides from a different cell.
  • the loop region can contain a sample barcode.
  • target specific amplification with DB primers can be performed in a separate reaction for each sample, and then samples can be combined before, or after, universal primer amplification.
  • DB primer amplification can be used to prepare, e.g., a sequencing library containing multiple samples and/or a multiple single-cell DB primer pair amplified targets while maintaining sample- and/or cell-specific tracking.
  • the barcode region can be 3' of a universal primer binding site (or its complement).
  • the loop region can contain two or three different barcode regions, e.g., two or three different barcode regions that are both 3' of a universal primer binding site (or its complement).
  • the loop region can contain a cell and a molecular barcode.
  • the loop region can contain a cell barcode, a molecular barcode, and a sample barcode.
  • the barcode region is about 3-12 nucleotides in length, or 3-5 nucleotides in length. In some cases, each barcode of the barcode region is about 3-12 nucleotides in length, or 3-5 nucleotides in length.
  • two different DB primers can be used as a DB primer pair for target-specific PC amplification.
  • the two different DB primers can be selected to have 5' and 3' arms that hybridize to different regions of a double-stranded target polynucleotide, such that the 3' arms hybridize to opposite strands of the double-stranded target polynucleotide and flank a region of interest (i.e., a region of genomic DNA to be sequenced).
  • the 5' arm of the first DB primer and the 5' arm of the second DB primer generally have different sequences.
  • the 3' arms have different sequences.
  • the loop regions are the DB primer pair are the same sequence, or are the same sequence except for within one or more barcode regions. While the above description of DB primer pairs refers to a double-stranded target polynucleotide, such DB primer pairs can be used to amplify a single-stranded target polynucleotide. In such cases, only one DB primer of the DB primer pair would hybridize and be extended in a first cycle of PC amplification to produce a hybridization substrate for the second DB primer of the pair.
  • a plurality of different DB primer pairs can be provided for simultaneous target-specific amplification of a plurality of target polynucleotide regions of interest.
  • composition, reaction mixture, or kit can be provided that contains from 1 to 50,000 or more DB primer pairs.
  • the composition, reaction mixture, or kit contains from 2 to 40,000, from 2 to 30,000; from 2 to 25,000; from 2 to 20,000; from 2 to 15,000; from 2 to 10,000; from 2 to 7,500; from 2 to 5,000; from 2 to 2,500; from 2 to 1,000; from 2 to 500; from 2 to 250; from 2 to 200; from 2 to 150; from 2 to 125; from 2 to 100; from 2 to 75; from 2 to 50; from 2 to 25, or from 2 to 10 different DB primer pairs.
  • the composition, reaction mixture, or kit contains from 10 to 40,000, from 10 to 30,000; from 10 to 25,000; from 10 to 20,000; from 10 to 15,000; from 10 to 10,000; from 10 to 7,500; from 10 to 5,000; from 10 to 2,500; from 10 to 1,000; from 10 to 500; from 10 to 250; from 10 to 200; from 10 to 150; from 10 to 125; from 10 to 100; from 10 to 75; from 10 to 50; from 10 to 25, or from 10 to 20 different DB primer pairs.
  • the composition, reaction mixture, or kit contains, contains about, contains at least, or contains at least about, 10; 20; 30; 40; 50; 75; 100; 200; 250; 300; 400; 500; 750 ;1,000; 2,000; 3,000; 4,000; 5,000; 7,500; 10,000; 15,000; or more different DB primer pairs.
  • the plurality of different DB primer pairs is a panel of DB primer pairs directed to a specific subset of target polynucleotides.
  • the DB primer pairs can be a cancer panel directed to, e.g., genomic, regions of DNA known to affect cancer risk of an organism.
  • the DB primer pairs can be an exome or transcriptome panel directed to a substantial portion, or all, of the exome sequences or transcriptome sequences of an organism of interest.
  • the DB primer pairs can be a panel of DB primer pairs for amplification of nucleic acids that indicate the presence of a pathogen, or a group of pathogens, and/or virulence markers associated with such a pathogen or group of pathogens.
  • the present invention provides DB primer extension products.
  • DB primer extension products are illustrated in, e.g., FIG. 3 and 5.
  • Such DB primer extension products can be produced by selectively hybridizing a DB primer to a target polynucleotide and incubating the hybridized DB primer under polymerization conditions (e.g., PCR amplification conditions).
