Improvements in next generation target enrichment performance

Abstract

The present disclosure relates to compositions and kits for PCR amplification. The present disclosure also relates to methods of amplifying nucleic acid molecules to improve coverage uniformity and / or reduce GC bias during downstream sequencing operations.

Description

Technical Field

[0001] The present disclosure relates to compositions and methods for improving coverage uniformity and / or reducing GC bias during sequencing. Background Art

[0002] Next-generation sequencing is a massively parallel sequencing technology that provides high throughput, scalability, and speed. Given its sensitivity and specificity, next-generation sequencing helps to study the molecular basis of diseases such as cancer.

[0003] Next-generation sequencing application data may result in poor performance, which may include low on-target rates, low genomic equivalent recovery (deduplication / unique depth), and low coverage uniformity. Low uniformity can be observed in target enrichment applications with high GC or high AT content. This is often described as GC bias. Coverage uniformity is a key metric and affects the ability to uniformly enrich and sequence target regions. Poor coverage uniformity results in lower sequencing coverage in areas of poor performance, which ultimately requires more sequencing to achieve target sequencing depth in these poorly performing areas. Low on-target rates also result in the need for more sequencing and ultimately lead to higher sequencing costs per sample.

[0004] Preparing nucleic acid molecules for next generation sequencing involves multiple library preparation steps. Generally, the target nucleic acid molecules are obtained, purified, fragmented, end-repaired, and A-tailed; adapters are then ligated; and the library is then purified and / or enriched, quantified, normalized, and loaded onto the instrument. GC bias can be introduced during PCR amplification of the nucleic acid library and / or during enrichment of the nucleic acid library. Therefore, there is a need in the art for a method of reducing GC bias during amplification and / or target enrichment in an NGS library preparation workflow. Summary of the invention

[0005] It is desirable to reduce the generation of GC bias during amplification and / or library enrichment. The present disclosure relates to compositions and methods for improving coverage uniformity and / or reducing GC bias during sequencing, especially reducing GC bias during sequencing of target enrichment samples. In particular, the applicant has unexpectedly found that the use of primers with high melting temperatures and / or compositions comprising one or more reinforcing agents improves coverage uniformity and / or reduces GC bias during one or more downstream sequencing operations. The applicant has also unexpectedly found that coverage uniformity can be improved by optimizing the temperature and time / time parameters during capture extension. These and other improvements will be further described herein.

[0006] A first aspect of the present disclosure is a composition comprising a polymerase, one or more primers, unmodified dNTPs, and at least one enhancer.

[0007] In some embodiments, the at least one enhancer is selected from betaine, dimethyl sulfoxide (DMSO), a disaccharide, and a single-stranded DNA binding protein (SSB).

[0008] In some embodiments, at least one enhancer is betaine. In some embodiments, the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM. In some embodiments, the concentration of betaine in the composition ranges from about 0.3 mM to about 0.6 mM. In some embodiments, the concentration of betaine in the composition is about 0.5 mM.

[0009] In some embodiments, at least one enhancer is DMSO. In some embodiments, the amount of DMSO in the composition ranges from about 1% (v / v) to about 10% (v / v). In some embodiments, the amount of DMSO in the composition ranges from about 2% (v / v) to about 9% (v / v).

[0010] In some embodiments, one or more primers have a melting temperature (Tm) ranging from about 57°C to about 95°C. In some embodiments, one or more primers have a Tm ranging from about 57°C to about 85°C. In some embodiments, one or more primers have a Tm ranging from about 57°C to about 75°C. In some embodiments, one or more primers have a Tm ranging from about 57°C to about 72°C. In some embodiments, one or more primers have a Tm ranging from about 57°C to about 69°C. In some embodiments, one or more primers have a Tm ranging from about 57°C to about 66°C. In some embodiments, one or more primers have a Tm ranging from about 57°C to about 63°C. In some embodiments, up to about 10% of the primers in the composition are high melting temperature and / or high GC content primers (e.g., preamplification primers, capture primers, release primers, amplification primers, etc.).

[0011] In some embodiments, one or more primers include one or more modified dNTPs. In some embodiments, one or more modified dNTPs are selected from modified dGTP and modified dATP. In some embodiments, one or more modified dNTPs are further included. In some embodiments, the concentration of one or more modified dNTPs in the composition is approximately the same as the concentration of unmodified dNTPs in the composition. In some embodiments, the concentration of one or more modified dNTPs in the composition ranges from about 0.1mM to about 0.5mM. In some embodiments, the concentration of one or modified dNTPs in the composition ranges from about 0.2mM to about 0.4mM. In some embodiments, one or more modified dNTPs are modified dGTP. In some embodiments, modified dGTP includes 7-deaza-2'-deoxyguanosine-5'-triphosphate. In some embodiments, one or more modified dNTPs are modified dATP. In some embodiments, modified dATP includes 2-amino-2'deoxyadenosine-5'-triphosphate.

[0012] In some embodiments, the composition further comprises a divalent cation. In some embodiments, the divalent cation is selected from the group consisting of: Co 2+ , Mn 2+ Mg 2+ 、Cd 2+ and Ca 2+ .

[0013] In some embodiments, the composition further comprises one or more buffering agents. In some embodiments, the composition further comprises one or more polyols.

[0014] In certain embodiments, one or more primers include a capture front forward primer and a capture front reverse primer. In certain embodiments, one or more primers include a capture primer. In certain embodiments, the capture primer includes a capture portion.

[0015] In some embodiments, the composition further comprises one or more nucleic acid molecules. In some embodiments, the one or more nucleic acid molecules comprise a nucleic acid molecule library, wherein each nucleic acid molecule in the nucleic acid molecule library comprises a first adapter and a second adapter. In some embodiments, the one or more primers comprise one or more capture primers, and wherein the one or more capture primers are capable of hybridizing with a target nucleic acid sequence in the nucleic acid molecule library.

[0016] A second aspect of the present disclosure is a composition comprising a polymerase, one or more primers, dNTPs, and optionally at least one enhancer, wherein the one or more primers include no restriction on the percentage of guanine or cytosine bases, and wherein at least one of the one or more primers has a melting temperature greater than 63°C. In some embodiments, at least one primer has a melting temperature greater than about 69°C. In some embodiments, at least one primer has a melting temperature greater than about 75°C. In some embodiments, at least one primer has a melting temperature greater than about 85°C. In some embodiments, at least one primer has a melting temperature greater than about 95°C. In some embodiments, at most about 10% of the primers in the composition are high melting temperature and / or high GC content primers (e.g., pre-amplification primers, capture primers, release primers, amplification primers, etc.).

[0017] In some embodiments, at least one optional enhancer is selected from betaine, DMSO, a disaccharide, and a single-stranded DNA binding protein (SSB). In some embodiments, at least one optional enhancer is betaine, and wherein the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM.

[0018] In some embodiments, dNTPs include a mixture of unmodified dNTPs and modified dNTPs. In some embodiments, one or more modified dNTPs include 7-deaza-2'-deoxyguanosine-5'-triphosphate. In some embodiments, one or more modified dNTPs include 2-amino-2'deoxyadenosine-5'-triphosphate. In some embodiments, the concentration of one or more modified dNTPs in the composition ranges from about 0.2mM to about 0.8mM. In some embodiments, one or more primers include one or more modified dNTPs. In some embodiments, one or more modified dNTPs are selected from the group consisting of 7-deaza-2'-deoxyguanosine-5'-triphosphate and 2-amino-2'deoxyadenosine-5'-triphosphate.

[0019] In some embodiments, the composition further comprises divalent cations. In some embodiments, the divalent cations are selected from the group consisting of Co2+, Mn2+, Mg2+, Cd2+ and Ca2+. In some embodiments, the composition further comprises one or more buffers. In some embodiments, the composition further comprises one or more polyols.

[0020] In some embodiments, one or more primers include a capture front forward primer and a capture front reverse primer. In some embodiments, one or more primers are capture primers. In some embodiments, the composition further includes one or more nucleic acid molecules.

[0021] In some embodiments, the one or more nucleic acid molecules comprise a first adaptor and a second adaptor. In some embodiments, the one or more primers comprise one or more capture primers, and wherein the one or more capture primers are capable of hybridizing with a target nucleic acid sequence of the nucleic acid molecule. In some embodiments, the one or more nucleic acid molecules comprise DNA.

[0022] A third aspect of the present disclosure is a reaction tube (or container, vial, reaction chamber, etc.) comprising the composition of any one of the first and second aspects of the present disclosure described above or as disclosed herein.

[0023] A fourth aspect of the present disclosure is the use of a composition, such as any of the compositions indicated in the first and second aspects of the present disclosure described above or as further described herein, in amplifying one or more nucleic acid molecules.

[0024] A fifth aspect of the present disclosure is a composition comprising one or more primers, an input nucleic acid molecule, and at least one enhancer selected from the group consisting of betaine, DMSO, a disaccharide, and a single-stranded DNA binding protein (SSB).

[0025] In some embodiments, at least one enhancer is betaine. In some embodiments, the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM. In some embodiments, the concentration of betaine in the composition ranges from about 0.3 mM to about 0.6 mM. In some embodiments, at least one enhancer is DMSO.

[0026] In some embodiments, the amount of DMSO in the composition ranges from about 1% (v / v) to about 10% (v / v). In some embodiments, the amount of DMSO in the composition ranges from about 2% (v / v) to about 8% (v / v).

[0027] In some embodiments, one or more primers have a Tm ranging from about 57°C to about 95°C. In some embodiments, one or more primers have a Tm ranging from about 57°C to about 85°C. In some embodiments, one or more primers have a Tm ranging from about 57°C to about 75°C. In some embodiments, one or more primers have a Tm ranging from about 57°C to about 72°C. In some embodiments, one or more primers have a Tm ranging from about 57°C to about 69°C. In some embodiments, one or more primers have a Tm ranging from about 57°C to about 66°C. In some embodiments, up to about 10% of the primers in the composition are high melting temperature and / or high GC content primers (e.g., preamplification primers, capture primers, release primers, amplification primers, etc.).

[0028] In some embodiments, the one or more primers comprise one or more modified dNTPs. In some embodiments, the one or more modified dNTPs are selected from the group consisting of: a modified dGTP and a modified dATP.

[0029] In some embodiments, the composition further comprises a divalent cation. In some embodiments, the divalent cation is selected from the group consisting of: Co 2+ , Mn 2+ Mg 2+ 、Cd 2+ and Ca 2+ In some embodiments, the composition further comprises one or more buffering agents.

[0030] In some embodiments, the input nucleic acid molecules comprise a prepared nucleic acid library. In some embodiments, the input nucleic acid molecules comprise captured nucleic acid molecules. In some embodiments, the captured nucleic acid molecules comprise a complex of nucleic acid molecules comprising a target nucleic acid sequence and an extended capture primer that hybridizes to at least a portion of the target nucleic acid sequence.

[0031] The sixth aspect of the present disclosure is a composition comprising an input nucleic acid molecule, one or more primers, and optionally at least one enhancer, wherein the one or more primers are not limited in the percentage of guanine or cytosine bases, and wherein at least one of the one or more primers has a melting temperature greater than 63°C. In some embodiments, at least one primer has a melting temperature greater than about 69°C. In some embodiments, at least one primer has a melting temperature greater than about 75°C. In some embodiments, at least one primer has a melting temperature greater than about 85°C. In some embodiments, at least one primer has a melting temperature greater than about 95°C. In some embodiments, at most about 10% of the primers in the composition are high melting temperature and / or high GC content primers (e.g., pre-amplification primers, capture primers, release primers, amplification primers, etc.).

[0032] In some embodiments, one or more primers comprise one or more modified dNTPs. In some embodiments, one or more modified dNTPs are selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate and 2-amino-2'deoxyadenosine-5'-triphosphate. In some embodiments, at least one optional enhancer is selected from betaine, DMSO, disaccharides, and single-stranded DNA binding proteins (SSBs).

[0033] In some embodiments, at least one optional enhancer is betaine, and wherein the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM. In some embodiments, the composition further comprises a divalent cation. In some embodiments, the divalent cation is selected from the group consisting of Co2+, Mn2+, Mg2+, Cd2+, and Ca2+. In some embodiments, the composition further comprises one or more buffers.

[0034] In some embodiments, the input nucleic acid molecule comprises a prepared nucleic acid library. In some embodiments, the input nucleic acid molecule comprises captured DNA. In some embodiments, the captured nucleic acid molecule comprises a complex of nucleic acid molecules comprising a target nucleic acid sequence and an extended capture primer hybridized to at least a portion of the target nucleic acid sequence.

[0035] A seventh aspect of the present disclosure is a reaction tube (or container, vial, reaction chamber, etc.) comprising a composition of any of the fifth or sixth aspects of the present disclosure as indicated above or as further described herein.

[0036] An eighth aspect of the present disclosure is use of the composition of any of the fifth aspect or sixth aspect of the present disclosure noted above or as described herein for preferentially enriching one or more target nucleic acid molecules in a nucleic acid molecule library.

[0037] The ninth aspect of the present invention is a kit comprising (i) a capture primer set; (ii) a release primer set; (iii) a polymerase; (iv) dNTPs; and (v) betaine or a derivative or analog thereof. In some embodiments, the dNTPs comprise a mixture of unmodified dNTPs and modified dNTPs. In some embodiments, the modified dNTPs comprise modified dGTP and modified dATP. In some embodiments, the capture primer set and the release primer set comprise one or more modified dNTPs. In some embodiments, the kit further comprises instructions for preparing a capture extension master mix, wherein the prepared capture extension master mix comprises betaine at a concentration of about 0.5 mM. In some embodiments, the kit further comprises one or more buffers. In some embodiments, the kit further comprises one or more divalent cations. In some embodiments, the kit further comprises one or more polyols. In some embodiments, at least one of the capture primer set and the release primer set has no restrictions on melting temperature and / or GC content. In some embodiments, both the capture primer set and the release primer set have no restrictions on melting temperature and / or GC content.

[0038] The tenth aspect of the present invention is a kit comprising (i) a capture primer set; (ii) a release primer set; (iii) a polymerase; and (iv) dNTPs; wherein at least one primer in the capture primer set has a melting temperature greater than about 65°C; and wherein at least one primer in the release primer set has a melting temperature greater than about 65°C. In some embodiments, at least one primer in the capture primer set has a melting temperature greater than about 72°C; and wherein at least one primer in the release primer set has a melting temperature greater than about 72°C. In some embodiments, at most about 10% of the capture primers and / or release primers in the kit are high melting temperature and / or high GC content primers (e.g., preamplification primers, capture primers, release primers, amplification primers, etc.). In some embodiments, at least one primer in the capture primer set comprises one or more modified dNTPs; and wherein at least one primer in the release primer set comprises one or more modified dNTPs. In some embodiments, the kit further comprises at least one enhancing agent selected from the group consisting of betaine or a derivative or analog thereof, DMSO, a single-stranded DNA binding protein, or a disaccharide.

[0039] The eleventh aspect of the present disclosure is a method for producing a capture primer extension complex comprising a target nucleic acid molecule and a capture primer, the method comprising: (a) hybridizing a capture primer to a portion of a target nucleic acid molecule in a nucleic acid molecule library, wherein each of the nucleic acid molecules in the nucleic acid molecule library has a first end comprising a first adaptor and a second end comprising a second adaptor; and (b) extending the hybridized capture primer with a first polymerase to produce a capture primer extension complex; wherein the capture primer has a melting temperature greater than 63°C. In some embodiments, the melting temperature of the capture primer is greater than 66°C. In some embodiments, the melting temperature of the capture primer is greater than 72°C. In some embodiments, the melting temperature of the capture primer is greater than 75°C.

[0040] In some embodiments, the capture primer hybridized to the target nucleic acid molecule is extended in a composition comprising at least one enhancer selected from the group consisting of betaine or a derivative or analog thereof, DMSO (or a solvent with similar chemical and / or physical properties, such as DMF), a single-stranded DNA binding protein (such as a thermostable single-stranded DNA binding protein), and a disaccharide (such as trehalose). In some embodiments, at least one enhancer is betaine, and the concentration of the betaine ranges from about 0.2 mM to about 0.8 mM. In some embodiments, at least one enhancer is betaine, and the concentration of the betaine ranges from about 0.3 mM to about 0.7 mM. In some embodiments, at least one enhancer is betaine, and the concentration of the betaine ranges from about 0.4 mM to about 0.6 mM.

[0041] In some embodiments, the capture primer hybridized to the target nucleic acid molecule is extended in a composition comprising one or more modified dNTPs. In some embodiments, the one or more modified dNTPs are modified dGTPs. In some embodiments, the modified dGTPs comprise 7-deaza-2'-deoxyguanosine-5'-triphosphate. In some embodiments, the one or more modified dNTPs are modified dATPs. In some embodiments, the modified dATPs comprise 2-amino-2'deoxyadenosine-5'-triphosphate. In some embodiments, the capture primer hybridized to the target nucleic acid molecule is extended in a composition comprising betaine and one or more modified dNTPs.

[0042] In some embodiments, the concentration of betaine in the composition is about 0.5 mM. In some embodiments, the method further comprises capturing the capture primer extension complex. In some embodiments, the method further comprises hybridizing a release primer to a target nucleic acid. In some embodiments, the release primer is hybridized to a target nucleic acid in a composition comprising at least one enhancer. In some embodiments, at least one enhancer is betaine. In some embodiments, the release primer is hybridized to a target nucleic acid in a composition comprising one or more dNTPs. In some embodiments, the release primer has a melting temperature greater than 63°C. In some embodiments, the melting temperature is greater than 65°C. In some embodiments, the melting temperature is greater than 72°C.