  • polymerization conditions e.g., PCR amplification conditions.
  • DB primer extension products have all the features of the DB primers described above, except that the 3' arm of the DB primer is an extended 3' arm. Accordingly, after selective degradation of SDNs, extended 3' arms of DB primer extension products can hybridize to complementary polynucleotides (e.g., complementary target polynucleotides or complementary primer extension products).
  • 3' arms of non-extended and selectively degraded DB primers can, in some cases, be too short to efficiently hybridize to target polynucleotides or interfere with downstream processes (e.g., universal amplification) (FIG. 5, bottom).
  • DB primer extension products can be any length that can be produced under PC amplification conditions.
  • Typical DB primer extension products include, but are not limited to, an extension product that is from about 1 nucleotide to about 10,000 nucleotides longer than the initial DB primer hybridized to the target polynucleotide. In some cases, the DB primer extension product is from 10 to 1,000 nucleotides longer than the initial DB primer hybridized to the target
  • the DB primer extension product is from 10 to 500 nucleotides longer than the initial DB primer hybridized to the target polynucleotide.
  • the DB primer extension product is from 25 to 10,000; from 25 to 5,000; from 25 to 3,000; from 25 to 2,000; from 25 to 1,500; from 25 to 1,000; from 25 to 500; from 25 to 300; from 25 to 250; from 25 to 200; from 25 to 175; from 25 to 150; from 25 to 125; from 25 to 100; from 50 to 10,000; from 50 to 5,000; from 50 to 3,000; from 50 to 2,000; from 50 to 1,500; from 50 to 1,000; from 50 to 500; from 50 to 300; from 50 to 250; from 50 to 200; from 50 to 175; from 50 to 150; from 50 to 125; from 50 to 100; or from 50 to 75 nucleotides longer than the initial DB primer hybridized to the target polynucleotide.
  • the DB primer extension products are from 50 to 10,000; from 50 to 5,000; from 50 to 3,000; from 50 to 2,000; from 50 to 1,500; from 50 to 1,000; from 50 to 500; from 50 to 300; from 50 to 250; from 50 to 200; from 50 to 175; from 50 to 150; or from 50 to 100 nucleotides in length.
  • the DB primer extension products are from 75 to 10,000; from 75 to 5,000; from 75 to 3,000; from 75 to 2,000; from 75 to 1,500; from 75 to 1,000; from 75 to 500; from 75 to 300; from 75 to 250; from 75 to 200; from 75 to 175; from 75 to 150; or from 75 to 100 nucleotides in length.
  • the DB primer extension products are from 100 to 10,000; from 100 to 5,000; from 100 to 3,000; from 100 to 2,000; from 100 to 1,500; from 100 to 1,000; from 100 to 500; from 100 to 300; from 75 to 250; from 75 to 200; from 75 to 175; from 75 to 150; or from 75 to 100 nucleotides in length.
  • DB primer extension products are produced under conditions that do not incorporate additional SDNs during the extension reaction.
  • Such DB primer extension products can contain SDNs positioned within regions at the 5' end corresponding to, e.g., the loop region, 5' arm, or loop region and 5' arm of the initial hybridized DB primer.
  • such DB primer extension products do not contain SDNs positioned within the 3' arm of the initial hybridized DB primer or within the extended nucleotides of the 3' arm, or the combination thereof.
  • the DB primer extension products are free of SDNs in at least the first 25; 50; 75; 100; 125; 150; 175; 200; 250; 300; 350; 400; 450; 500; 550; 600; 700; 800; 1,000; 1,500; 2,000; 3,000; 4,000; 5,000; 7,500; or 10,000 nucleotides from the 3' end, provided that such nucleotides do not include SDNs from the loop region or 5' arm of the initial hybridized DB primer.
  • At least the first 25; 50; 75; 100; 125; 150; 175; 200; 250; 300; 350; 400; 450; 500; 550; 600; 700; 800; 1,000; 1,500; 2,000; 3,000; 4,000; 5,000; 7,500; or 10,000 nucleotides from the 3' end of the DB primer extension products consist of A, C, G, and T.
  • At least the first 25; 50; 75; 100; 125; 150; 175; 200; 250; 300; 350; 400; 450; 500; 550; 600; 700; 800; 1,000; 1,500; 2,000; 3,000; 4,000; 5,000; 7,500; or 10,000 nucleotides from the 3' end of the DB primer extension products contain only SDNs provided by the initial hybridized DB primer.
  • DB primer extension products can contain a universal primer binding site or a complement thereof in the region corresponding to the loop region of the initial hybridized primer.
  • the universal primer binding site or its complement can contain an SDN positioned at the 3'- most nucleotide of the universal primer binding site or its complement.
  • the DB primer extension products can contain a barcode, such as a cell barcode, a sample barcode, or a molecular barcode, or a combination of two or all thereof. In some cases, the DB primer extension product contains both a cell and a molecular barcode.
  • DB primer amplicons can be produced by hybridizing two different DB primers [i.e., a DB primer pair) to a double-stranded target polynucleotide, such that the two different primers hybridize to opposite strands of the target polynucleotide with their 3' arms oriented towards each other and flanking a region of interest [i.e., a region of a target polynucleotide to be amplified) and extending the hybridized DB primer pair with a polymerase in a target-specific PC amplification reaction.
  • two different DB primers i.e., a DB primer pair
  • a region of interest i.e., a region of a target polynucleotide to be amplified
  • the second strand of a double-stranded target polynucleotide is a DB primer extension product that is generated in a first DB primer extension reaction by hybridizing a first DB primer to a single-stranded target polynucleotide and extending the first DB primer to form a double-stranded target polynucleotide having a first and second end, where the first end contains the 3' arm, loop region, and 5' arm of the first DB primer and the second end contains a second DB primer binding site.
  • DB primer amplicons contain a first and a second strand, wherein the first strand is a first DB primer extension product and the second strand is a second DB primer extension product that is complementary to and hybridized to the first DB primer extension product.
  • the DB primer amplicon is a double-stranded amplicon wherein one strand of the double-stranded amplicon contains a DNA polymerase extension product of a first DB primer having a structure according to any one of the DB primers described herein, and an opposite strand of the double-stranded amplicon contains a DNA polymerase extension product of a second DB primer having a structure according to any one of the DB primers described herein, and wherein the first and second DB primer extension products are reverse complements of each other.
  • the double-stranded amplicon contains: i) a first end region containing at least two SDNs on the one strand; and ii) a second end region containing at least two SDNs on the opposite strand.
  • the double stranded amplicon contains a region of interest flanked by the first end region and second end region, wherein the region of interest consists of nucleotides selected from A, C, G, and T.
  • DB primer amplicons can contain features of DB primers and DB primer extension products described above. Such features include, but are not limited to, one or more barcodes, universal primer binding sites, SDNs, and regions corresponding to 3' arms, 5' arms, and loop regions. Although loop regions of the DB primers are generally selected such that they do not hybridize to target polynucleotide, they still serve as a template for polymerase-mediated extension of the opposite strand. Thus, DB primer amplicon regions corresponding to DB primer loop regions are complementary and hybridized to the opposite strand of the DB primer amplicon.
  • the DB primer amplicon can contain two universal primer binding sites (i.e., one in each end region of the amplicon), where the two universal primer binding sites are on opposite strands of the amplicon.
  • the universal primer binding sites can be used to hybridize a pair of universal PC amplification primers for universal amplification of a plurality of target-specific DB primer amplicons. In this way, the regions of interest of the target polynucleotides flanked by DB primer pairs are selectively amplified.
  • the selectively degradable nucleotides (SDNs) of a DB primer amplicon are typically positioned close to each end of the amplicon.
  • the SDNs can be positioned at a first end on a first strand and at a second end on the opposite strand.
  • at least one, at least two, or all of the SDNs of the first strand are within 200 nucleotides of the 5' end of the first strand.
  • at least one, at least two, or all of the SDNs of the first strand are within 200 nucleotides of the 5' end of the second strand.
  • At least one, at least two, or all of the SDNs of the amplicon are within 175, 150, 125, 100, 75, 50, 45, 40, 35, 30, 25, 20, 18, 15, or 12 nucleotides of the 5' end of the strand in which they are positioned.
  • a DB primer amplicon can be selectively degraded to remove one or more, or all, SDNs in the amplicon, thereby providing a selectively degraded DB primer amplicon.
  • the selectively degraded DB primer amplicon can contain a single-stranded nucleotide on the opposite strand of each single-nucleotide gap corresponding to the position of the removed SDN. In some cases, the gap is adjacent to a 3' blocking moiety, such that the adjacent 5' strand, if present cannot be extended.