[0043] In some embodiments, the method further comprises extending the release primer hybridized to the target nucleic acid with a second polymerase.

[0044] The twelfth aspect of the present disclosure is a method for amplifying one or more nucleic acid molecules, the method comprising: (i) obtaining a plurality of nucleic acid molecules; and (ii) performing a first amplification reaction in a first composition comprising the obtained plurality of nucleic acid molecules and a first set of primers, wherein at least one primer in the first set of primers has a melting temperature greater than 63°C. In some embodiments, each nucleic acid molecule in the obtained plurality of nucleic acid molecules comprises a first adapter and a second adapter. In some embodiments, the first composition further comprises betaine. In some embodiments, wherein the concentration of betaine in the first composition is about 0.5 mM. In some embodiments, the method further comprises enriching the obtained plurality of nucleic acid molecules for one or more target nucleic acid molecules. In some embodiments, the method further comprises performing a second amplification reaction in a second composition comprising one or more target nucleic acid molecules and a second set of primers, wherein at least one primer in the second set of primers has a melting temperature greater than 63°C. In some embodiments, the second composition further comprises betaine. In some embodiments, the concentration of betaine in the human composition is about 0.5 mM. In some embodiments, the obtained plurality of nucleic acid molecules is a target enrichment library comprising a plurality of target nucleic acid molecules. In some embodiments, the method further comprises sequencing the amplified target enrichment library.

[0045] The thirteenth aspect of the present disclosure is a method for amplifying one or more nucleic acid molecules, the method comprising: (i) obtaining a plurality of nucleic acid molecules; (ii) performing a first amplification reaction in a first composition comprising a plurality of nucleic acid molecules, betaine, and a mixture of unmodified dNTPs and modified dNTPs. In some embodiments, the concentration of betaine in the first composition is about 0.5 mM. In some embodiments, at least one primer in the first set of primers has a high melting temperature and / or a high GC content. In some embodiments, each of the plurality of nucleic acid molecules obtained comprises a first adapter and a second adapter, and wherein the method further comprises enriching the plurality of nucleic acid molecules obtained for one or more target nucleic acid molecules. In some embodiments, the method further comprises performing a second amplification reaction in a second composition comprising one or more target nucleic acid molecules, betaine, and a mixture of unmodified dNTPs and modified dNTPs. In some embodiments, the plurality of nucleic acid molecules obtained are a target enrichment library comprising a plurality of target nucleic acid molecules. In some embodiments, further comprising sequencing the amplified target enrichment library.

[0046] The fourteenth aspect of the present disclosure is a kit for enriching at least one target nucleic acid in a nucleic acid molecule library, the kit comprising: a first oligonucleotide complementary to a target nucleic acid in a nucleic acid molecule library, each of the nucleic acid molecules in the nucleic acid molecule library having a first end comprising a first adapter and a second end comprising a second adapter; a second oligonucleotide complementary to the target nucleic acid; a first amplification primer; and a second amplification primer; and wherein at least one of the first oligonucleotide or the second oligonucleotide has a high melting temperature and / or a high GC content. In some embodiments, both the first oligonucleotide and the second oligonucleotide have a high melting temperature and / or a high GC content.

[0047] The fifteenth aspect of the present disclosure is a composition comprising: a nucleic acid molecule library comprising at least one target nucleic acid, each of the nucleic acid molecules in the nucleic acid molecule library having a first end comprising a first adaptor, a second end comprising a second adaptor, and a target region between the first adaptor and the second adaptor; an extended first oligonucleotide hybridized to the target region of the target nucleic acid, the extended first oligonucleotide comprising at least one capture portion; a solid support bound to at least one capture portion; a second oligonucleotide hybridized to the target nucleic acid at the 5' position of the first extended oligonucleotide; and a polymerase associated with the 3' end of the second oligonucleotide; wherein at least one of the first oligonucleotide or the second oligonucleotide has a high melting temperature and / or a high GC content.

[0048] The sixteenth aspect of the present disclosure is a composition comprising a polymerase, one or more primers with no restrictions on melting temperature and / or GC content, a mixture of unmodified dNTPs and modified dNTPs, and at least one enhancer. In some embodiments, at least one enhancer is betaine. In some embodiments, the composition further comprises one or more nucleic acid molecules.

[0049] The seventeenth aspect of the present disclosure is a composition that is essentially composed of a polymerase, one or more primers that are not limited to melting temperature and / or GC content, a mixture of unmodified dNTPs and modified dNTPs, and at least one reinforcing agent. In some embodiments, at least one reinforcing agent is betaine. In some embodiments, the composition further comprises one or more nucleic acid molecules.

[0050] The eighteenth aspect of the present disclosure is a composition comprising a polymerase, one or more primers with no restrictions on melting temperature and / or GC content, a mixture of unmodified dNTPs and modified dNTPs, and at least one enhancer. In some embodiments, at least one enhancer is betaine. In some embodiments, the composition further comprises one or more nucleic acid molecules.

[0051] The nineteenth aspect of the present disclosure is a reaction tube comprising the composition of any one of the sixteenth aspect, seventeenth aspect or eighteenth aspect of the present disclosure as noted above or as described herein.

[0052] The twentieth aspect of the present disclosure is the use of a composition of any of the sixteenth, seventeenth or eighteenth aspects of the present disclosure as indicated above or as described herein in amplifying one or more nucleic acid molecules (such as a library of nucleic acids or captured nucleic acid molecules).

[0053] The twenty-first aspect of the present disclosure is a method of amplifying one or more nucleic acid molecules, wherein the amplification is performed in the presence of any of the compositions of the sixteenth, seventeenth, or eighteenth aspects of the present disclosure as indicated above or as described herein. In some embodiments, the thermal cycling step is performed for a period of time ranging between about 25 minutes to about 35 minutes, such as about 34 minutes. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with one or more color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0055] For a general understanding of the features of the present disclosure, reference is made to the drawings, in which like reference numerals are used throughout to identify like elements.

[0056] Figure 1 A flow chart illustrating an embodiment of a method for enriching at least one target nucleic acid molecule in a nucleic acid molecule library according to the present disclosure is provided. The steps shown can be performed in the presence of any of the compositions described herein.

[0057] Figure 2A Depicted is the limited availability of primers in target regions with high %GC when primer %GC is constrained between 20% and 80% and primer Tm is constrained between 57° C. and 63° C. Due to these constraints on primer %GC and primer Tm, primer coverage on the primary target is incomplete.

[0058] Figure 2B It is shown that by removing restrictions on primer %GC content and primer Tm, primer availability in target regions with high %GC content is increased and primer coverage in these regions is improved.

[0059] Figure 2C It was shown that increasing the maximum primer Tm allowed during primer design reduces the number of target bases not covered by primers.

[0060] Figure 3AShown are samples enriched with a primer test set designed with primer Tm ranges between about 57°C to about 63°C (current primer database), between about 57°C to about 66°C (maximum Tm 66), between about 57°C to about 69°C (maximum Tm 69), between about 57°C to about 62°C (maximum Tm 72°C), and between about 57°C to "no Tm limit" (no maximum Tm). Samples enriched with the "no maximum Tm" test set had the highest percentage of bases within a 2-fold range and the percentage of group exonic regions >= 1000X, which are measures of sequencing coverage uniformity across the target.

[0061] Figure 3B Shown are samples enriched with a primer test set designed with primer Tm between about 57°C to about 63°C (current primer database), between about 57°C to about 66°C (maximum Tm 66), between about 57°C to about 69°C (maximum Tm 69), between about 57°C to about 62°C (maximum Tm 72°C), and between about 57°C to unlimited (no maximum Tm). The normalized sequencing coverage of high %GC targets (65%, 75%, and 77%) was improved the most in samples enriched with the no maximum Tm test set compared to samples enriched with the current database

[0062] Figure 4 Samples prepared as libraries with and without betaine (PCR1+B, PCR1-B) and 7-deaza-dGTP (PCR1+7, PCR1-7) are depicted. The libraries were then used as input to HyperPETE with or without betaine in the capture extension (CapExt+B, CapExt-B). The yield of PCR1+B with PCR1-7 pre-capture input library was higher than that of PCR1-B with PCR1-7 pre-capture input library. The yield of PCR1+7 pre-capture input library was relatively low

[0063] Figure 5Samples prepared as libraries with and without betaine (PCR1+B, PCR1-B) and 7-deaza dGTP (PCR1+7, PCR1-7) are shown. The libraries were then used as input to HyperPETE with or without betaine in the capture extension (CapExt+B, CapExt-B). The PCR1+B plus PCR1-7 samples had higher deduplication depth and average fragment length compared to the PCR1-B samples. The PCR1-7 samples with CapExt+B had a higher percentage of bases in 2x and a higher percentage of group exon regions >=1000x compared to the CapExt-B samples. Regardless of whether betaine was used in PCR1 or the capture extension, the PCR1+7 samples had a higher percentage of bases in 2x and a higher percentage of group exon regions >=1000x.

[0064] Fig. 6A Samples prepared with and without 7-deaza dGTP as libraries are shown (PCR1+7-deaza dGTP, PCR1-7-deaza dGTP). The libraries were then used as input to HyperPETE with or without betaine in capture extension (capture extension+betaine, capture extension-betaine). The normalized coverage of the three high %GC targets for the PCR1+7 sample was more uniform compared to the PCR1-7 sample.

[0065] Figure 6B Samples prepared with and without 7-deaza dGTP as libraries are shown (PCR1 + 7-deaza dGTP, PCR1 - 7-deaza dGTP). The libraries were then used as input to HyperPETE with or without betaine in capture extension (capture extension + betaine, capture extension - betaine). For the PCR1 - 7-deaza dGTP samples, the normalized coverage of the three high % GC targets was more uniform for the capture extension + betaine samples than for the capture extension - betaine samples.

[0066] Figure 7 Samples prepared as libraries with and without betaine (PCR1+betaine, PCR1-betaine) and 7-deazadGTP (PCR1+7-deazadGTP, PCR1-7-deazadGTP) are shown. The libraries were then used as input to HyperPETE with or without betaine in capture extension (capture extension+betaine, capture extension-betaine). The PCR1+7-deazadGTP sample had the highest normalized coverage for high %GC targets (75%, 80%), followed by the PCR1-7-deazadGTP+capture extension+betaine sample. The PCR1+betaine sample had higher coverage in the low %GC (30%) target compared to all other sample groups.

[0067] Figure 8Depicted are samples enriched using different capture extension test programs detailed in Tables 3 to 6 herein and a control capture extension program following the HyperPETE Instructions for Use (IFU). All samples enriched using the test programs (Programs 1 to 4) had higher read on-target percentages and deduplication depths compared to the IFU. The average fragment lengths of the IFU samples were much higher compared to the test program samples. Program 3 samples had the highest percentage of bases within 2-fold range and the percentage of group exon regions >= 1000x.

[0068] Fig. 9 Shown are samples enriched using the different capture extension test procedures detailed in Tables 3 to 6 herein and a control capture extension procedure following the HyperPETE Instructions for Use (IFU). Compared to the IFU samples, samples enriched using the test procedures (Procedures 1 to 4) have higher normalized coverage in high %GC targets (75%, 80%), with the Program 3 sample having the highest coverage.

[0069] Fig.10 It is shown that in samples prepared as libraries containing 7-deazadGTP and enriched with a set of primers designed with no %GC and maximum Tm restrictions (no max Tm+PCR1 w / 7-deazadGTP), the normalized coverage in the three high %GC targets (65%, 75%, 77%) was the highest among all conditions tested to improve coverage in high %GC targets, with all three targets exceeding 90%.

[0070] Fig.11 Sequencing quality control metrics are depicted.

[0071] Fig.12 Normalized position-deduplication coverage of high GC percentage target regions is shown.

[0072] Fig.13 Normalized position-deduplicated coverage of the target region is shown.

[0073] Fig.14 The sequencing quality control indicators are described.

[0074] Fig.15 Normalized position-deduplication coverage of high GC percentage target regions is shown.

[0075] Fig.16 Normalized position-deduplicated coverage of the target region is shown.

[0076] Fig.17 Sequencing quality control metrics for DMSO titration using the 1.7 Mb panel are provided.

[0077] Fig.18DMSO titration performed with the 1.7 Mb set is shown, showing normalized positional deduplication coverage and high GC percentage. DETAILED DESCRIPTION

[0078] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the described order of the steps or actions of the method, unless indicated to the contrary.

[0079] definition

[0080] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The term "comprising" is defined as inclusive, such as "comprising A or B" means comprising A, B, or A and B.

[0081] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as being inclusive, for example, at least one element in a number of elements or a list of elements, but also including more than one element, and optionally including additional unlisted items. Only terms that indicate the contrary, such as "only one of" or "exactly one of", or "consisting of..." used in the claims, will refer to the inclusion of exactly one element in a number of elements or a list of elements. In general, the term "or" used herein should be interpreted as indicating an exclusive alternative (e.g., "one or the other, but not two") only when it is preceded by an exclusive term such as "any", "one of", "only one of", or "exactly one of". "Substantially consisting of..." used in the claims should have the ordinary meaning used in the field of patent law.

[0082] The terms "including", "comprising", "having", etc. are used interchangeably and have the same meaning. Similarly, "including", "comprising", "having", etc. are used interchangeably and have the same meaning. Specifically, the definition of each term is consistent with the definition of "including" under ordinary U.S. patent law, so each term can be understood as an open term, which means "at least the following" and can also be interpreted as not excluding additional features, limitations, aspects, etc. Therefore, for example, "a device having components a, b and c" means that the device includes at least components a, b and c. Similarly, the phrase: "a method involving steps a, b and c" means that the method includes at least steps a, b and c. In addition, although the steps and processes may be outlined in a particular order herein, those skilled in the art will recognize that the order steps and processes may vary.

[0083] As used herein in the specification and claims, with respect to a list of one or more elements, the phrase "at least one" should be understood as at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, nor does it exclude any combination of elements in the list of elements. In addition to the elements specifically identified in the list of elements to which the phrase "at least one" refers, the definition also allows other elements to be optionally present, whether or not they are related to the specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently, "at least one of A and / or B") can refer to at least one, optionally including more than one A, without B (and optionally including elements other than B) in one embodiment; in another embodiment, at least one, optionally including more than one B, without A (and optionally including elements other than A); in yet another embodiment, at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements); and so on.

[0084] References throughout this specification to "one embodiment," "an embodiment," etc., mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in different places in this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0085] As used herein, the term "adapter" refers to a nucleotide sequence that can be added to another sequence to impart additional properties to the other sequence. An adaptor can be single-stranded or double-stranded, or can have both single-stranded and double-stranded portions.

[0086] As used herein, "amplification" refers to the process of increasing the number of copies. Amplification can be a process in which replication occurs repeatedly over time to form multiple copies of a template. As the amplification proceeds, the amplification can produce an exponential or linear increase in the number of copies. Exemplary amplification strategies include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), rolling circle replication (RCA), cascade RCA, nucleic acid-dependent amplification (NASBA), etc. In addition, amplification can apply linear or circular templates. Amplification can be performed under any suitable temperature conditions, such as thermal cycling or isothermal. In addition, amplification can be performed in an amplification mixture (or reagent mixture), and the amplification mixture (or reagent mixture) is any composition capable of amplifying a nucleic acid target (if any) in a mixture. PCR amplification relies on repeated cycles of heating and cooling (i.e., thermal cycling) to achieve continuous multiple rounds of replication. PCR can be performed by thermal cycling between two or more temperature set points (such as a higher denaturation temperature and a lower annealing / extension temperature) or between three or more temperature set points (such as a higher denaturation temperature, a lower annealing temperature, and an intermediate extension temperature, etc.). PCR can be performed using a thermostable polymerase, such as Taq DNA polymerase. PCR typically produces an exponential increase in the amount of the product amplicon in successive cycles. PCR is described in, e.g., U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,683,195; U.S. Pat. No. 4,000,159; U.S. Pat. No. 4,965,188; U.S. Pat. No. 5,176,995), the disclosures of which are incorporated herein by reference in their entirety.

[0087] As used herein, the term "complementary" generally refers to the ability of accurate pairing between two nucleotides. The term "complementary" refers to the ability to form favorable thermodynamic stability and specific pairing between the bases of two nucleotides under appropriate temperature and ion buffer conditions. Complementarity is achieved by different interactions between the nucleobases adenine, thymine (uracil in RNA), guanine and cytosine, wherein adenine is paired with thymine or uracil, and guanine is paired with cytosine. For example, if the nucleotides at a given position of a nucleic acid can be hydrogen-bonded with the nucleotides of another nucleic acid, it is considered that the two nucleic acids are complementary to each other at that position. The complementarity between two single-stranded nucleic acid molecules can be "partial" complementarity, i.e., only part of the nucleotides are combined, or it can also be completely complementary when there is overall complementarity between single-stranded molecules. If a first nucleotide sequence is complementary to a second nucleotide sequence, the first nucleotide sequence can be said to be the "complementary sequence" of the second sequence. If a first nucleotide sequence is complementary to the reverse (i.e., the order of the nucleotides is reversed) sequence of the second sequence, the first nucleotide sequence can be said to be the "reverse complementary sequence" of the second sequence.