  • the blocking moiety can be a product of the selective degradation method used to remove the SDN.
  • Such blocking moieties include, but are not limited to, a 3' phosphate, 3' phosphate ester, or a 3' ring-opened sugar such as 3 ' -phospho-a, ⁇ -unsaturated aldehyde (PA).
  • the gap is adjacent to a 5' phosphate.
  • the selectively degraded DB primer amplicon can contain a single- stranded region on the strand opposite from the gap produced by melting of such fragments from the degraded amplicon.
  • This single stranded region can include a universal primer binding site.
  • the selectively degraded DB primer amplicon can contain exposed single-stranded universal primer binding sites on opposite strands.
  • the gap opposite the universal primer binding site can be adjacent to a 5' phosphate and thus suitable for ligation.
  • hybridization of a universal primer to the exposed single-stranded universal primer binding site can produce a substrate for ligation (e.g., ligation by a DNA ligase such as T4 DNA ligase).
  • the substrate for ligation can include a 3' end of the hybridized universal primer and a 5' phosphate of a nucleotide of the amplicon adjacent to the exposed single-stranded region.
  • the gap opposite the universal primer binding site can be adjacent to a 3' blocking moiety.
  • the remaining oligonucleotide corresponding to the region 5' to the 5'-most SDN of the amplicon on one or both strands has a lower melting temperature than the temperature of the amplicon and is no longer hybridized to the amplicon.
  • the selectively degraded DB primer amplicon has a structure as schematically diagrammed in FIG. 8.
  • the selectively degraded DB primer amplicon can be a construct having the following elements with reference to the structural element labels in FIG.
  • the construct further contains a fourth nucleic acid (8) hybridized to the second nucleic acid at positions that result in a gap (9) between a 3'-most nucleotide of the fourth nucleic acid (10) and a 5'-most nucleotide of the third nucleic acid (11), wherein the 3'-most nucleotide of the fourth nucleic acid is blocked at the 3' -end with a 3' blocking group and the 5'-most nucleotide of the third nucleic acid is 5'-phosphorylated, and wherein the second nucleic acid has an exposed second single-stranded universal primer binding site (12).
  • a fourth nucleic acid hybridized to the second nucleic acid at positions that result in a gap (9) between a 3'-most nucleotide of the fourth nucleic acid (10) and a 5'-most nucleotide of the third nucleic acid (11), wherein the 3'-most nucleotide of the fourth nucleic acid is blocked at the
  • the selectively degraded DB primer amplicon can be a construct in which the polynucleotides corresponding to the 5' arms of the DB primer pair do not anneal to the amplicon under the conditions (e.g., temperature) in which the reaction mixture containing the amplicon is incubated.
  • the selectively degraded DB primer amplicon can be a construct having the following elements with reference to the structural element labels in FIG.
  • the construct can further contains a 5'-most nucleotide of the third nucleic acid (11), wherein the 5'- most nucleotide of the third nucleic acid is 5'-phosphorylated, and wherein the second nucleic acid has an exposed second single-stranded universal primer binding site (12), wherein the fourth nucleic acid (8) is not hybridized to the second nucleic acid, but may be present in the reaction mixture.
  • a construct may be annealed to one or more universal primers, optionally ligated, and universally amplified.
  • a selectively degraded DB primer amplicon can be hybridized to a universal primer or a pair of different universal primers.
  • at least one of the universal primers contains a barcode.
  • at least one of the universal primers can contain a sample barcode in a reaction mixture containing DB primer amplicons from a single sample.
  • at least one of the universal primers can contain a cell barcode in a reaction mixture containing DB primer amplicons from a single cell.
  • universal primer amplification can be used to prepare, e.g., a sequencing library containing multiple samples and/or a multiple single-cell DB primer pair amplified targets while maintaining sample- and/or cell-specific tracking.
  • this hybridization structure results in a product containing 4 different oligonucleotide molecules.
  • the amplicon can contain a first strand (1) containing a first universal primer binding site near a 3' end and hybridized to a second strand (2) containing a second universal primer binding site near a 3' end, where the first universal primer binding site is hybridized to a first universal primer (3) and the second universal primer binding site is hybridized to a second universal primer (4).