[0088] As used herein, the term "enrichment" refers to the process of increasing the relative abundance of a population of molecules (e.g., nucleic acid molecules) in a sample relative to the total amount of molecules initially present in the sample prior to processing. Thus, the enrichment step provides a percentage or fractional increase, rather than directly increasing the number of copies of, for example, a target nucleic acid sequence as in amplification methods such as polymerase chain reaction.

[0089] As used herein, the term "hybridization" refers to base pairing between different nucleic acid molecules identical to their nucleotide sequences.

[0090] As used herein, the term "modified dNTP" or "modified nucleoside triphosphate" refers to any molecule suitable for replacing a corresponding unmodified or classical dNTP. Such a modified nucleotide must be able to undergo the same or similar base pair matching as the unmodified dNTP it replaces. A modified nucleotide or modified dNTP may have a chemical structure similar to the corresponding nucleotide or dNTP, but differs from the nucleotide or dNTP in at least one atom or at least one bond type.

[0091] As used herein, the term "nucleic acid" or "polynucleotide" (used interchangeably herein) refers to a polymeric form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any known or unknown function. Unless otherwise specified, the term encompasses nucleic acids or polynucleotides comprising known analogs of natural nucleotides that have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Non-limiting examples of polynucleotides include coding or non-coding regions of genes or gene fragments, loci / locus defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, synthetic polynucleotides, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.

[0092] Polynucleotides may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure may be modified before or after polymer assembly. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified, such as by being conjugated with a labeling component. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon replacements), alleles, homologous gene sequences, SNPs and complementary sequences, and sequences explicitly indicated. Specifically, degenerate codon replacements may be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)).

[0093] As used herein, the term "oligonucleotide" refers to an oligomer of nucleotide or nucleoside monomer units, wherein the oligomer optionally includes non-nucleotide monomer units and / or other chemical groups connected at internal and / or external positions of the oligomer. The oligomer can be natural or synthetic, and can include naturally occurring oligonucleotides, or include oligomers of nucleosides with non-naturally occurring (or modified) bases, sugar moieties, phosphodiester-analog bonds and / or alternative monomer unit chirality and isomeric structures (e.g., 5'-bonds to 2'-bonds, L-nucleosides, α-anomeric nucleosides, β-isomer nucleosides, locked nucleic acids (LNA), peptide nucleic acids (PNA)). For example, the oligonucleotide can be 10 to 20, 11 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, or 150 to 200 nucleotides in length.

[0094] As used herein, the term "polymerase" refers to an enzyme that performs template-directed synthesis of polynucleotides. DNA polymerases can only add free nucleotides at the 3' end of the newly formed chain. This causes the newly formed chain to elongate in the 5'-3' direction. No known DNA polymerase can start a new chain (from scratch). DNA polymerases can only add nucleotides on pre-existing 3'-OH groups, so primers are needed where they can add the first nucleotide.

[0095] As used herein, the term "primer" refers to an oligonucleotide that binds to a specific region of a single-stranded template nucleic acid molecule and initiates nucleic acid synthesis via a polymerase-mediated enzymatic reaction (extending from the 3' end of the primer) and is complementary to the sequence of the template molecule. PCR amplification primers may be referred to as "forward" and "reverse" primers, one of which is complementary to a nucleic acid strand and the other is complementary to the complementary sequence of the strand. Typically, a primer includes less than about one hundred nucleotides, and preferably includes less than about 50 nucleotides. Exemplary primers range from about five to about 25 nucleotides. Primers may include, for example, RNA and / or DNA bases, as well as non-naturally occurring bases.

[0096] As used herein, the term "unmodified dNTP" or "unmodified nucleoside triphosphates" refers to the four deoxyribonucleotide triphosphates commonly used as building blocks for DNA synthesis: dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate), and dTTP (deoxythymidine triphosphate).

[0097] As used herein, the term "sequence" when used to refer to a nucleic acid molecule refers to the order of nucleotides (or bases) in a nucleic acid molecule. In the case of different types of nucleotides in a nucleic acid molecule, the sequence includes the identification of the types of nucleotides (or bases) at various positions in the nucleic acid molecule. A sequence is a characteristic of all or part of a nucleic acid molecule. The term can be similarly used to describe the order and positional identity of monomeric units in other polymers (e.g., amino acid monomeric units of protein polymers).

[0098] As used herein, the term "sequencing" refers to determining the order and position of bases in a nucleic acid molecule. More specifically, the term "sequencing" refers to a biochemical method for determining the order of nucleotide bases, adenine, guanine, cytosine, and thymine in a DNA oligonucleotide. As used herein, sequencing may include, but is not limited to, parallel sequencing or any other sequencing method known to those skilled in the art, such as chain termination, rapid DNA sequencing, wandering-spot analysis, Maxam-Gilbert sequencing, dye terminator sequencing, or use any other modern automated DNA sequencing instrument.

[0099] As used herein, the term "target" or "target sequence" refers to nucleic acid molecule sequences of interest, such as those to which an oligonucleotide probe hybridizes.

[0100] As used herein, the term "universal primer" refers to a primer that can hybridize with a target polynucleotide with a common complementary universal primer binding site and support amplification. Similarly, the term "universal primer pair" refers to a forward and reverse primer pair that can hybridize and support the pcr amplification of a target polynucleotide with a common complementary forward and reverse universal primer binding site. Such one or more universal primers and one or more universal primer binding sites can allow a single primer or dual primer-mediated universal amplification (e.g., universal PCR) in a polynucleotide region of interest. The title provided herein is only for convenience and is not intended to explain the scope or meaning of the disclosed embodiments of the invention.

[0101] Overview

[0102] The present disclosure relates to compositions including one or more components, which contribute to improving coverage uniformity and / or reducing GC bias during one or more downstream sequencing operations. In certain embodiments, compositions described herein can be used to prepare target enrichment samples and / or amplify such target enrichment samples. The method for preparing target enrichment samples and / or amplifying target enrichment samples is disclosed in U.S. Patent Publication Nos. 2018 / 0016630 and 2020 / 0032244 and International Application Nos. PCT / US2017 / 041748 and PCT / EP2018 / 08527, and its disclosure is hereby incorporated herein in its entirety. In certain embodiments, compositions of the present disclosure can be used to prepare one or more steps of target enrichment samples, including amplification before capture, capture extension and release primer hybridization and / or extension period, and subsequently during amplification of prepared target enrichment samples after capture.

[0103] The present disclosure also relates to methods for preparing target enrichment samples and methods for amplifying one or more targets in prepared target enrichment samples. In some embodiments, the steps of pre-capture, capture extension, release primer hybridization and extension, and / or capture post-amplification are performed in the presence of one or more components, which improve coverage uniformity and / or reduce GC bias during one or more downstream sequencing operations. In some embodiments, one or more components that increase coverage uniformity and / or reduce GC bias during sequencing are enhancers, including but not limited to betaine or its derivatives or analogs, DMSO (or similar solvents with similar chemical and / or physical properties), single-stranded DNA binding proteins and disaccharides (such as trehalose). In other embodiments, one or more components that increase coverage uniformity and / or reduce GC bias during sequencing include high melting temperature primers or primers with high GC content. In yet other embodiments, one or more components that increase coverage uniformity and / or reduce GC bias are dNTP mixtures, which include unmodified dNTPs and one or more modified dNTPs; or primers including one or more modified dNTPs. Of course, as described in further detail herein, any combination of any of these components may be used during any hybridization and / or extension reaction.

[0104] The present disclosure also provides methods for improving coverage uniformity and / or reducing GC bias during sequencing by optimizing certain parameters of certain incubation steps used during the preparation of target enrichment samples. For example, the present disclosure provides methods for incubating samples at higher temperatures and / or longer durations compared to control incubation procedures. These and other embodiments are further described herein.

[0105] Composition

[0106] As described above, the present disclosure relates to compositions for hybridization and / or extension reactions, wherein the compositions include one or more components that contribute to improving coverage uniformity during sequencing and / or reducing GC bias. In some embodiments, the one or more components that contribute to improving coverage uniformity during sequencing include (i) primers with high GC content and / or high melting temperature; (ii) primers comprising one or modified dNTPs; (iii) enhancers; and / or (iv) dNTP mixtures comprising unmodified dNTPs and modified dNTPs. In some embodiments, enhancers include, but are not limited to, betaine, DMSO, single-stranded DNA binding proteins, disaccharides (e.g., trehalose), and combinations thereof. Each of these components is described herein.

[0107] In some embodiments, the compositions of the present disclosure include (a) primers with high GC content and / or high melting temperature; and (b) both one or more enhancers. In other embodiments, the compositions of the present disclosure include (a) primers with high GC content and / or high melting temperature; and (b) both primers including one or more modified dNTPs. In other embodiments, the compositions of the present disclosure include (a) primers including one or modified dNTPs; and (b) both one or more enhancers. In further embodiments, the compositions of the present disclosure include (a) one or more enhancers; and (b) a mixture of modified dNTPs and unmodified dNTPs. In even further embodiments, the compositions of the present disclosure include (a) primers with high GC content and / or high melting temperature; (b) primers including one or modified dNTPs; and (c) one or more enhancers. In yet further embodiments, the compositions of the present disclosure include (a) primers having high GC content and / or high melting temperature; (b) primers including one or modified dNTPs; and (c) a mixture of unmodified dNTPs and modified dNTPs.

[0108] In some embodiments, the composition includes one or more substrates; one or more polymerases; deoxynucleoside triphosphates (dNTPs) (e.g., unmodified dNTPs or a mixture of unmodified dNTPs and modified dNTPs); and at least one enhancer. In other embodiments, the composition includes one or more primers, wherein at least one of the one or more primers has a high melting temperature (or high GC content) and / or includes one or more modified dNTPs; one or more polymerases; dNTPs (e.g., unmodified dNTPs or a mixture of unmodified dNTPs and modified dNTPs); and optionally at least one enhancer. In some embodiments, the composition further includes one or more input nucleic acid molecules; one or more buffers; one or more divalent cations; one or more cofactors; and / or one or more polyols.

[0109] In some embodiments, the composition includes one or more substrates; one or more nucleic acid molecules; and at least one enhancer. In other embodiments, the composition includes one or more primers, wherein at least one of the one or more primers has a high melting temperature (or high GC content) and / or includes one or more modified dNTPs (e.g., unmodified dNTPs or a mixture of unmodified dNTPs and modified dNTPs); one or more nucleic acid molecules; and optionally one or more enhancers. In some embodiments, the composition further includes one or more polymerases; dNTPs; one or more buffers; one or more divalent cations; one or more cofactors; and / or one or more polyols.

[0110] Primers

[0111] Primers, including any one of the capture pre-primers (e.g., capture pre-forward primers and capture pre-reverse primers), capture primers, release primers and / or capture post-amplification primers described herein, can be synthesized to include at least one modified dNTP, including any one of the modified dNTPs described herein. Similarly, primers, including any one of the capture pre-primers (e.g., capture pre-forward and capture pre-reverse primers), capture primers, release primers and / or capture post-amplification primers described herein, can be synthesized to include high GC content and / or have high melting temperature. In addition, primers, including any one of the capture pre-primers (e.g., capture pre-forward and capture pre-reverse primers), capture primers, release primers and / or capture post-amplification primers described herein, can be synthesized to include (i) high GC content and / or have high melting temperature; and (ii) one or more modified dNTPs.

[0112] In certain embodiments, compositions of the present disclosure include one or more primers, wherein at least one primer in one or more primers has high GC content and / or high melting temperature." high GC content " refers to that at least one primer in one or more primers has a GC content greater than about 70%. For example, high GC content primers can have at least about 75%, at least about 80%, at least about 81% GC content, at least about 82% GC content, at least about 83% GC content, at least about 84% GC content, at least about 85% GC content, at least about 86% GC content, at least about 87% GC content, at least about 88% GC content, at least about 89% GC content, at least about 90% GC content, at least about 91% GC content, at least about 92% GC content, at least about 93% GC content, at least about 94% GC content, at least about 95% GC content, at least about 96% GC content, at least about 97% GC content, at least about 98% GC content or at least about 99% GC content. In some embodiments, high GC primers have no restrictions on GC content.

[0113] "High melting temperature" means that at least one primer of the one or more primers has a melting temperature greater than 63°C. In some embodiments, at least one of the one or more primers has a melting temperature greater than about 64°C, greater than about 65°C, greater than about 66°C, greater than about 67°C, greater than about 68°C, greater than about 69°C, greater than about 70°C, greater than about 71°C, greater than about 72°C, greater than about 73°C, greater than about 74°C, greater than about 75°C, greater than about 76°C, greater than about 77°C, greater than about 78°C, greater than about 79°C, greater than about 80°C, greater than about 81°C, greater than about 82°C, greater than about 83°C, greater than about 84°C, greater than about 85°C, greater than about 86°C, greater than about 87°C, greater than about 88°C, greater than about 89°C, greater than about 90°C, greater than about 91°C, greater than about 92°C, greater than about 93°C, greater than about 94°C, greater than about 95°C, greater than about 96°C, greater than about 97°C, greater than about 98°C, greater than about 99°C, greater than about 100°C, or the like.

[0114] In some embodiments, the primers included in any primer set may have a range of between about 57°C to about 63°C, between about 57°C to about 64°C, between about 57°C to about 65°C, between about 57°C to about 66°C, between about 57°C to about 67°C, between about 57°C to about 68°C, between about 57°C to about 69°C, between about 57°C to about 70°C, between about 57°C to about 71°C, between about 57°C to about 72°C, between about 57°C to about 73°C, between about 57°C to about 74°C, between about 57°C to about 75°C, between about 57°C to about 76°C, between about 57°C to about 77°C, between about 57°C to about 78°C, between about 57°C to about 79°C, between about 57°C to about 80°C, between about 57°C to about 81°C, between about 57°C to about 82°C, between about 57°C to about 83°C, between about 57°C to about 84°C ℃ to about 81 ℃, between about 57 ℃ to about 82 ℃, between about 57 ℃ to about 83 ℃, between about 57 ℃ to about 84 ℃, between about 57 ℃ to about 85 ℃, between about 57 ℃ to about 86 ℃, between about 57 ℃ to about 87 ℃, between about 57 ℃ to about 88 ℃, between about 57 ℃ to about 89 ℃, between about 57 ℃ to about 90 ℃, between about 57 ℃ to about 91 ℃, between about 57 ℃ to about 92 ℃, between about 57 ℃ to about 93 ℃, between about 57 ℃ to about 94 ℃, between about 57 ℃ to about 95 ℃, between about 57 ℃ to about 96 ℃, between about 57 ℃ to about 97 ℃, between about 57 ℃ to about 98 ℃, between about 57 ℃ to about 99 ℃, between about 57 ℃ to about 100 ℃, and the like. In some embodiments, at least one primer in the primer set having any of the above temperature ranges has a melting temperature greater than 63° C. Of course, one skilled in the art will appreciate that any of the primers within the primer set (including at least one primer having a melting temperature greater than 63° C.) may include one or more modified dNTPs, e.g., one or more modified dGTPs or dATPs.

[0115] In some embodiments, at most about 1% of the primers in any primer set (in any composition) have high GC content and / or high melting temperature. In some embodiments, at most about 2% of the primers in any primer set have high GC content and / or high melting temperature. In some embodiments, at most about 3% of the primers in any primer set have high GC content and / or high melting temperature. In some embodiments, at most about 4% of the primers in any primer set have high GC content and / or high melting temperature. In some embodiments, at most about 5% of the primers in any primer set have high GC content and / or high melting temperature. In some embodiments, at most about 6% of the primers in any primer set have high GC content and / or high melting temperature. In some embodiments, at most about 7% of the primers in any primer set have high GC content and / or high melting temperature. In some embodiments, at most about 8% of the primers in any primer set have high GC content and / or high melting temperature. In some embodiments, at most about 9% of the primers in any primer set have high GC content and / or high melting temperature. In some embodiments, at most about 10% of the primers in any primer set have high GC content and / or high melting temperature. In some embodiments, at most about 15% of the primers in any primer set have high GC content and / or high melting temperature. In some embodiments, at most about 20% of the primers in any primer set have high GC content and / or high melting temperature. In some embodiments, at most about 25% of the primers in any primer set have high GC content and / or high melting temperature. In some embodiments, at most about 30% of the primers in any primer set have high GC content and / or high melting temperature.

[0116] In some embodiments, two or more modified dNTPs may be used in any one of the primers in any one of the compositions described herein. In some embodiments, a primer may include two or more identical modified dNTPs. In other embodiments, a primer may include two or more different modified dNTPs. In some embodiments, a forward primer and a reverse primer may include identical or different dNTP analogs. In some embodiments, up to about 5% of the nucleoside triphosphates included in any primer are modified dNTPs. In some embodiments, up to about 10% of the nucleoside triphosphates included in any primer are modified dNTPs. In other embodiments, up to about 15% of the nucleoside triphosphates included in any primer are modified dNTPs. In yet other embodiments, up to about 20% of the nucleoside triphosphates included in any primer are modified dNTPs. In further embodiments, up to about 25% of the nucleoside triphosphates included in any primer are modified dNTPs. In even further embodiments, no more than about 30% of the nucleoside triphosphates included within any primer are modified dNTPs. In yet further embodiments, no more than about 35% of the nucleoside triphosphates included within any primer are modified dNTPs.

[0117] In some embodiments, at least one substrate in any composition may simultaneously have (i) high GC content and / or high melting temperature; and (ii) may further include one or more modified dNTPs, such as two or more modified dNTPs, three or more modified dNTPs, four or more modified dNTPs, six or more modified dTNPs, seven or more modified dNTPs, etc. The plurality of primers included in any composition may simultaneously have (i) high GC content and / or high melting temperature; and (ii) may further include one or more modified dNTPs, such as two or more modified dNTPs, three or more modified dNTPs, four or more modified dNTPs, six or more modified dTNPs, seven or more modified dNTPs, etc.

[0118] In certain embodiments, the primer is a target-specific primer having a sequence complementary to the target nucleic acid sequence. An example of a target-specific primer is a gene-specific primer designed to hybridize with a target gene (e.g., cDNA, genomic DNA) or its vicinity (e.g., upstream or 5'). The target nucleic acid can be RNA, DNA, or a combination thereof.

[0119] dNTP

[0120] In some embodiments, the compositions of the present disclosure include one or more dNTPs, including unmodified dNTPs and / or modified dNTPs. In some embodiments, dNTPs are selected from the group consisting of unmodified dNTPs (dCTP, dATP, dGTP, dTTP, and dUTP). In other embodiments, one or more modified dNTPs are used to replace unmodified dNTPs or one or more modified dNTPs are used in addition to unmodified dNTPs.

[0121] Modified dNTPs include, but are not limited to, 5-aminoallyl-2'-dCTP, 5-(3-aminoallyl)-2'-deoxycytidine-5'-triphosphate (5-aminoallyl-2'-dCTP), 2'-deoxycytidine-5'-O-(1-thiotriphosphate) ((1-thio)-2'-dCTP), 2'-deoxy-5-methylcytidine 5'-triphosphate (5-methyl-2'-dCTP), 2-thio-2'-deoxycytidine-5'-triphosphate (2-thio-2'-dCTP), 5-iodo-2'-deoxycytidine-5'-triphosphate (5-iodo-2'-dCTP), 2-amino-2'-deoxyadenosine-5'-triphosphate (2-amino-2'-dATP), 2-thiothymidine-5'-triphosphate (5-thiothymidine-5'-triphosphate). phosphate (thio-TTP), 5-propynyl-2'-deoxycytidine-5'-triphosphate (5-propynyl-2'-dCTP), N4-methyl-2'-deoxycytidine-5'-triphosphate (N4-methyl-2'-dCTP), 7-deaza-2'-deoxyadenosine-5'-triphosphate (7-deaza-2'-dATP), 2'-deoxyguanosine-5'-O-(1-thiotriphosphate) ((1-thio)-2'-dGTP), 2'-deoxyadenosine-5'-O-(1-thiotriphosphate) ((1-thio)-2'-dATP), 5-bromo-2'-deoxycytidine-5'-triphosphate (5-bromo-2'-dCTP) and 7-deaza-2'-deoxyguanosine-5'-triphosphate (7-deaza-dGTP). In some embodiments, the modified dNTP is selected from one of (1-thio)-2'-dCTP, N4-methyl-2'-dCTP, 7-deaza-2'-dATP, 2-amino-2'-dATP, (1-thio)-2'dGTP and 7-deaza-dGTP. In other embodiments, the modified dNTP is selected from 2-amino-2'-dATP and 7-deaza-dGTP.

[0122] In some embodiments, the compositions of the present disclosure are mixtures of unmodified dNTPs and modified dNTPs. In some embodiments, the ratio between the modified dNTPs and the corresponding unmodified dNTPs can range from about 1:3, about 1:2, about 1:1, about 2:1, and about 3:1. In some embodiments, the ratio of unmodified dNTPs to modified dNTPs is about 1:1.

[0123] In some embodiments, the composition may include between about 0.1 mM and about 0.7 mM of unmodified dNTPs; and may further include one or more modified dNTPs. In other embodiments, the composition may include between about 0.1 mM and about 0.6 mM of unmodified dNTPs; and may further include one or more modified dNTPs. In still other embodiments, the composition may include between about 0.2 mM and about 0.5 mM of unmodified dNTPs; and may further include one or more modified dNTPs.

[0124] In some embodiments, the composition may comprise an unmodified dNTP between about 0.1 mM and about 0.7 mM; and may further comprise one or more modified dNTPs between about 0.1 mM and about 0.7 mM. In other embodiments, the composition may comprise an unmodified dNTP between about 0.1 mM and about 0.6 mM; and may further comprise one or more modified dNTPs between about 0.1 mM and about 0.6 mM. In other embodiments, the composition may comprise an unmodified dNTP between about 0.2 mM and about 0.5 mM; and may further comprise one or more modified dNTPs between about 0.2 mM and about 0.5 mM. In further embodiments, the composition may comprise an unmodified dNTP between about 0.2 mM and about 0.4 mM; and may further comprise one or more modified dNTPs between about 0.2 mM and about 0.4 mM. In even further embodiments, the composition may comprise about 0.3 mM of unmodified dNTPs; and may further comprise between about 0.2 mM and about 0.5 mM of one or more modified dNTPs. In even further embodiments, the composition may comprise about 0.3 mM of unmodified dNTPs; and may further comprise about 0.3 mM of one or more modified dNTPs.

[0125] In some embodiments, the composition may include between about 0.1 mM and about 0.7 mM of unmodified dNTPs; and may further include between about 0.1 mM and about 0.8 mM of one or more modified dNTPs. In other embodiments, the composition may include between about 0.1 mM and about 0.6 mM of unmodified dNTPs; and may further include between about 0.2 mM and about 0.7 mM of one or more modified dNTPs. In other embodiments, the composition may include between about 0.2 mM and about 0.5 mM of unmodified dNTPs; and may further include between about 0.2 mM and about 0.6 mM of one or more modified dNTPs.

[0126] Enhancer

[0127] The compositions of the present disclosure may optionally include one or more enhancers selected from betaine (or any derivative or analog thereof), DMSO (or a solvent with similar chemical and / or physical properties, such as DMF), single-stranded DNA binding proteins, disaccharides (e.g., trehalose, sucrose, lactose, maltose, etc.), and combinations thereof. In some embodiments, the compositions of the present disclosure may include one enhancer. In other embodiments, the compositions of the present disclosure may include two enhancers. In yet other embodiments, the compositions of the present disclosure may include three enhancers. In further embodiments, the compositions of the present disclosure may include four enhancers.

[0128] In some embodiments, the enhancer is betaine (or a derivative or analog thereof) and the concentration of betaine in any composition ranges between about 0.05 mM and about 1 mM. In other embodiments, the enhancer is betaine and the concentration of betaine in any composition ranges between about 0.1 mM to about 0.9 mM. In other embodiments, the enhancer is betaine and the concentration of betaine in any composition ranges between about 0.2 mM to about 0.8 mM. In other embodiments, the enhancer is betaine and the concentration of betaine in any composition ranges between about 0.3 mM to about 0.7 mM. In other embodiments, the enhancer is betaine and the concentration of betaine in any composition ranges between about 0.4 mM to about 0.6 mM. In some embodiments, the enhancer is betaine, and the concentration of betaine in any composition is at least about 0.1 mM, at least about 0.2 mM, at least about 0.3 mM, at least about 0.4 mM, at least about 0.5 mM, at least about 0.6 mM, at least about 0.7 mM, at least about 0.8 mM, at least about 0.9 mM, etc.

[0129] In some embodiments, the enhancer is DMSO and DMSO may be present in any composition in an amount of up to about 15% (v / v). In some embodiments, the enhancer is DMSO and DMSO may be present in any composition in an amount of up to about 10% (v / v). In some embodiments, the enhancer is DMSO and DMSO may be present in any composition in an amount of up to about 9% (v / v). In some embodiments, the enhancer is DMSO and DMSO may be present in any composition in an amount of up to about 8% (v / v). In some embodiments, the enhancer is DMSO and DMSO may be present in any composition in an amount of up to about 7% (v / v). In some embodiments, the enhancer is DMSO and DMSO may be present in any composition in an amount of up to about 6% (v / v). In some embodiments, the enhancer is DMSO and DMSO may be present in any composition in an amount of up to about 5% (v / v). In some embodiments, the enhancer is DMSO and DMSO may be present in any composition in an amount of up to about 4% (v / v). In some embodiments, the enhancer is DMSO and DMSO may be present in any composition in an amount up to about 3% (v / v). In some embodiments, the enhancer is DMSO and DMSO may be present in any composition in an amount up to about 2% (v / v). In some embodiments, the enhancer is DMSO and DMSO may be present in any composition in an amount up to about 1 (v / v).

[0130] In some embodiments, the enhancer is DMSO and the amount of DSMO in any composition ranges from about 0.1% (v / v) to about 10% (v / v) of the composition. In other embodiments, the enhancer is DMSO and the amount of DSMO in any composition ranges from about 0.5% (v / v) to about 10% (v / v) of the composition. In other embodiments, the enhancer is DMSO and the amount of DSMO in any composition ranges from about 1% (v / v) to about 10% (v / v) of the composition. In other embodiments, the enhancer is DMSO and the amount of DSMO in any composition ranges from about 2% (v / v) to about 8% (v / v) of the composition. In other embodiments, the enhancer is DMSO and the amount of DSMO in any composition ranges from about 3% (v / v) to about 7% (v / v) of the composition.

[0131] In some embodiments, the single-stranded DNA binding protein is a thermostable binding protein, such as some derived from hyperthermophilic microorganisms (e.g., ET SSB, available from New England BioLabs, Inc.). In other embodiments, the single-stranded DNA binding protein is derived from Escherichia coli, Drosophila or Xenopus, from the 32, 41, 44, 45 or 61 gene proteins of T4 phage, or RPA proteins in eukaryotes. Non-limiting examples of single-stranded DNA binding proteins are gene 2.5 proteins of phage T7 and equivalent such T7-type phage proteins or other proteins. Examples of T7-type phages include T7, T3, ΦII, H, W31, gh-1, Y, A1122 and SP6. In some embodiments, the single-stranded DNA binding protein can be present in an amount ranging from 0 ng / μL to about 50 ng / μL. In some embodiments, the single-stranded DNA binding protein can be present in an amount ranging from 1 ng / μL to about 50 ng / μL. In some embodiments, the single-stranded DNA binding protein can be present in an amount ranging from 5 ng / μL to about 50 ng / μL. In some embodiments, the single-stranded DNA binding protein can be present in an amount ranging from 10 ng / μL to about 50 ng / μL. In some embodiments, the single-stranded DNA binding protein can be present in an amount ranging from 5 ng / μL to about 40 ng / μL.

[0132] In some embodiments, the compositions of the present disclosure include disaccharides, such as trehalose. In some embodiments, the disaccharide is present in a concentration ranging from about 0 M to about 1 M. In some embodiments, the disaccharide is present in a concentration ranging from about 0.1 M to about 1 M. In some embodiments, the disaccharide is present in a concentration ranging from about 0.1 M to about 0.8 M. In some embodiments, the disaccharide is present in a concentration ranging from about 0.2 M to about 0.8 M.

[0133] Polymerase

[0134] In some embodiments, the compositions of the present disclosure include one or more polymerases. Non-limiting examples of polymerases include prokaryotic DNA polymerases (e.g., Pol I, Pol II, Pol III, Pol IV, and Pol V), eukaryotic DNA polymerases, archaeal DNA polymerases, telomerases, reverse transcriptases, and RNA polymerases. Reverse transcriptases are RNA-dependent DNA polymerases that can synthesize DNA from RNA templates. The reverse transcriptase family contains DNA polymerase function and RNase H function, which can degrade paired RNA bases into DNA. RNA polymerases are enzymes that use DNA as a template to synthesize RNA during gene transcription. RNA polymerases polymerize ribonucleotides at the 3' end of RNA transcripts.

[0135] In some embodiments, polymerases from the following can be used for polymerase-mediated primer extension, terminal modification (e.g., terminal transferase, degradation, or blunting), or amplification reactions: Archaea (e.g., Thermococcus litoralis (Vent, GenBank: AAA72101), Pyrococcus furiosus (Pfu, GenBank: D12983, BAA02362), Pyrococcus woesii, Pyrococcus GB-D (Deep Vent, GenBank: AAA67131), Thermococcus kodakaraensis KODI (KOD, GenBank: BD175553, BAA06142; Thermococcus strain KOD (Pfx, GenBank: AAE68738), Thermococcus caucasus (Thermococcus kodakaraensis KODI, GenBank: BD175553, BAA06142), Thermococcus caucasus (Thermococcus caucasus) (Pfx, GenBank: AAE68738), Pyrococcus woesii (Pyrococcus woesii, Pyrococcus GB-D (Deep Vent, GenBank: AAA67131), Thermococcus kodakaraensis KODI (KOD, GenBank: BD175553, BAA06142), Pyrococcus caucasus (Thermococcus caucasus) (Pfx, GenBank: AAE68738), Pyrococcus caucasus (Pyrococ ... gorgonarius)(Tgo,Pdb:4699806), Sulfolobus solataricus(GenBank:NC002754,P26811), Aeropyrumpernix(GenBank:BAA81109), Archaeglobus fulgidus(GenBank:029753), Pyrobaculum aerophilum(GenBank:AAL63952), Pyrodictium occultum(GenBank:BAA07579,BAA07580), Thermococcus 9 degrees Nm(GenBank:AAA88769,Q56366), Thermococcus fumicolans (GenBank: CAA93738, P74918), Thermococcus hydrothermalis (GenBank: CAC18555), Thermococcus GE8 (GenBank: CAC12850), Thermococcus JDF-3 (GenBank: AX135456, WO0132887), Thermococcus TY (GenBank: CAA73475), Pyrococcus abyssi (GenBank: P77916), Pyrococcusglycovorans)(GenBank:CAC12849), Pyrococcus horikoshii(GenBank:NP 143776), Pyrococcus GE23(GenBank:CAA90887), Pyrococcus ST700(GenBank:CAC 12847), Thermococcus pacificus(GenBank:AX411312.1), Thermococcus zilligii(GenBank:DQ3366890), Thermococcus aggregans, Thermococcus barossii, Thermococcus celer(GenBank:DD259850.1), Thermococcus profundus)(GenBank:E14137), Thermococcus siculi(GenBank:DD259857.1), Thermococcusthioreducens, Thermococcus onnurineusNA1, Sulfolobus acidocaldarium, Sulfolobus tokodaii, Pyrobaculum calidifontis, Pyrobaculum islandicum(GenBank:AAF27815), Methanococcus jannaschii jannaschii) (GenBank: Q58295), Desulfurococcus species TOK, Desulfurococcus, Pyrolobus, Pyrodictium, Staphylothermus, Vulcanisaetta, Methanococcus (GenBank: P52025, and other archaeal B polymerases, such as GenBank AAC62712, P956901, BAAA07579)), thermophilic bacteria Thermus species (e.g., Thermus flavus, Thermus ruber, Thermus thermophilus, Thermus lactis, Thermuslacteus), Thermus rubens, Thermus aquaticus), Bacillus stearothermophilus, Thermotogamaritima, Methanothermus fervidus, KOD polymerase, TNA1 polymerase, Pyrococcus 9 degrees N-7, T4, T7, phi29, Pyrococcus furiosus), P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, Thermococcus GT, Pyrococcus GB-D, KOD, Pfu, T. gorgonarius, T. zilligii, T. litoralis, and Thermococcus sp. 9N-7 polymerase.

[0136] In certain embodiments, the compositions of the present disclosure include one or more thermostable polymerases. As used herein, the term "thermostable polymerase" refers to a thermostable enzyme, which is heat-resistant, and after the time required for achieving double-stranded nucleic acid denaturation at high temperature, it retains enough activity to achieve subsequent polynucleotide extension reactions, and will not be irreversibly denatured (inactivated). The necessary heating conditions for nucleic acid denaturation are well known in the art and are illustrated in, for example, U.S. Patent Nos. 4,683,202, 4,683,195 and 4,965,188, which are incorporated herein by reference. As used herein, thermostable polymerases are suitable for use in temperature cycling reactions such as polymerase chain reaction ("PCR"), primer extension reactions or end modification (e.g., terminal transferase, degradation or error correction (polishing)) reactions. Irreversible denaturation for the purposes of this article refers to permanent and complete loss of enzyme activity. For thermostable polymerases, enzyme activity refers to the catalytic effect of combining nucleotides in an appropriate manner to form polynucleotide extension products complementary to the template nucleic acid chain. Thermostable DNA polymerases include DNA polymerases from Thermotoga maritima, Thermus aquaticus, Thermus thermophilus, Thermus flavus, Thermus filiformis, Thermus species sps17, Thermus species Z05, Thermus caldophilus, Bacillus caldotenax, Thermotoga neopolitana, Thermosipho africanus, and other thermostable DNA polymerases disclosed above.

[0137] One example of a polymerase is Taq or a Taq-derived polymerase (eg, KAPA2G polymerase from KAPA BIOSYSTEMS). Another example of a polymerase is a B-family DNA polymerase (eg, KAPAHIFI polymerase from KAPA BIOSYSTEMS).