  • this hybridization structure contains 5 or 6 different oligonucleotide molecules, including the four described above and a first 5' degradation fragment adjacent to the gap exposing the first universal primer binding site (5), or a second 5' degradation fragment adjacent to the gap exposing the second universal primer binding site, or the combination thereof (5 and 6).
  • the 3' end of the 5' degradation fragments can be blocked with a blocking moiety such as an OH, phosphate, or 3' ring-opened sugar.
  • One or more of the foregoing hybridization structures can be contacted with a ligase to produce a ligated DB amplicon.
  • the present invention provides a reaction mixture containing a target polynucleotide and a DB primer or a DB primer pair.
  • the DB primer compositions are used for highly multiplex target-specific amplification of target polynucleotides from a sample.
  • the reaction mixture contains from 10 to 50,000 different DB primer pairs.
  • the reaction mixture further contains a DNA polymerase that is suitable for PC amplification.
  • the DNA polymerase can be a polymerase that can use an SDN as a template in a primer extension reaction.
  • the DNA polymerase is an archael polymerase, such as an archael polymerase that recognizes uracil template nucleotides described in Wardle et al. Nucleic Acids Res. 2008 Feb; 36(3): 705-711, or a variant thereof.
  • the DNA polymerase is PfuV93Q or PfuV93Q-S7.
  • the DNA polymerase is PFU TURBO CX, or a hot-start variant thereof.
  • the reaction mixture further contains nucleotide triphosphates, salts, buffers, and divalent cations (e.g., Mg 2+ ) at concentrations and at a pH suitable for DNA polymerase- mediated DB primer extension.
  • the reaction mixture can contain DB primer extension products or DB primer amplicons, or the combination thereof.
  • the reaction mixture is incubated under conditions suitable for DB primer hybridization and extension.
  • the reaction mixture is incubated under thermal cycling conditions suitable for DB primer-mediated PCR.
  • the reaction mixture further contains one or more universal primers (e.g., a pair of universal primers) and the reaction mixture is incubated under thermal cycling conditions suitable for universal amplification.
  • the reaction mixture contains an enzyme or mixture of enzymes for selective degradation of SDNs.
  • the reaction mixture can contain APE I, Endo III, Endo IV, Endo V, DNA glycosylase-lyase Endonuclease VIII, Fpg, hOGGI, hNEILl, T7 Endo I, T4 PDG, Uracil DNA glycosylase (UDG), Afu, a mixture of UDG or Afu and the DNA glycosylase-lyase Endonuclease VIII, or a combination thereof.
  • the mixture of enzymes for selective degradation of SDNs is uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII.
  • the reaction mixture further contains a DNA ligase, such as T4 DNA ligase.
  • the reaction mixture contains a degraded DB primer amplicon.
  • the degraded DB primer amplicon is hybridized to a universal primer or a pair of universal primers.
  • the reaction mixture contains a ligated DB primer amplicon.
  • the DB primer compositions are used for highly multiplex target-specific amplification of target polynucleotides from a sample.
  • the reaction mixture contains from 10 to 50,000 different DB primer pairs, from 10 to 50,000 different DB primer amplicons, from 10 to 50,000 different universal amplification products, or a combination thereof, wherein the term "different" refers to primer pairs or amplicons having a different polynucleotide sequence, other than one or more barcode regions if present.
  • multiple different DB primer pairs target different regions of the same target polynucleotide in a reaction mixture.
  • the reaction mixture contains from 10 to 50,000 different DB primer pairs, amplification products thereof, or selective degradation products thereof, and from 1 to 5,000 different target polynucleotides.
  • methods for amplifying a target polynucleotide in a PC reaction using a pair of target-specific DB primers.
  • the method includes: providing a composition containing a double-stranded target polynucleotide, a forward DB primer and a reverse DB primer.
  • the forward DB primer hybridizes under hybridization conditions to a first site on one strand of the double-stranded target polynucleotide and the reverse DB primer hybridizes under hybridization conditions to a second site on an opposite strand of the double-stranded target polynucleotide.
  • the first and second sites flank a region of interest of the double-stranded target polynucleotide.
  • the composition can be in a reaction mixture and subjected to amplification conditions, thereby amplifying the region of interest and forming a DB primer amplicon.
  • the method includes amplifying from 10-15,000 or more different target polynucleotide regions of interest in a PCR reaction using from 10-15,000 or more different pairs of target-specific DB primers.
  • the method can include providing at least 100 different double-stranded polynucleotides, at least 1,000 different forward DB primers, and at least 1,000 different reverse DB primers, wherein the reverse DB primers and forward DB primers together make at least 1,000 different DB primer pairs, wherein individual different primer pairs hybridize to opposite strands of a double-stranded target polynucleotide under hybridization conditions and flank a region of interest, wherein the at least 1,000 different DB primer pairs flank at least 1,000 different regions of interest.
  • the method includes providing from 10-15,000 different double-stranded polynucleotides, from 10-15,000 different forward DB primers, from 10-15,000 different reverse DB primers, wherein the reverse DB primers and forward DB primers together make from 10-15,000 different DB primer pairs, wherein individual different primer pairs hybridize to opposite strands of a double-stranded target polynucleotide under hybridization conditions and flank a region of interest, wherein the 10-15,000 different DB primer pairs flank 10- 15,000 different regions of interest.
  • the plurality of DB primer pairs and target polynucleotides can be in a reaction mixture and subjected to amplification conditions, thereby amplifying the plurality of regions of interest and thereby forming a plurality of different DB primer amplicons.
  • the one or more different DB primer pairs are incubated in a reaction mixture with the one or more different target polynucleotides under amplification reaction conditions for a small number of DB primer PCR amplification cycles.
  • DB primer PCR amplification can be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles of a 3-step denaturing, annealing, and extending amplification method or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles of a 2-step denaturing and annealing/extending method.
  • DB primer PCR amplification is performed for 2-10, 2-8, 2-5, 3-10, 3-8, or 3-5 cycles.
  • DB primer PCR amplification is performed for 2-25, 2-20, 2-15, 2-12, 3-25, 3-20, 3-15, or 3-12 cycles.
  • initial DB primer PCR amplification is performed in the presence of universal primers (e.g., during one or more initial rounds of amplification DB primer and universal primer amplification occurs simultaneously). In other cases, initial DB primer PCR amplification is performed in the absence of universal primers. Generally, after the initial DB primer PCR amplification (e.g., in the presence of universal primers or in the absence of universal primers), DB primers and/or DB amplicons can be selectively degraded and subsequent universal amplification can be performed. In some cases, subsequent universal amplification is performed for 10-25, 10-35, 15-30, or 15-35 cycles to provide sufficient material for high-throughput sequencing analysis.
  • DB primer amplification is performed using a 2-step or 3-step amplification method with a first annealing temperature and 2-step or 3-step universal amplification is performed subsequently with a second annealing temperature.
  • the second annealing temperature is higher than the first annealing temperature.
  • the amplification is performed as a first set of 2-3, 2-4, 2-5, or 3-5 cycles with a first annealing temperature, a second set of 2-3, 2-4, 2-5, or 3-5 cycles with a second annealing temperature, and a final set of 10-20, 10-30, 15-30, or 15-35 cycles with a final annealing temperature.
  • the initial DB primer PC amplification reaction further includes first and second universal primers that selectively hybridize to forward and reverse universal primer binding sites encoded by the loop regions of the forward and reverse DB primers of a DB primer pair.
  • the method includes a first phase of DB primer PCR amplification that includes universal primers and DB primers to produce a mixture of amplicons having SDNs and amplicons that do not have SDNs.
  • the DB primers and amplicons having SDNs can be degraded in a selective degradation step and universal PCR can be performed in a second amplification phase.
  • additional universal primers are added to the reaction mixture for the second amplification.
  • the additional universal primers have the same sequence as those used in the initial DB primer PCR amplification phase. In some cases, the additional universal primers have a different sequence. For example, the additional universal primers can be longer or have a higher Tm. In such cases, the second amplification phase can be performed at a higher annealing or extension temperature to further increase the specificity of the universal amplification reaction.
  • the method includes providing a reaction mixture containing a first universal primer hybridized to a single-stranded universal primer binding site on a first strand of a selectively degraded double-stranded amplicon such that the 3'-most nucleotide of the first universal primer is adjacent to a 5' end of the second strand of the selectively degraded double- stranded amplicon.
  • the 5' end of the second strand adjacent to the 3' end of the hybridized first universal primer is phosphorylated.
  • the method further includes ligating the 3' end of the first universal primer to the 5'-phosphorylated end of the second strand.
  • the reaction mixture further contains a second universal primer hybridized to a single-stranded universal primer binding site on the second strand of the selectively degraded double-stranded amplicon.