[0138] Input nucleic acid molecules

[0139] In some embodiments, the input nucleic acid molecule or the input nucleic acid molecule library is included in the composition of the present disclosure. In some embodiments, the nucleic acid molecule in the nucleic acid library obtained is selected from a DNA molecule, an RNA molecule, a genomic DNA molecule, a cDNA molecule, an mRNA molecule, an rRNA molecule, an mtDNA, an siRNA molecule or a combination thereof. In some embodiments, a plurality of nucleic acid molecules comprise a single-stranded polynucleotide. In some embodiments, a plurality of nucleic acid molecules are derived from a tissue sample, such as a tissue sample derived from a mammalian subject. In other embodiments, a plurality of nucleic acid molecules are derived from a paraffin-embedded sample or a cytological sample fixed with formalin, such as a cytological sample derived from a mammalian subject.

[0140] In some embodiments, the nucleic acid library obtained includes multiple target nucleic acid molecules and / or multiple non-target nucleic acid molecules. In some embodiments, non-target nucleic acid molecules are highly abundant compared to target nucleic acid molecules in the nucleic acid library. In some embodiments, in the nucleic acid library obtained, non-target nucleic acid molecules account for at least 70% of nucleic acid molecules. In other embodiments, in the nucleic acid library obtained, non-target nucleic acid molecules account for at least 75% of nucleic acid molecules. In some other embodiments, in the nucleic acid library obtained, non-target nucleic acid molecules account for at least 80% of nucleic acid molecules. In further embodiments, in the nucleic acid library obtained, non-target nucleic acid molecules account for at least 85% of nucleic acid molecules. In further embodiments, in the nucleic acid library obtained, non-target nucleic acid molecules account for at least 90% of nucleic acid molecules. In further embodiments, in the nucleic acid library obtained, non-target nucleic acid molecules account for at least 95% of nucleic acid molecules. In some embodiments, in the nucleic acid library obtained, non-target nucleic acid molecules account for at least 96% of nucleic acid molecules. In some embodiments, in the nucleic acid library obtained, non-target nucleic acid molecules account for at least 97% of nucleic acid molecules. In some embodiments, in the nucleic acid library obtained, non-target nucleic acid molecules account for at least 98% of nucleic acid molecules. In some embodiments, non-target nucleic acid molecules account for at least 99% of the nucleic acid molecules in the resulting nucleic acid library.

[0141] In some embodiments, the nucleic acid molecules in any library include the first adapter and the second adapter connected. For example, in some embodiments, nucleic acid fragments are first prepared from biological samples, such as tissue samples and / or cytological samples. DNA sequencing libraries can be constructed by genomic DNA for genome analysis, or by cDNA prepared from RNA or mRNA for transcriptome analysis, and it can be constructed by DNA or cDNA of any organism species, and these nucleic acids can be extracted from any organism species. In some embodiments, the sample obtained is sheared into fragments to provide a nucleic acid fragment group. In some embodiments, mechanical (e.g., atomization or ultrasound) and / or enzymatic fragmentation (e.g., restriction endonuclease) are used to achieve the shearing of the genomic sample obtained. In some embodiments, the nucleic acid fragments generated are random sizes. In some embodiments, the nucleic acid fragments generated have a length of less than about one thousand base pairs. In other embodiments, the nucleic acid fragments generated include sequence fragments with a length range of about one hundred to about one thousand base pairs. In yet other embodiments, the nucleic acid fragments generated include sequence fragments with a length range of about five hundred to about 750 base pairs.

[0142] In some embodiments, the adapter is then added to the population of nucleic acid molecules via a ligation reaction. In some embodiments, the adapter comprises one or more barcode sequences. Methods for connecting adapters to nucleic acid molecules are described in U.S. Patent Publication Nos. 2017 / 0037459, 2018 / 0334709, 2018 / 0016630 and in PCT Publication No. WO2017021449, the disclosures of which are incorporated herein by reference in their entirety.

[0143] In some embodiments, the input nucleic acid molecule includes a complex comprising a target nucleic acid molecule hybridized with at least one primer. For example, the input nucleic acid molecule can include a complex of a target nucleic acid hybridized with a capture primer. In some embodiments, the capture primer of the complex can be extended.

[0144] In other embodiments, the input nucleic acid molecule comprises a complex comprising a target nucleic acid molecule hybridized to a first primer and a second primer (including primers with a high melting temperature and / or no restriction on GC content). For example, the input nucleic acid molecule can comprise a complex of a target nucleic acid molecule and an extended capture primer; and wherein the complex further comprises a release primer.

[0145] In other embodiments, the input nucleic acid molecule can comprise a complex of a target nucleic acid molecule and an extended released primer.

[0146] In some embodiments, the input nucleic acid molecules are target enriched, i.e., include a plurality of target nucleic acid molecules and lack or substantially lack non-target nucleic acid molecules.

[0147] Buffer

[0148] The compositions of the present disclosure may include one or more buffers. Non-limiting examples of buffers include citric acid, potassium dihydrogen phosphate, boric acid, diethyl barbituric acid, piperazine-N, N'-bis (2-ethanesulfonic acid), dimethyl arsenic acid, 2- (N-morpholino) ethanesulfonic acid, tris (hydroxymethyl) methylamine (TRIS), 2- (N-morpholino) ethanesulfonic acid (TAPS), N, N-bis (2-hydroxyethyl) glycine (Bicine), N-tris (hydroxymethyl) methylglycine (Tricine), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), 2-{[tris (hydroxymethyl) methyl] amino} ethanesulfonic acid (TES) and combinations thereof. In other embodiments, the buffer may be composed of tris(hydroxymethyl)methylamine (TRIS), 2-(N-morpholino)ethanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methylglycine (Tricine), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), or a combination thereof. Additional washing solutions, transfer solutions, acid solutions, and alkaline solutions are described in U.S. Patent Application Publication No. 2016 / 0282374, the disclosure of which is incorporated herein by reference in its entirety.

[0149] Divalent cations

[0150] In some embodiments, the compositions of the present disclosure include one or more divalent cations. In some embodiments, the divalent cation is selected from Co 2+ , Mn 2+ Mg 2+ 、Cd 2+ and Ca 2+ .

[0151] In some embodiments, any of the compositions of the present disclosure can include a divalent cation concentration of at least about 0.01 mM, about 0.02 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.5 mM, about 1 mM, about 2 mM, about 5 mM, about 8 mM, about 10 mM, about 12 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, etc. In some embodiments, any of the compositions of the present disclosure can have a CoCl concentration of at least about 0.01 mM, about 0.02 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.5 mM, about 1 mM, about 2 mM, about 5 mM, about 8 mM, about 10 mM, about 12 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, etc. In some embodiments, any of the present disclosure can have a MnCl concentration of at least about 0.01 mM, about 0.02 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.5 mM, about 1 mM, about 2 mM, about 5 mM, about 8 mM, about 10 mM, about 12 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, etc. In some embodiments, any of the compositions of the present disclosure can have a MgCl concentration of at least about 0.01 mM, about 0.02 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.5 mM, about 1 mM, about 2 mM, about 5 mM, about 8 mM, about 10 mM, about 12 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, etc. In some embodiments, any of the compositions of the present disclosure can have a CdCl concentration of at least about 0.01 mM, about 0.02 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.5 mM, about 1 mM, about 2 mM, about 5 mM, about 8 mM, about 10 mM, about 12 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, etc.In some embodiments, any of the compositions of the present disclosure can have a CaCl concentration of at least about 0.01 mM, about 0.02 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.5 mM, about 1 mM, about 2 mM, about 5 mM, about 8 mM, about 10 mM, about 12 mM, about 15 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, etc.

[0152] Polyols

[0153] In some embodiments, any of the compositions of the present disclosure includes one or more polyols. Suitable polyols include 1,2-ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 2-methyl-1,3-propylene glycol, 2-methyl-1,2-propylene glycol, 2,2-dimethyl-1,3-propylene glycol, 2,2-diethyl-1,3-propylene glycol, 2-methyl-2-propyl-1,3-propylene glycol, 2-butyl-2-ethyl-1,3-propylene glycol, dihydroxyacetone, 2,2-dibutyl-1,3-propylene glycol, 3-methoxy-1,3-propylene glycol, 3-methoxy-1,2-propylene glycol, 3-methoxy-2,3-propylene glycol, 2-methoxymethyl-1,3-propylene glycol, 3-ethoxy-1,3-propylene glycol, 3-ethoxy-1,2-propylene glycol, 3-ethoxy-2, 3-Propanediol, 3-allyloxy-1,2-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-dimethyl-2,3-butanediol, 3,3-dimethyl-1,2-butanediol, 1,2-pentanediol, 1,3-pentanediol, 1,4-pentanediol, 1,5-pentanediol, 2,3-pentanediol, 2,4-pentanediol, 2-methyl-2,4-pentanediol, 2,4-dimethyl-2,4-pentanediol, 2,2,4-trimethyl-1,3-pentanediol, 1,2-hexanediol, 1,3-hexanediol, 1,4-hexanediol, 1,5-hexanediol, 1,6-hexanediol, 2,3-hexanediol, 2,4-hexanediol, 2,5-hexanediol alcohol, 3,4-hexanediol, 2,5-dimethyl-2,5-hexanediol, 2-ethyl-1,3-hexanediol, 1,2-heptanediol, 1,3-heptanediol, 1,4-heptanediol, 1,5-heptanediol, 1,6-heptanediol, 1,7-heptanediol, 1,8-octanediol, 1,2-octanediol, 1,3-octanediol, 1,4-octanediol, 1,5-octanediol, 1,6-octanediol, 1,7-octanediol, 1,2-nonanediol, 1,9-nonanediol, 1,10-decanediol, 1,2-decanediol, 1,2-undecanediol, 1,11-undecanediol, 1,12-dodecanediol, 1,2-dodecanediol, diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol , tetrapropylene glycol, pentaethylene glycol, pentapropylene glycol, hexaethylene glycol, hexapropylene glycol, heptaethylene glycol, heptapropylene glycol, octaethylene glycol, octapropylene glycol, nonaethylene glycol, nonapropylene glycol, decaethylene glycol, decapropylene glycol, cis- or trans-1,2-cyclopentanediol, cis- or trans-1,3-cyclopentanediol, cis- or trans-1,2-cyclohexanediol, cis- or trans-1,3-cyclohexanediol, cis- or trans-1,4-cyclohexanediol, cis- or trans-1,2-cycloheptanediol, cis- or trans-1,3-cycloheptanediol, cis- or trans-1,4-cycloheptanediol, 1,2,3-cyclopentanetriol, 1,2,4-cyclopentanetriol, 1,2,3-cyclohexanetriol, 1,2,4-cyclohexanetriol, 1,2,3-cycloheptantriol, 1,2,4-cycloheptantriol, 1,2,3-propanetriol, 3-ethyl-2-hydroxymethyl-1,3-propanediol, 2-hydroxymethyl-2-methyl-1,3-propanediol, 2-hydroxymethyl-2-methyl-1,3-propanediol, 1,2,3-butanetriol, 1,2,4-butanetriol, 2-methyl-1,2,3-butanetriol, 2-methyl-1,2,4-butanetriol, 1,2,3-pentanetriol, 1,2,4-pentanetriol, 1,2,5-pentanetriol, 2,3,4-pentanetriol, 1,3,5-pentanetriol, 3-methyl-1,3,5-pentanetriol, 1,2,3-hexanetriol, 1,2,4-hexanetriol, 1,2,5-hexanetriol, 1,2,6-hexanetriol, 2,3,4-hexanetriol, 2,3,5-hexanetriol, 1,2,3-heptanetriol, 1,2,7-heptanetriol, 1,2,3-octantriol, 1,2,8-octantriol, 1,2,3-nonanetriol, 1,2,9-nonanetriol, 1 ,2,3-decantriol, 1,2,10-decantriol, 1,2,3-undecanetriol, 1,2,11-undecanetriol, 1,2,3-dodecanetriol, 1,1,12-dodecanetriol, 2,2-bis(hydroxymethyl)-1,3-propanediol, 1,2,3,4-butanetetrol, 1,2,3,4-pentanetetrol, 1,2,3,5-pentanetetrol, 1,2,3,4-hexanetetrol, 1,2,3,6-hexanetetrol, 1,2,3, 4-heptetrol, 1,2,3,7-heptetrol, 1,2,3,4-octanetrol, 1,2,3,8-octanetrol, 1,2,3,4-nonanetrol, 1,2,3,9-nonanetrol, 1,2,3,4-decanetrol, 1,2,3,10-decanetrol, trimethylolpropanol, pentaerythritol, sugar alcohols such as mannitol, sorbitol or arabitol, hexapentol, 1,2,3,4,5-pentapentol and 1,2,3,4,5,6-hexapentol.

[0154] In some embodiments, the polyol is 1,2,3-propanetriol (also known as glycerol).

[0155] In some embodiments, the polyol may be present in an amount ranging from about 1% to about 10% by total volume of the composition; from about 2% to about 8% by total volume of the composition; from about 3% to about 7% by total volume of the composition; from about 4% to about 6% by total volume of the composition.

[0156] Examples of compositions

[0157] An example of a composition includes a primer set; a polymerase; and betaine. In some embodiments, betaine is present at a concentration ranging from about 0.3mM to about 0.7mM. In some embodiments, the concentration of betaine is about 0.5mM. In some embodiments, at least one primer in the primer set has a high melting temperature and / or a high GC content (e.g., a melting temperature greater than 63°C). In some embodiments, each of the multiple primers in the primer set has a high melting temperature and / or a high GC content (e.g., a melting temperature greater than 63°C). In some embodiments, there is no restriction or limitation on the melting temperature and / or GC content of any one of the primers in the primer set. In some embodiments, at most about 10% of the primers in the composition have a high melting temperature and / or a high GC content. In some embodiments, the composition further includes a mixture of modified dNTPs and modified dNTPs. In some embodiments, the ratio of modified dNTPs to unmodified dNTPs is about 1:1. In some embodiments, the composition further includes at least one of a buffer, a divalent cation, and a polyol. In certain embodiments, composition comprises at least two of buffer, divalent cation and polyol.In certain embodiments, composition comprises each of buffer, divalent cation and polyol.In certain embodiments, composition can be used for amplification of nucleic acid molecules, such as nucleic acid molecules present in nucleic acid library.In certain embodiments, composition can be used for capture front amplification.In certain embodiments, composition can be used for one or more steps of target enrichment, such as during hybridization and extension of capture primer (as further described herein).

[0158] Another example of a composition comprises a primer set; a polymerase; and optionally a betaine; wherein at least one primer in the primer set has a high melting temperature and / or a high GC content. In some embodiments, at least one primer in the primer set has a melting temperature greater than about 64°C, greater than about 65°C, greater than about 66°C, greater than about 67°C, greater than about 68°C, greater than about 69°C, greater than about 70°C, greater than about 71°C, greater than about 72°C, greater than about 73°C, greater than about 74°C, greater than about 75°C, greater than about 76°C, greater than about 77°C, greater than about 78°C, greater than about 79°C, greater than about 80°C, greater than about 81°C, greater than about 82°C, greater than about 83°C, greater than about 84°C, greater than about 85°C, greater than about 86°C, greater than about 87°C, greater than about 88°C, greater than about 89°C, greater than about 90°C, greater than about 91C, greater than about 92°C, greater than about 93°C, greater than about 94°C, greater than about 95°C, greater than about 96°C, greater than about 97°C, greater than about 98°C, greater than about 99°C, greater than about 100°C, or the like. In some embodiments, the multiple primers in the primer set each have a high melting temperature and / or a high GC content (e.g., a melting temperature greater than 63°C). In some embodiments, there is no restriction or limitation on the melting temperature and / or GC content of any one of the primers in the primer set. In some embodiments, at most about 10% of the primers in the composition have a high melting temperature and / or a high GC content. In some embodiments, betaine is present in a concentration ranging from about 0.3mM to about 0.7mM. In some embodiments, the composition further comprises a mixture of modified dNTPs and modified dNTPs. In some embodiments, the ratio of modified dNTPs to unmodified dNTPs is about 1:1. In some embodiments, the composition further comprises at least one of a buffer, a divalent cation, and a polyol. In some embodiments, the composition comprises at least two of a buffer, a divalent cation, and a polyol. In some embodiments, the composition comprises each of a buffer, a divalent cation, and a polyol. In some embodiments, the composition can be used to amplify nucleic acid molecules, such as nucleic acid molecules present in a nucleic acid library. In some embodiments, the composition can be used to capture pre-amplification. In some embodiments, the compositions can be used in one or more steps of target enrichment, such as during hybridization and extension of capture primers (as further described herein).

[0159] method

[0160] The present disclosure also relates to methods of enriching a sample for a plurality of target nucleic acid molecules (e.g., between about one to about ten thousand target nucleic acid molecules, or between about one to about five thousand target nucleic acid molecules, or between about one to about 1000 target nucleic acid molecules).

[0161] The present disclosure also relates to a method for amplifying a target enriched sample, such as a target enriched sample prepared using any of the methods described herein. In addition, the present disclosure also relates to a method for sequencing using a target enriched sample, such as a target enriched sample prepared using any of the methods described herein. The method disclosed herein can be used as part of a sequencing scheme, including a high-throughput single-molecule sequencing scheme. In some embodiments, the method disclosed herein generates an enriched library of target nucleic acid molecules to be sequenced. The target nucleic acid molecules enriched in the library can be optionally incorporated into a barcode for molecular identification and sample identification, such as described in U.S. Publication No. 2020 / 0032244 and U.S. Patent Nos. 7,393,665, 8,168,385, 8,481,292, 8,685,678 and 8,722,368, the disclosure of which is incorporated herein by reference in its entirety.

[0162] refer to Figure 1 , the method 100 for target enrichment by unidirectional dual probe primer extension includes a step 102 of preparing a nucleic acid library for enrichment. In some embodiments, step 102 includes fragmentation (optional), adapter connection (connected to each end of the nucleic acid molecule) and pre-capture amplification. Ultimately, the product of step 102 produces a nucleic acid molecule library, wherein each of the nucleic acid molecules in the nucleic acid molecule library has a first end comprising a first adapter and a second end comprising a second adapter. In some embodiments, the first adapter and the second adapter may be the same or different.

[0163] In some aspects, nucleic acid libraries can be prepared from any nucleic acid molecule source including one or more target nucleic acid molecules. Generally speaking, the target nucleic acid will include a target region or sequence, and method 100 helps to preferentially enrich one or more target nucleic acid molecules relative to non-target nucleic acid molecules in the nucleic acid library for downstream detection and analysis of those target regions or sequences.

[0164] After connecting the adapter, the adapter-connected (and optionally fragmented) nucleic acid molecules are amplified (capture pre-amplification). In some embodiments, capture pre-amplification occurs using any of the compositions described herein. For example, a suitable composition (e.g., capture pre-amplification master mixture composition) may include one or more primers; one or more polymerases; dNTPs (e.g., unmodified dNTPs and a mixture of unmodified and modified dNTPs); and at least one enhancer. As another example, the capture pre-master mixture composition may include one or more primers, wherein at least one of the one or more primers has a high melting temperature (or high GC content) and / or includes one or more modified dNTPs; one or more polymerases; dNTPs; and optionally at least one enhancer (e.g., betaine, dimethyl sulfoxide (DMSO), trehalose, and / or single-stranded DNA binding proteins, such as in amounts / concentrations described herein).

[0165] In some embodiments, a capture pre-primer set (e.g., a forward capture pre-primer and a reverse capture pre-primer) can be used for capture pre-amplification, wherein at least one primer in the capture pre-primer set has a high GC content and / or a high melting temperature (e.g., a melting temperature greater than 63°C, greater than about 66°C, greater than about 69°C, greater than about 72°C, greater than about 75°C, etc.). In some embodiments, the introduced capture pre-primer may have no restrictions on GC content and / or melting temperature. In some embodiments, at most about 25% of the primers used during capture pre-amplification are high melting temperatures and / or high GC content primers. In some embodiments, at most about 20% of the primers used during capture pre-amplification are high melting temperatures and / or high GC content primers. In some embodiments, at most about 15% of the primers used during capture pre-amplification are high melting temperatures and / or high GC content primers. In some embodiments, at most about 12% of the primers used during capture pre-amplification are high melting temperatures and / or high GC content primers. In some embodiments, at most about 10% of the primers used during the pre-capture amplification are high melting temperature and / or high GC content primers. In some embodiments, at most about 9% of the primers used during the pre-capture amplification are high melting temperature and / or high GC content primers. In some embodiments, at most about 8% of the primers used during the pre-capture amplification are high melting temperature and / or high GC content primers. In some embodiments, at most about 7% of the primers used during the pre-capture amplification are high melting temperature and / or high GC content primers. In some embodiments, at most about 6% of the primers used during the pre-capture amplification are high melting temperature and / or high GC content primers. In some embodiments, at most about 5% of the primers used during the pre-capture amplification are high melting temperature and / or high GC content primers. In some embodiments, at most about 4% of the primers used during the pre-capture amplification are high melting temperature and / or high GC content primers. In some embodiments, at most about 3% of the primers used during the pre-capture amplification are high melting temperature and / or high GC content primers. In some embodiments, at most about 2% of the primers used during pre-capture amplification are high melting temperature and / or high GC content primers. In some embodiments, at most about 1% of the primers used during pre-capture amplification are high melting temperature and / or high GC content primers.

[0166] In other embodiments, the pre-capture amplification can be performed in the presence of a pre-capture primer set (e.g., a forward pre-capture primer and a reverse pre-capture primer), wherein at least one primer in the primer set has (i) a high GC content and / or a high melting temperature; and (ii) it includes one or more modified dNTPs (7-deaza dGTP and / or 2-amino dATP). For example, the introduced primers can have a melting temperature greater than 63°C, greater than about 66°C, greater than about 69°C, greater than about 72°C, greater than about 75°C, etc.; and wherein the primers include one or more modified dNTPs. In some embodiments, the introduced primers can have no restrictions on GC content and / or melting temperature; and wherein the primers also include one or more modified dNTPs.

[0167] In other embodiments, the pre-capture amplification occurs in the presence of one or more enhancers, such as betaine, DMSO, a single-stranded DNA binding protein, a disaccharide, or any combination thereof. For example, a composition comprising betaine (or a derivative or analog thereof) can be used for pre-capture amplification, such as a betaine comprising a concentration ranging from about 0.1 mM to about 1 mM (e.g., a concentration of about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, etc.).

[0168] In further embodiments, the pre-capture amplification occurs in a composition comprising one or more pre-capture primers (e.g., a forward pre-capture primer and a reverse pre-capture primer) having a high GC content and / or a high melting temperature; and wherein the composition further comprises at least one enhancer. In some embodiments, the enhancer is betaine (or a derivative or analog thereof) and the concentration of betaine in the composition ranges from about 0.2mM to about 0.8mM. In other embodiments, the enhancer is betaine (or a derivative or analog thereof) and the concentration of betaine in the composition ranges from about 0.3mM to about 0.7mM. In other embodiments, the enhancer is betaine (or a derivative or analog thereof) and the concentration of betaine in the composition ranges from about 0.4mM to about 0.6mM. In further embodiments, the enhancer is betaine (or a derivative or analog thereof) and the concentration of betaine in the composition is about 0.5mM.

[0169] In even further embodiments, amplification before capture can be performed in a composition comprising a mixture of unmodified dNTPs and modified dNTPs. In some embodiments, the range of the ratio of unmodified dNTPs to modified dNTPs in any composition is between about 2:1 and about 1:2. In some embodiments, the ratio of unmodified dNTPs to modified dNTPs is about 1:1. In some embodiments, amplification before capture can occur in a composition comprising one or more modified dNTPs (such as a concentration range between about 0.2mM to about 0.8mM, a concentration range between about 0.3mM to about 0.7mM, a concentration range between about 0.3mM to about 0.6mM, etc.). In some embodiments, the concentration of each different modified dNTP in the composition is about 0.3mM. In some embodiments, one or more modified dNTPs include modified dGTP and / or modified dATP. In some embodiments, one or more modified dNTPs are 7-deaza-2'-deoxyguanosine-5'-triphosphate and / or 2-amino-2'deoxyadenosine-5'-triphosphate. In some embodiments, the concentration of 7-deaza-2'-deoxyguanosine-5'-triphosphate and / or 2-amino-2'deoxyadenosine-5'-triphosphate is about the same as the concentration of unmodified dNTPs in the composition.

[0170] In some embodiments, the pre-capture amplification can occur in the presence of a polymerase, betaine, and a mixture of unmodified dNTPs and modified dNTPs. In these embodiments, betaine is present at a concentration ranging from about 0.4 mM to about 0.6 mM; and wherein the ratio of unmodified dNTPs to modified dNTPs is about 1:1. In some embodiments, the modified dNTPs are selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate or 2-amino-2'deoxyadenosine-5'-triphosphate.

[0171] In some embodiments, amplification before capture can occur in the presence of a polymerase, betaine, an optional mixture of unmodified dNTPs and modified dNTPs, one or more buffers, and one or more divalent cations. In these embodiments, betaine is present at a concentration ranging from about 0.4 mM to about 0.6 mM; and wherein the ratio of unmodified dNTPs to modified dNTPs is about 1: 1. In some embodiments, betaine is present at a concentration ranging from about 0.4 mM to about 0.6 mM; the ratio of unmodified dNTPs to modified dNTPs is about 1: 1; and one or more divalent cations are present at a concentration of about 0.1 mM to about 0.4 mM. In some embodiments, the modified dNTP is selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate or 2-amino-2'deoxyadenosine-5'-triphosphate.

[0172] In some embodiments, the amplification before capture can occur in the presence of a polymerase, a mixture of unmodified dNTPs and modified dNTPs, one or more buffers, and one or more divalent cations. In these embodiments, the ratio of unmodified dNTPs to modified dNTPs is about 1:1. In some embodiments, the ratio of unmodified dNTPs to modified dNTPs is about 1:1; and wherein one or more divalent cations are present at a concentration of about 0.1 mM to about 0.4 mM. In some embodiments, the modified dNTP is selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate or 2-amino-2'deoxyadenosine-5'-triphosphate.

[0173] An example of a pre-capture amplification master mix is ​​shown in Table 1 below:

[0174] Table 1: Pre-capture amplification master mix that can be used when performing step 102 of method 100.

[0175]

[0176]

[0177] After the capture pre-amplification of step 102, step 104 of method 100 includes hybridizing the capture primer with the target nucleic acid present in the nucleic acid molecule library to form an unextended capture primer-target complex. In some embodiments, the capture primer is a target-specific primer having a defined sequence complementary to the target nucleic acid sequence. An example of a target-specific primer is a gene-specific primer designed to hybridize with a target gene (e.g., cDNA, genomic DNA) or its vicinity (e.g., upstream or 5'). The target nucleic acid can be RNA, DNA, or a combination thereof. The capture primer can be an oligonucleotide primer consisting of ribonucleic acid, deoxyribonucleic acid, modified nucleic acid (e.g., biotinylated, locked nucleic acid, inosine, Seela bases, etc.) or other nucleic acid analogs known in the art.

[0178] In certain embodiments, the capture primer may include one or more modified bases, capture moieties, or combinations thereof. In the case where the capture primer includes a capture moiety, before the step 104 of hybridizing the capture primer with the target nucleic acid, the capture primer may be attached to a solid support or free in solution (i.e., not combined with or otherwise attached to a solid support). Other suitable capture moieties and their applications are described in the U.S. Publication No. 2020 / 0032244, the disclosure of which is hereby incorporated herein by reference in its entirety). In the embodiment where the capture primer including the capture moiety is not attached to a solid support via the capture moiety, step 104 may be performed in solution. In the embodiment where the capture primer including the capture moiety is attached to a solid support via the capture moiety, step 104 may be performed in situ. It is noteworthy that in the case of in situ reaction, the primer-target complex not extended by the obtained will be attached to a solid support. Any non-target nucleic acid molecules or target nucleic acid molecules not annealed with the capture primer retained in the solution may be removed by separating the solution from the solid support to which the primer-target complex is bound.

[0179] In step 106, a capture primer extension reaction is performed. In some embodiments, step 106 includes extending the hybridized capture primer with a first polymerase, thereby generating a capture primer extension product or complex, which includes a 3' region of the extended capture primer, which region includes a reverse complement of at least a portion of the target nucleic acid template. In some embodiments, the hybridization and extension reactions (steps 104 and 106) are optionally performed simultaneously, while in other embodiments, the hybridization and extension reactions are performed separately (e.g., sequentially) and can be separated by a washing step to remove unannealed and uncaptured target nucleic acid molecules from the reaction mixture.

[0180] In embodiments where the capture primer is hybridized and extended in the same step (referred to as "capture extension"), capture extension can occur using any of the compositions described herein, for example, a composition comprising one or more capture primers; one or more polymerases; dNTPs (including unmodified dNTPs or a mixture of unmodified dNTPs and modified dNTPs); and at least one enhancer (e.g., betaine). In other embodiments, capture extension occurs in a composition comprising one or more capture primers (wherein at least one of the one or more capture primers has a high melting temperature) and / or comprising one or more modified dNTPs; one or more polymerases; dNTPs (including unmodified dNTPs or a mixture of unmodified dNTPs and modified dNTPs); and optionally at least one enhancer (e.g., betaine).

[0181] In some embodiments, capture extension can be performed in the presence of a capture primer set, wherein at least one primer in the capture primer set has a high GC content and / or a high melting temperature (e.g., a melting temperature greater than 63° C., greater than about 66° C., greater than about 69° C., greater than about 72° C., greater than about 75° C., etc.). In some embodiments, the introduced capture primers can have no restrictions on GC content and / or melting temperature.

[0182] In some embodiments, the capture extension can be performed in the presence of a capture pre-primer set (e.g., a forward capture pre-primer and a reverse capture pre-primer), wherein at least one primer in the capture pre-primer set has a high GC content and / or a high melting temperature (e.g., a melting temperature greater than 63° C., greater than about 66° C., greater than about 69° C., greater than about 72° C., greater than about 75° C., etc.). In some embodiments, the introduced capture pre-primer may have no restrictions on GC content and / or melting temperature.

[0183] In some embodiments, at most about 25% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers. In some embodiments, at most about 20% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers. In some embodiments, at most about 15% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers. In some embodiments, at most about 12% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers. In some embodiments, at most about 10% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers. In some embodiments, at most about 9% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers. In some embodiments, at most about 8% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers. In some embodiments, at most about 7% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers. In some embodiments, at most about 6% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers. In some embodiments, at most about 5% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers. In some embodiments, at most about 4% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers. In some embodiments, at most about 3% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers. In some embodiments, at most about 2% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers. In some embodiments, at most about 1% of the capture primers in any capture primer set are high melting temperature and / or high GC content capture primers.

[0184] In other embodiments, capture extension can be performed in the presence of a capture primer set, wherein at least one primer in the primer set has (i) a high GC content and / or a high melting temperature; and (ii) it includes one or more modified dNTPs. For example, the introduced primers can have a melting temperature greater than 63°C, greater than about 66°C, greater than about 69°C, greater than about 72°C, greater than about 75°C, etc.; and wherein the primers include one or more modified dNTPs. In some embodiments, the introduced primers can have no restrictions on GC content and / or melting temperature; and wherein the primers also include one or more modified dNTPs.

[0185] In other embodiments, capture extension can occur in the presence of one or more enhancers, such as betaine, DMSO, single-stranded DN binding protein, disaccharide, or any combination thereof. For example, capture extension can be performed using a composition comprising betaine (or a derivative or analog thereof), such as a betaine comprising a concentration ranging from about 0.1 mM to about 1 mM (e.g., a concentration of about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, etc.).

[0186] In further embodiments, capture extension can be performed in a composition comprising one or more capture primers having a high GC content and / or a high melting temperature; and wherein the composition further comprises at least one enhancer. In some embodiments, the enhancer is betaine (or a derivative or analog thereof) and the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM. In other embodiments, the enhancer is betaine (or a derivative or analog thereof) and the concentration of betaine in the composition ranges from about 0.3 mM to about 0.7 mM. In other embodiments, the enhancer is betaine (or a derivative or analog thereof) and the concentration of betaine in the composition ranges from about 0.4 mM to about 0.6 mM. In further embodiments, the enhancer is betaine (or a derivative or analog thereof) and the concentration of betaine in the composition is about 0.5 mM.

[0187] In even further embodiments, capture extension can be performed in a composition comprising a mixture of unmodified dNTPs and modified dNTPs. In some embodiments, the range of the ratio of unmodified dNTPs to modified dNTPs in any composition is between about 2:1 and about 1:2. In some embodiments, the ratio of unmodified dNTPs to modified dNTPs is about 1:1. In some embodiments, capture extension can occur in a composition comprising one or more modified dNTPs (such as a concentration range between about 0.2mM to about 0.8mM, a concentration range between about 0.3mM to about 0.7mM, a concentration range between about 0.3mM to about 0.6mM, etc.). In some embodiments, one or more modified dNTPs include modified dGTP and / or modified dATP. In some embodiments, one or more modified dNTPs are 7-deaza-2'-deoxyguanosine-5'-triphosphate and / or 2-amino-2'deoxyadenosine-5'-triphosphate. In some embodiments, the concentration of 7-deaza-2'-deoxyguanosine-5'-triphosphate and / or 2-amino-2'deoxyadenosine-5'-triphosphate is about the same as the concentration of unmodified dNTPs in the composition.

[0188] In some embodiments, capture extension can occur in the presence of a polymerase, betaine, and a mixture of unmodified dNTPs and modified dNTPs. In these embodiments, betaine is present at a concentration ranging from about 0.4 mM to about 0.6 mM; and wherein the ratio of unmodified dNTPs to modified dNTPs is about 1:1. In some embodiments, the modified dNTP is selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate or 2-amino-2'deoxyadenosine-5'-triphosphate.

[0189] In some embodiments, capture extension can occur in the presence of a polymerase, betaine, an optional mixture of unmodified dNTPs and modified dNTPs, one or more buffers, and one or more divalent cations. In these embodiments, betaine is present at a concentration ranging from about 0.4mM to about 0.6mM; and wherein the ratio of unmodified dNTPs to modified dNTPs is about 1:1. In some embodiments, betaine is present at a concentration ranging from about 0.4mM to about 0.6mM; the ratio of unmodified dNTPs to modified dNTPs is about 1:1; and one or more divalent cations are present at a concentration of about 0.1mM to about 0.4mM. In some embodiments, the modified dNTP is selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate or 2-amino-2'deoxyadenosine-5'-triphosphate. In some embodiments, the divalent cation is present at a concentration of about 0.1mM to about 0.4mM.

[0190] In some embodiments, capture extension can occur in the presence of a polymerase, a mixture of unmodified dNTPs and modified dNTPs, one or more buffers, and one or more divalent cations. In these embodiments, the ratio of unmodified dNTPs to modified dNTPs is about 1:1. In some embodiments, the ratio of unmodified dNTPs to modified dNTPs is about 1:1; and one or more divalent cations are present at a concentration of about 0.1 mM to about 0.4 mM. In some embodiments, the modified dNTP is selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate or 2-amino-2'deoxyadenosine-5'-triphosphate.