  • the second universal primer is hybridized to the selectively degraded double-stranded amplicon such that the 3'-most nucleotide of the second universal primer is adjacent to a 5' end of the first strand of the selectively degraded double- stranded amplicon.
  • the 5' end of the first strand adjacent to the 3' end of the hybridized second universal primer is phosphorylated.
  • the method further includes ligating the 3' end of the second universal primer to the 5'-phosphorylated end of the first strand.
  • reagents for DB primer PCR amplification are provided into a reaction mixture, DB primer PCR amplification is performed, and then the reagents for selective degradation of SDNs are introduced in the reaction mixture and selective degradation of SDNs is performed.
  • the reagents for DB primer PCR amplification are provided into a reaction mixture, DB primer PCR amplification is performed, and then the reagents for selective degradation of SDNs, universal primer ligation, and universal amplification are introduced in the reaction mixture and selective degradation of SDNs is performed.
  • the reaction mixture is incubated under universal amplification conditions.
  • the reagents for DB primer PCR amplification and simultaneous universal amplification are provided into a reaction mixture, simultaneous universal and DB primer amplification is performed, and then the reagents for selective degradation are added into the reaction mixture, and the reaction mixture is incubated under conditions suitable for selective degradation of SDNs.
  • additional universal primers or different universal primers are added to the reaction mixture at the same time as the selective degradation reagents.
  • the reaction mixture is incubated under conditions suitable for selective degradation of SDNs and then incubated under conditions suitable for universal amplification.
  • additional universal primers or different universal primers are added to the reaction mixture after selective degradation is performed.
  • the method includes a purification step after one or more of DB primer PCR amplification, simultaneous DB primer and universal primer amplification, selective degradation of SDNs, ligation of universal primers hybridized to selectively degraded DB primer amplicons, or universal primer amplification of ligated double-stranded amplicons, or a combination thereof.
  • the present invention provides the following method for enriching or amplifying a region of interest of a target polynucleotide from a sample: a) amplifying the target polynucleotide from the sample with a first (e.g., forward) and a second (e.g., reverse) DB primer, thereby forming a double-stranded DB primer amplicon; b), degrading the SDNs of the DB primer amplicon to produce a degraded double-stranded amplicon containing a first single-stranded universal primer binding site near a 3' end of a first strand of the DB primer amplicon and a second single-stranded universal primer binding site near a 3' end of a second strand of the DB primer amplicon; c) hybridizing a first universal primer to the first single-stranded universal primer binding site and hybridizing a second universal primer to the second single-stranded universal primer binding site; d)
  • the amplifying is performed by incubating the ligated double-stranded amplicon under PCR conditions in the presence of a molar excess of the first and second universal primers. In some cases, the amplifying is performed by incubating the ligated double-stranded amplicon under PCR conditions in the presence of third and fourth universal primers that anneal to the ligated double-stranded amplicon with a higher Tm relative to the first and second universal primers.
  • the present invention provides the following method for enriching or amplifying a region of interest of a target polynucleotide from a sample: a) providing a reaction mixture containing the sample, wherein the target polynucleotide in the sample is single- or double- stranded, and a DB amplification primer pair, i.e., a first and a second DB primer, wherein the first and second DB primers hybridize to opposite strands of the target polynucleotide if double-stranded and flank the region of interest under hybridization conditions.
  • a DB amplification primer pair i.e., a first and a second DB primer
  • the method further includes: b) heating the reaction mixture to denature target polynucleotide and DB primers and then cooling the reaction mixture to thereby hybridize the first DB primer to the single-stranded target polynucleotide or first and second DB primer to opposite strands of the double-stranded target polynucleotide under hybridization conditions.
  • the method further includes: c) extending the hybridized first DB primer or hybridized first and second DB primer with a polymerase (e.g., a polymerase capable of utilizing an SDN as a template nucleotide).
  • a polymerase e.g., a polymerase capable of utilizing an SDN as a template nucleotide.