[0191] Examples of capture extension master mixes that may be used when performing step 106 are listed in Table 2 below.

[0192] Table 2: Capture extension master mix that can be used when performing the capture extension step of method 100.

[0193]

[0194] In addition to providing a composition for performing step 106 of method 100, the present disclosure also provides a method for incubating a sample. In particular, the present disclosure provides an incubation program (including denaturation, annealing, and extension portions) with varying temperatures and times that can be used to improve coverage uniformity and / or reduce GC bias during one or more downstream sequencing operations. Of course, any of the incubation programs described herein can be used in combination with any of the compositions disclosed herein.

[0195] In some embodiments, the denaturation, annealing, and extension portions of the incubation can be performed over a cumulative time period ranging from about 20 minutes to about 40 minutes, such as between about 25 minutes and about 35 minutes. For example, the cumulative time period for performing the denaturation, annealing, and extension portions of step 106 of method 100 can be about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33 minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37 minutes, about 38 minutes, about 39 minutes, about 40 minutes, etc.

[0196] In some embodiments, the cumulative time period for the annealing and extension portion of the incubation ranges from about 20 minutes to about 25 minutes. In some embodiments, the cumulative time period for the annealing and extension portion of the incubation is about 23 minutes. In other embodiments, the cumulative time period for the annealing and extension portion of the incubation ranges from about 30 minutes to about 34 minutes. In other embodiments, the cumulative time period for the annealing and extension portion of the incubation is about 32 minutes.

[0197] In some embodiments, annealing and extension are performed at a temperature ranging from about 60°C to about 80°C, such as shown in Tables 3 to 7. In some embodiments, the incubation programs of the present disclosure include one or more pauses between annealing temperatures compared to a control incubation program. For example, Program 1 adds pauses at fixed intervals between 80°C and 60°C while keeping the entire program length the same as the control program (Program 5). Similarly, Program 2 adds pauses at higher temperature intervals between 80°C and 60°C while keeping the entire program length the same as the control program (Program 5). Program 3 adds pauses at higher temperature intervals between 80°C and 60°C while matching the total incubation time to the control program (Program 5), i.e., incubating at 60°C for 10 minutes, thereby increasing the total program time by 10 minutes. Program 4 adds pauses at fixed intervals between 80°C and 60°C while matching the incubation time to the control program (Program 5), i.e., incubating at 60°C for 10 minutes, thereby increasing the total program time by 10 minutes.

[0198] Table 3 - Exemplary incubation program with a total time of approximately 25 minutes.

[0199]

[0200]

[0201] Table 4 - Exemplary incubation program with a total time of approximately 25 minutes.

[0202]

[0203] Table 5 - Exemplary incubation program with a total time of approximately 34 minutes.

[0204]

[0205] Table 6 - Exemplary incubation program with a total time of approximately 34 minutes.

[0206]

[0207]

[0208] Table 7 - Exemplary incubation program with a total time of approximately 24 minutes.

[0209]

[0210] Method 100 further includes a step 108 of capturing a capture primer extension complex. The capture of the capture primer extension complex can be achieved in a variety of ways disclosed herein, and can be achieved before, simultaneously or after step 104 and step 106 of method 100. As described above, the capture oligonucleotide can include a capture portion, which can be used to capture the capture primer on a solid support before, during or after step 104 or step 106 of method 100. In another example, the extension of the capture primer after hybridization with the target nucleic acid includes the incorporation of one or more modified nucleotides. The modified nucleotide can include a capture portion or can be configured to enable downstream modification of the modified nucleotide to attach or otherwise incorporate the capture portion into the extension portion of the capture primer extension complex. Therefore, the capture primer extension complex can be captured by a capture portion associated with one or more modified nucleotides during or after step 106. Whether to capture the target nucleic acid, the annealed primer-target complex or the primer complex extended by the target further determines whether the steps 104 and 106 of the method are performed in solution or in situ.

[0211] Step 110 of method 110 includes enriching the captured primer extension complex. In some embodiments, step 110 includes one or more purification and enrichment steps for recovering the captured primer extension complex from non-target nucleic acid molecules and other molecules in the library, such as unused reaction components (e.g., nucleotides, primer molecules, ATP, etc.), enzymes, buffers, etc. In some embodiments, step 110 includes enzymatic digestion, size exclusion-based purification, affinity-based purification, etc. or a combination thereof. In some embodiments, enrichment involves increasing the concentration of the target nucleic acid by consuming (i.e., removing) other members of the nucleic acid molecule library of non-target nucleic acid molecules.

[0212] Hybridization of the release primer with the target nucleic acid occurs in step 112 of method 100. In some embodiments, the release primer is a target-specific primer that binds to the target region within the target nucleic acid (in contrast to hybridization or complementation with one or both of the first adapter and the second adapter introduced in step 102). In some embodiments, the target nucleic acid is a part of the capture primer extension complex synthesized in step 112. For example, the release primer can hybridize with the target nucleic acid at the 5' position (i.e., upstream) relative to the capture primer extension complex extended by the capture primer. In this case, the unextended release primer-target complex obtained includes the extended capture primer, the target nucleic acid hybridized with the extended capture primer, and the second (unextended) oligonucleotide primer. In the case where the capture primer extension product is attached to a solid support during step 112, the unextended release primer-target complex will be similarly attached to the solid support. In other embodiments, (e.g., after removing non-target nucleic acid molecules from the reaction mixture), the capture primer extension product is released from the solid support and is in a solution so that the release primer can hybridize in the solution in step 112.

[0213] Step 114 of method 100 includes performing a release primer extension reaction. After the release primer is hybridized with the target nucleic acid template in step 112, the release primer is extended by the second polymerase, thereby generating a release primer extension product or complex including the target nucleic acid. The extended release primer includes a 3' region of the reverse complement of at least a portion of the target nucleic acid template. In some embodiments, the release primer is extended with a second polymerase to release the extended capture primer from the complex with the target nucleic acid. The extended capture oligonucleotide can be released from the capture primer extension complex and can include one or more of strand displacement (e.g., by polymerase) or digestion (e.g., by nuclease). For example, the release of the extended capture oligonucleotide can be achieved with an enzyme having at least one of strand displacement activity, 5' to 3' exonuclease activity and flap endonuclease activity. In some embodiments, the first polymerase and the second polymerase are identical. In other embodiments, the first polymerase and the second polymerase are different.

[0214] Steps 112 and 114 can be performed sequentially or simultaneously. No matter whether steps 112 and 114 are performed sequentially or simultaneously, any one of the release primers used can have a high melting temperature and / or a high GC content. In certain embodiments, at least one release primer in the release primer set has a melting temperature greater than 63°C, greater than about 66°C, greater than about 69°C, greater than about 72°C, greater than about 75°C, greater than about 80°C, etc. In certain embodiments, a plurality of release primers in the release primer set have a melting temperature greater than 63°C, greater than about 66°C, greater than about 69°C, greater than about 72°C, greater than about 75°C, greater than about 80°C, etc. In certain embodiments, there is no restriction on the melting temperature and / or GC content of the release primer. In certain embodiments, the release primer can include one or more modified dNTPs, such as two or more modified dNTPs, three or more modified dNTPs, four or more modified dNTPs, etc.

[0215] In certain embodiments, at most about 25% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers. In certain embodiments, at most about 20% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers. In certain embodiments, at most about 15% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers. In certain embodiments, at most about 12% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers. In certain embodiments, at most about 10% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers. In certain embodiments, at most about 9% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers. In certain embodiments, at most about 8% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers. In certain embodiments, at most about 7% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers. In certain embodiments, at most about 6% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers. In certain embodiments, at most about 5% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers. In certain embodiments, at most about 4% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers. In certain embodiments, at most about 3% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers. In certain embodiments, at most about 2% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers. In certain embodiments, at most about 1% of the release primers in any release primer set are high melting temperatures and / or high GC content release primers.

[0216] In embodiments where steps 112 and 114 are performed sequentially, step 112 may be performed in the presence of at least one enhancer selected from betaine (or a derivative or analog thereof), DMSO, a disaccharide, and a single-stranded DNA binding protein (SSB). In some embodiments, the at least one enhancer is betaine, and the betaine concentration in any release primer hybridization master mix used during step 112 includes a betaine concentration of about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.25 mM, about 0.3 mM, about 0.35 mM, about 0.4 mM, about 0.5 mM, about 0.55 mM, about 0.6 mM, about 0.65 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, etc.

[0217] In embodiments where steps 112 and 14 are performed simultaneously, the combined release primer hybridization and extension can be performed in the presence of one or more enhancers, such as betaine, DMSO, a single-stranded DN binding protein, a disaccharide, or any combination thereof. For example, a composition comprising betaine (or a derivative or analog thereof) can be used to perform the combined release primer hybridization and extension, such as a betaine comprising a concentration ranging from about 0.1 mM to about 1 mM (e.g., a concentration of about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, etc.).

[0218] In some embodiments, the release primer hybridization and extension of the combination can be carried out in a composition comprising a mixture of unmodified dNTPs and modified dNTPs. In some embodiments, the range of the ratio of unmodified dNTPs to modified dNTPs in any composition is between about 2:1 and about 1:2. In some embodiments, the ratio of unmodified dNTPs to modified dNTPs is about 1:1. In some embodiments, one or more modified dNTPs include modified dGTP and / or modified dATP. In some embodiments, one or more modified dNTPs are 7-deaza-2'-deoxyguanosine-5'-triphosphate and / or 2-amino-2'deoxyadenosine-5'-triphosphate. In some embodiments, the concentration of 7-deaza-2'-deoxyguanosine-5'-triphosphate and / or 2-amino-2'deoxyadenosine-5'-triphosphate is about the same as the concentration of unmodified dNTPs in the composition.

[0219] In other embodiments, the combined release primer hybridization and extension occurs in a composition comprising one or more capture extensions having high GC content and / or high melting temperature; and wherein the composition further comprises at least one enhancer. In some embodiments, the enhancer is betaine (or a derivative or analog thereof) and the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM. In other embodiments, the enhancer is betaine (or a derivative or analog thereof) and the concentration of betaine in the composition ranges from about 0.3 mM to about 0.7 mM. In other embodiments, the enhancer is betaine (or a derivative or analog thereof) and the concentration of betaine in the composition ranges from about 0.4 mM to about 0.6 mM. In further embodiments, the enhancer is betaine (or a derivative or analog thereof) and the concentration of betaine in the composition is about 0.5 mM.

[0220] In other embodiments, the combined release primer hybridization and extension can occur in the presence of a polymerase, betaine, and a mixture of unmodified dNTPs and modified dNTPs. In these embodiments, betaine is present at a concentration ranging from about 0.4 mM to about 0.6 mM; and wherein the ratio of unmodified dNTPs to modified dNTPs is about 1:1. In some embodiments, the modified dNTP is selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate or 2-amino-2'deoxyadenosine-5'-triphosphate.

[0221] In some embodiments, the release primer hybridization and extension of the combination can occur in the presence of a polymerase, betaine, an optional mixture of unmodified dNTPs and modified dNTPs, one or more buffers, and one or more divalent cations. In these embodiments, betaine is present at a concentration ranging from about 0.4mM to about 0.6mM; and wherein the ratio of unmodified dNTPs to modified dNTPs is about 1:1. In some embodiments, betaine is present at a concentration ranging from about 0.4mM to about 0.6mM; the ratio of unmodified dNTPs to modified dNTPs is about 1:1; and one or more divalent cations are present at a concentration of about 0.1mM to about 0.4mM. In some embodiments, the modified dNTP is selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate or 2-amino-2'deoxyadenosine-5'-triphosphate. In some embodiments, the divalent cation is present at a concentration of about 0.1mM to about 0.4mM.

[0222] In some embodiments, the combined release primer hybridization and extension can occur in the presence of a polymerase, a mixture of unmodified dNTPs and modified dNTPs, one or more buffers, and one or more divalent cations. In these embodiments, the ratio of unmodified dNTPs to modified dNTPs is about 1:1. In some embodiments, the ratio of unmodified dNTPs to modified dNTPs is about 1:1; and one or more divalent cations are present at a concentration of about 0.1 mM to about 0.4 mM. In some embodiments, the modified dNTP is selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate or 2-amino-2'deoxyadenosine-5'-triphosphate.

[0223] In some embodiments, step 114 may further include termination of the primer extension reaction to control the length of the extended release primer. It is noteworthy that the length of the extended release primer product can be actively controlled by technology (such as inactivating the polymerase added in step 114), or passively controlled by allowing the reaction to complete (such as by consuming the limiting reactant) or by controlling / selecting the size of the nucleic acid molecule fragments in the nucleic acid molecule library.

[0224] In the case where the extended capture primer includes one or more capture moieties attached to a solid support, the release of the extended capture oligonucleotide in step 114 releases the primer extension complex free in solution, rather than attached to the solid support. Thus, as described in step 110 of method 100, one or more purification techniques may be performed after step 114 to recover the unbound second extension product or complex including the target nucleic acid from the support-attached extended capture primer, the second polymerase, other reaction components, etc., and combinations thereof.

[0225] Method 100 further includes a step 116 of amplification after capture, involving linear or exponential amplification (e.g., PCR). In general, step 116 includes amplifying the target nucleic acid molecule with a third polymerase, a first amplification primer, and a second amplification primer. In some embodiments, the design of the first amplification primer and the second amplification primer is designed to be complementary to the adapter sequence incorporated into the target nucleic acid molecule in the nucleic acid molecule library in step 102. For example, the first amplification primer can have a 3' end complementary to the first adapter, and the second amplification primer can have a 3' end complementary to the second adapter. However, the amplification primer can include any sequence (e.g., gene / target specific primers, universal primers, etc.) present in the target nucleic acid to be amplified, and can support the synthesis of one or two chains (i.e., both the upper and lower chains of the double-stranded nucleic acid molecules corresponding to the template of the amplification reaction). In some embodiments, amplification after capture can be carried out in a composition similar to the composition used in the amplification before capture, including any one of those compositions described herein.

[0226] In some embodiments, step 116 enables selective amplification of target nucleic acid molecules from a nucleic acid molecule library, which is opposite to any of the extended capture or release primers derived from the target nucleic acid. In one example, uracil compatible polymerase and dUTP are included in one or both of the extension reactions performed in step 106 and step 114. The extended amplification primer produced by the reaction will include at least one uracil nucleotide, and the target nucleic acid template can be a DNA template without uracil nucleotides. Thereafter, uracil incompatible polymerase is included in step 116 to amplify the target nucleic acid. Uracil incompatible polymerase can amplify target nucleic acid without uracil nucleotides; however, uracil incompatible polymerase will not be able to replicate the extended oligonucleotide primer containing uracil. Alternatively or in addition, the product containing uracil can be selectively digested or otherwise degraded, thereby leaving only the original molecule from the nucleic acid molecule library.

[0227] After the amplification step 116, the method 100 may include a step 118 of analyzing the amplified target nucleic acid molecules. Step 116 may include any method for determining the nucleic acid sequence of one or more products of the method 100. Step 116 may further include sequence alignment, identification of sequence variations, counting of unique primer extension products, the like, or a combination thereof.

[0228] In some embodiments, the amplicon generated after step 116 is sequenced, for example, using next generation sequencing. As used herein, the term "next generation sequencing" refers to a sequencing technique with high-throughput sequencing compared to traditional methods based on Sanger and capillary electrophoresis, wherein the sequencing process is performed in parallel, such as generating thousands or millions of relatively small sequence reads at a time. Some examples of next generation sequencing techniques include but are not limited to sequencing while synthesis, sequencing while connection, and sequencing while hybridization. These technologies produce shorter reads (from about 25 to about 500bp), but produce hundreds of thousands or millions of reads in a relatively short time. Examples of such sequencing devices that can be obtained from Illumina (San Diego, CA) include but are not limited to iSEQ, MiniSEQ, MiSEQ, NextSEQ, NoveSEQ. It is believed that Illumina next generation sequencing technology uses clonal amplification and synthetic sequencing (SBS) chemistry to achieve rapid sequencing. The process simultaneously identifies DNA bases while incorporating them into nucleic acid chains. Each base emits a unique fluorescent signal when added to the growing chain for determining the order of the DNA sequence. Non-limiting examples of sequencing devices available from ThermoFisher Scientific (Waltham, MA) include the Ion Personal Genome Machine TM (PGM TM) system. It is believed that Ion Torrent sequencing measures the direct release of H+ (proton) when a DNA polymerase binds to a single base. Non-limiting examples of sequencing devices available from Pacific Biosciences (Menlo Park, California) include PacBio Sequel Systems. Non-limiting examples of sequencing devices available from Roche (Pleasanton, CA) are Roche 454. Next-generation sequencing methods can also include nanopore sequencing methods. In general, three nanopore sequencing methods have been used: chain sequencing, in which the bases of DNA are identified as they pass through the nanopore one by one; exonuclease-based nanopore sequencing, in which nucleotides are enzymatically cut from the DNA molecule one by one and monitored as they are captured by the nanopore and pass through the nanopore; and nanopore sequencing by synthesis (SBS) methods, in which identifiable polymer tags are attached to nucleotides and aligned in the nanopore during enzyme-catalyzed DNA synthesis. Common to all these methods is the need to precisely control the reaction rate in order to determine each base in sequence. Chain sequencing requires methods for slowing down the speed at which DNA passes through the nanopore and decoding multiple bases within the channel; to this end, ratchet methods using molecular motors have been developed. Exonuclease-based sequencing requires the release of each nucleotide when it is close enough to the hole to ensure its capture and pass through the hole at a slow enough rate to obtain an effective ionic current signal. In addition, both methods rely on the distinction between four natural bases, namely two relatively similar purines and two similar pyrimidines. The nanopore SBS method utilizes synthetic polymer tags attached to nucleotides, which are specially designed to produce unique and easily distinguishable ionic current blocking marks for sequence determination. In some embodiments, nucleic acid molecule sequencing includes sequencing via nanopores, including preparing nanopore sequencing complexes and determining polynucleotide sequences. Methods for preparing nanopores and nanopore sequencing are described in U.S. Patent Application Publication No. 2017 / 0268052 and PCT Publications WO2014 / 074727, WO2006 / 028508, WO2012 / 083249, and WO / 2014 / 074727, the disclosures of which are hereby incorporated by reference in their entirety. In some embodiments, labeled nucleotides can be used to determine polynucleotide sequences (see, e.g., PCT Publications WO / 2020 / 131759, WO / 2013 / 191793, and WO / 2015 / 148402, the disclosures of which are hereby incorporated by reference in their entirety). Analysis of the data generated by sequencing is typically performed using software and / or statistical algorithms that perform various data conversions, e.g., converting signal emissions into base calls, converting base calls into consensus sequences of nucleic acid templates, etc.Such software, statistical algorithms, and their use are described in detail in U.S. Patent Application Publication Nos. 2009 / 0024331, 2017 / 0044606, and PCT Publication No. WO / 2018 / 034745, the disclosures of which are hereby incorporated by reference in their entirety.