  • the method further includes: d) repeating the melting and hybridizing of b) and the extending of c) from about 3 to about 5 times to thereby generate a selectively degradable double-stranded DB primer amplification product containing a primer extension product of the first DB primer hybridized to a primer extension product of the second DB primer, wherein the selectively degradable double-stranded DB primer amplification product has a first end region and a second end region, wherein: i) the first end region has a 3' end region of a first strand and a 5' end region of a second strand, wherein the 5' end region of the second strand has the SDNs of the first DB primer, and the 3' end region of the first strand has a first universal primer binding site; and ii) the second end region has a 3' end region of the second strand and a 5' end region of the first strand, wherein the 5' end region of the first strand has the SDNs of the first
  • the method further includes: e) selectively degrading the SDNs of: i) first and second DB primers that have not been extended by a DNA polymerase, if present; and ii) the selectively degradable double-stranded DB primer amplification product, wherein the degrading produces a degraded double-stranded polynucleotide having a first single-stranded region containing the first universal primer binding site of the first strand and a second single-stranded region having the second universal primer binding site of the second strand of the degraded double- stranded polynucleotide, wherein the first strand includes a 5' terminal phosphate and the second strand includes a 5' terminal phosphate.
  • the method further includes: f) introducing into the reaction mixture a first universal primer and a second universal primer, and hybridizing the first universal primer under hybridization conditions to the first universal primer binding site and the second universal primer under hybridization conditions to the second universal primer binding site, wherein the hybridizing: i) positions a 3' -end of the hybridized first universal primer adjacent to the 5' terminal phosphate of the second strand of the degraded double-stranded polynucleotide; and ii) positions a 3'-end of the hybridized second universal primer adjacent to the 5' terminal phosphate of the first strand of the degraded double-stranded polynucleotide.
  • the method further includes: g) ligating the 3' end of the hybridized first universal primer to the 5' terminal phosphate of the second strand of the degraded double-stranded polynucleotide and the 3' end of the second universal primer to the 5' terminal phosphate of the first strand of the degraded double-stranded polynucleotide, thereby generating a ligated double-stranded product.
  • the method further includes: h) amplifying the ligated double-stranded product by universal PCR in the presence of a molar excess of first and second universal primers or amplifying the ligated double-stranded product by universal PCR in the presence of a molar excess of third and fourth universal primers, thereby enriching region of interest of the target polynucleotide from the sample.
  • the selectively degrading of e), the introducing and hybridizing of a first universal primer and a second universal primer of f), and the ligating of g) are performed simultaneously in the reaction mixture.
  • the introducing into the reaction mixture a first universal primer and a second universal primer of f) is performed simultaneously with the providing the reaction mixture containing the target polynucleotide and the DB primer pair.
  • the target polynucleotide, DB primer pair, and universal primers are mixed together in a reaction mixture containing a polymerase, amplification is performed and then an enzyme or enzyme mixture is introduced into the reaction mixture to selectively degrade SDNs.
  • all the reagents for DB primer PCR amplification, selective degradation of SDNs, ligation of hybridized universal primers, and universal primer amplification are mixed together in a single reaction tube before DB primer PCR amplification is performed.
  • Example 1 DB primer Amplification and Selective Degradation
  • Bubble Primer PCR was performed in 50 ⁇ reaction volumes containing IX PfuCx reaction buffer, 10 ng NA19238 human genome, 0.2 or 1 pmol of bubble primer gene sets, and 0.03 U/uL PfuCx polymerase.

Abstract

La présente invention concerne des procédés, des compositions, et des kits destinés à l'enrichissement de polynucléotides cibles à partir d'un échantillon. De tels procédés, compositions, et kits peuvent être utilisés pour préparer des bibliothèques de séquençage à haut débit pour l'analyse des polynucléotides cibles. Les procédés, les compositions, et les kits peuvent permettre l'enrichissement rapide, efficace, et sensible d'un grand nombre (par exemple, de 10 à 15 000) de différentes séquences polynucléotidiques cibles à partir d'un échantillon.
PCT/US2017/040917 2016-07-07 2017-07-06 Enrichissement rapide de cible par pcr relais multiplexée avec des amorces à bulles modifiées WO2018009677A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114134219A (zh) * 2021-12-14 2022-03-04 广州市金圻睿生物科技有限责任公司 一种多重核酸检测系统及其制备方法与应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016102956A1 (fr) * 2014-12-22 2016-06-30 Dnae Group Holdings Ltd Amorces à bulle

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016102956A1 (fr) * 2014-12-22 2016-06-30 Dnae Group Holdings Ltd Amorces à bulle

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114134219A (zh) * 2021-12-14 2022-03-04 广州市金圻睿生物科技有限责任公司 一种多重核酸检测系统及其制备方法与应用

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