[0229] Examples

[0230] Example 1 - Effect of various primer maximum melting temperature (TM) limits on enrichment of high %GC targets

[0231] Material

[0232] NA12878 cell line DNA

[0233] Hot Spot Group

[0234] HyperPlus Library Preparation Kit

[0235] KAPA UDI Primer Mix

[0236] KAPA Universal UMI Adapters

[0237] method

[0238] Panels were designed with three high %GC targets, no restriction on %GC, a minimum Tm of 57°C, and various maximum Tm restrictions (eg, 66°C, 69°C, 72°C, no restriction).

[0239] Twenty-four libraries were made according to the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 Chapter 4. Libraries were prepared from DNA using 10 ng of non-formalin damaged NA24143 cell line DNA, with the exceptions listed below: During step 5, post-ligation 0.8X purification was performed using KAPA HyperPure beads, adapter-ligated DNA samples were pooled, and the pool was aliquoted into 20 μL per sample and carried into step 6.

[0240] Ten libraries were subjected to primer extension target enrichment according to Chapter 5 of the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 (the disclosure of which is hereby incorporated herein by reference in its entirety). Primer extension target enrichment (PETE) was performed (described herein) with the following exceptions: During step 2 (capture extension reaction), 5 μL of five small custom capture test sets targeting three high %GC ROIs with different maximum Tm limits (Table 8) were added to four samples in each condition. 5 μL of Hot Spot capture set was added, but water was not included in the capture extension reaction master mix. During step 6 (release primer hybridization), 20 μL (instead of 30 μL) of 1X wash and resuspension buffer and 10 μL of Hot Spot release set were added to the release primer hybridization master mix. 10 μL of the five test capture sets were added to the corresponding four samples in each condition.

[0241] Primer extension target enrichment libraries were sequenced and analyzed on a NextSeq 550. Normalized coverage at each position was calculated for each sample by dividing the coverage at a certain position by the average coverage across the sample.

[0242] Table 8: Experimental settings.

[0243]

[0244] result

[0245] The results of the current primer database are as follows Figure 2A Similarly, the results for the “No Maximum Tm Group” are shown in Figure 2B shown. Figure 2C Primer availability is depicted. Increasing the maximum primer Tm increases primer availability in high %GC regions (a) and reduces the number of targeted bases not covered by the capture and release primer ranges (b). Figure 3A As shown, the samples enriched with the no maximum Tm test set had the highest percentage of bases within 2-fold, and the percentage of exonic regions of the set>=1000X. It was found that the higher primer maximum Tm improved the normalized coverage of targets>65% GC compared to the current primer database with a maximum Tm of 57°C (see Figure 3D). The small custom set designed with no maximum primer Tm had the highest normalized coverage in high %GC targets.

[0246] in conclusion

[0247] Applicants have discovered that target enrichment in the presence of primers synthesized without restrictions on melting temperature and / or GC content improves the enrichment of high GC target nucleic acid molecules, ultimately resulting in improved uniformity of coverage when sequencing high GC target enriched nucleic acid molecules.

[0248] Example 2-7 - Effects of deaza-DGTP and betaine on PCR and capture extension

[0249] Purpose

[0250] The effects of the addition of 7-deaza-dGTP in the library preparation amplification and betaine in both the library preparation amplification and the capture extension reaction on the enrichment of high %GC targets were tested.

[0251] Material

[0252] NA24143 cell line DNA

[0253] Hot Spot Group

[0254] Small Custom Group (3 High %GC ROI)

[0255] HyperPlus Library Preparation Kit

[0256] KAPA UDI Primer Mix

[0257] KAPA Universal UMI Adapters

[0258] method

[0259] Sixteen libraries were made according to KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 Chapter 4 (the disclosure of which is hereby incorporated by reference in its entirety). Libraries were prepared from DNA using 10 ng of non-formalin-damaged NA12878 cell line DNA, with the exceptions listed below: During step 5, 12 μL (instead of 20 μL as indicated in the instructions) 10 mM Tris-HCl pH 8.0 was used to elute the adapter-ligated DNA samples after 0.8X purification using KAPA HyperPure beads. The adapter-ligated DNA samples were combined and the combined product was aliquoted into 12 μL per sample and entered into step 6, during which 3 μL of water was added to eight samples and 3 μL of 5 mM 7-deaza dGTP was added to the other eight samples using the KAPA UDI primer mix for amplification (also referred to as PCR1). Then, in each set of eight samples, 5 μL of water was added to four samples and 5 μL of 5M betaine was added to the other four samples. During step 7 (1X purification after amplification using KAPA HyperPure beads), the pre-capture library was eluted using 15 μL of 10 mM Tris-HCl pH 8.0 (instead of 25 μL as indicated in the instructions).

[0260] Sixteen libraries were subjected to primer extension target enrichment according to the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 Chapter 5 Primer Extension Target Enrichment (PETE), with the exceptions listed below: During step 2 (capture extension reaction), 5 μL (instead of the indicated 10-15 μL) of each pre-capture input library was used, which resulted in less than 500 ng of pre-capture input library per sample for some samples. In addition to the 5 μL Hot Spot capture set, 5 μL of a small custom capture set targeting three high %GC ROIs was added to the capture extension reaction master mix. Water was not included in the capture extension reaction master mix, and instead 5 μL of water or 5M betaine was added to the 5 μL pre-capture input library, resulting in two replicates for each condition combination (Table 9). During step 6 (release primer hybridization), 20 μL (instead of 30 μL) of 1X wash and resuspension buffer was added to the release primer hybridization master mix, and 10 μL of the small custom release set was added along with the 10 μL Hot Spot release set.

[0261] Primer extension target enrichment libraries were sequenced and analyzed on a NextSeq 550. Normalized coverage at each position was calculated for each sample by dividing the coverage at a certain position by the average coverage across the sample.

[0262] Table 9: Experimental settings.

[0263]

[0264] result

[0265] The concentration of pre-capture input library was lower in samples spiked with 7-deaza-dGTP (PCR1+7) compared to samples without 7-deaza-dGTP (PCR1-7) (see Figure 4 ). Due to the low pre-capture input library yield, the PETE input was also lower for samples containing 7-deaza-dGTP. The PETE library yield was lower for samples containing betaine in the capture extension reaction (CapExtx+B) compared to samples without betaine in the capture extension under shared PCR1 conditions (see Figure 4 ).

[0266] The percentage of on-target reads was similar between all conditions tested. For samples containing betaine in PCR1, the deduplication depth was higher and the average fragment length was larger. For samples containing 7-deaza-dGTP, the percentage of bases within 2-fold, the percentage of exonic regions >= 1000X, and the percentage of errors were higher. For samples without 7-deaza-dGTP, samples containing betaine in the capture extension reaction had a higher percentage of bases within 2-fold and the percentage of exonic regions >= 1000X (see Figure 5 ).

[0267] Fig. 6A and 6B Normalized coverage on high %GC targets is shown. In particular, Fig. 6A Depicted is an even more uniform coverage across the three high %GC ROIs targeted by a small custom panel for samples containing 7-deaza-dGTP compared to samples without 7-deaza-dGTP. Figure 6B It is shown that for samples without 7-deaza-dGTP, coverage across the three high %GC ROIs targeted by the small custom panel is even more uniform for samples containing betaine in the capture extension reaction than for samples without betaine in the capture extension reaction.

[0268] Figure 7 Normalized coverage of target %GC is shown. Samples containing 7-deaza dGTP have higher normalized coverage of target >70% GC than samples without 7-deaza dGTP. For samples without 7-deaza dGTP, when betaine was included in the capture extension reaction, normalized coverage of target >70% GC was higher than samples without betaine in the capture extension reaction.

[0269] in conclusion

[0270] Applicants have discovered that the addition of 7-deaza dGTP in library preparation or betaine in capture extension can be used to improve high %GC target enrichment. Further testing is needed to determine the role of variant identification in the higher error rate percentages observed in samples containing 7-deaza dGTP.

[0271] Example 3 - Capture extension program optimization

[0272] Purpose

[0273] The effects of different capture extension incubation times and temperatures on the enrichment of high %GC targets were tested.

[0274] Material

[0275] NA24143 cell line DNA

[0276] Hot Spot Group

[0277] Small Custom Group (3 High %GC ROI)

[0278] HyperPlus Library Preparation Kit

[0279] KAPA UDI Primer Mix

[0280] KAPA Universal UMI Adapters

[0281] method

[0282] Ten libraries were made using 10 ng of non-formalin-damaged NA24143 cell line DNA according to the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 Chapter 4 Library Preparation from DNA with the exceptions listed below: During step 5, post-ligation 0.8X purification was performed using KAPA HyperPure beads, adapter-ligated DNA samples were pooled, and the pool was aliquoted into 20 μL per sample and proceeded to step 6.

[0283] Ten libraries were subjected to primer extension target enrichment according to KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 Chapter 5 (the disclosure of which is hereby incorporated herein by reference in its entirety). Primer extension target enrichment (PETE) with the following exceptions: During step 2 (capture extension reaction), 5 μL Hot Spot capture set, 5 μL small custom capture set targeting three high % GC ROIs were added to the capture extension reaction master mix. Water was not included in the capture extension reaction master mix. Two samples were incubated in each of the four test capture extension programs or control capture extension programs detailed in Table 10 according to the thermal cycler program in step 2.2. During step 6 (release primer hybridization), 20 μL (instead of 30 μL) 1X wash and resuspension buffer was added to the release primer hybridization master mix, and 10 μL small custom release set was added with 10 μL HotSpot release set.

[0284] Primer extension target enrichment libraries were sequenced and analyzed on a NextSeq 550. Normalized coverage at each position for each sample was calculated by dividing the coverage at a position by the average coverage across the sample.

[0285] Table 10: Capture program tests.

[0286]

[0287] Figure 8 Sequencing quality control metrics are depicted. Samples tested for programs 1 to 4 had higher percentage of reads on target, deduplication depth, percentage of bases within 2-fold range, and percentage of group exon regions >= 1000X compared to the program indicated in the instructions for use (IFU) samples. The average fragment length was higher for the IFU program. Program 3 samples had the highest percentage of bases within 2-fold range and percentage of group exon regions >= 1000X.

[0288] Fig. 9 Normalized coverage for each target is depicted. Test Programs 1 to 4 samples had higher normalized coverage for targets >70% GC compared to IFU samples, with Program 3 samples having the highest normalized coverage for targets >75% GC. Test Programs 1 to 4 samples had lower normalized coverage for targets <40% GC compared to IFU samples, with Program 1 samples having the lowest coverage for targets <35% GC.

[0289] in conclusion

[0290] Test procedure 3 (Table 10), with longer incubation times at higher temperatures, produced the greatest improvements in normalized coverage of high %GC targets, percentage of bases within a 2-fold range, and percentage of group exonic regions >= 1000X compared to the other test procedures and the IFU procedure. Applicants have found that longer incubation times and higher temperatures can result in improvements in normalized coverage of high %GC targets.

[0291] Example 4-7-Denitrogenated DGTP, 2-amino DATP, DMSO, trehalose, betaine and single-chain binding protein for capture delay The influence of stretch

[0292] The aim of this study was to test the effects of the addition of 7-deaza-dGTP and 2-amino-dATP to library preparation amplifications, and DMSO, trehalose, betaine, and single-stranded DNA binding proteins to capture extension reactions on the enrichment of high %GC targets.

[0293] Material

[0294] NA12878 cell line DNA

[0295] No maximum Tm group

[0296] KAPA HyperPETE Hotspot Group

[0297] 2-Amino dATP

[0298] 7-deaza-dGTP

[0299] ssDNA binding protein

[0300] Betaine

[0301] Trehalose

[0302] DMSO

[0303] KAPA HyperPETE Kit

[0304] KAPA HyperCapture Bead Kit

[0305] KAPA HyperPlus Library Preparation Kit

[0306] KAPA UDI Primer Mix

[0307] KAPA Universal UMI Adapters

[0308] method

[0309] Sixteen libraries were made according to the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 Chapter 4. Libraries were prepared from DNA using 10 ng of non-formalin damaged NA24143 cell line DNA, with the exceptions listed below: During step 5, KAPA HyperPure beads were used for post-ligation 0.8X purification, adapter-ligated DNA samples were pooled, and the pool was aliquoted into 20 μL per sample and entered into step 6. In four replicates, 2-amino-dATP was added to the PCR amplification (PCR1) at a concentration of 0.3 mM. In another four replicates, 2-amino-dATP and 7-deaza-dGTP were each added to the PCR1 amplification at a concentration of 0.3 mM. In another four replicates, 7-deaza-dGTP was added to the PCR1 amplification at a concentration of 0.3 mM (Table 11). In the last four replicates, additives were not included in the PCR1 amplification.

[0310] Primer extension target enrichment (PETE) was performed on 10 μL of each of the 16 libraries according to the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 Chapter 5. Primer Extension Target Enrichment (PETE) with the following exceptions: During step 2 (capture extension reaction), equal concentrations of the No Max Tm High % GC ROI Capture Set and the Hot Spot Capture Set were added to the Capture Extension Reaction Master Mix. During step 6 (release primer hybridization), the volume of 1X Wash and Resuspension Buffer was reduced to accommodate equal concentrations of the Hot Spot Release Set and the No Max Tm Release Set in the Release Primer Hybridization Master Mix.

[0311] Twenty-four libraries were made using 10 ng of non-formalin-damaged NA24143 cell line DNA according to the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 Chapter 4 Library Preparation from DNA with the exceptions listed below: During step 5, post-ligation 0.8X purification was performed using KAPA HyperPure beads, adapter-ligated DNA samples were pooled, and the pool was aliquoted into 20 μL per sample and carried into step 6.

[0312] Primer extension target enrichment was performed for each of the 16 libraries in 10 μL according to the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 Chapter 5 Primer Extension Target Enrichment (PETE), with the exceptions listed below: During step 2 (capture extension reaction), equal concentrations of the No Maximum Tm High % GC ROI capture group and the Hot Spot capture group were added to the capture extension reaction master mix. Single-stranded DNA binding protein was added to three replicates at 100 ng, 200 ng, or 400 ng, respectively. Betaine was added to three replicates to a final concentration of 0.5 M. Trehalose was added to three replicates to a final concentration of 0.1 M. DMSO was added to three replicates at a final concentration of 5% or 10%, respectively (Table 111). There was no capture extension additive in three replicates. During step 6 (release primer hybridization), the volume of the 1X wash and resuspension buffer was reduced to accommodate equal concentrations of the Hot Spot release group and the No Maximum Tm release group in the release primer hybridization master mix.

[0313] Primer extension target enrichment libraries were sequenced on a NextSeq 500 and analyzed using Roche internal pipelines with a total subsampled read count of 8 M. Normalized coverage at each position was calculated for each sample by dividing the coverage at a position by the average coverage across the sample.

[0314] Table 11 - Experimental setup.

[0315] <![CDATA[ additive ]]> <![CDATA[ Final concentration ]]> <![CDATA[ Repeated determination ]]> 2-Amino-dATP 0.3mM 4 Both 2-amino-dATP and 7-deaza-dGTP 0.3mM each 4 7-deaza-dGTP 0.3mM 4 Single-stranded DNA binding protein (total 100ng) 2ng / μL 3 Single-stranded DNA binding protein (total 200ng) 4ng / μL 3 Single-stranded DNA binding protein (400 ng total) 8ng / μL 3 Betaine 0.5M 3 Trehalose 0.1M 3 DMSO 10％ 3 DMSO 5％ 3

[0316] result

[0317] The percentage of reads on target was similar between the 7-deazadGTP only (7DdGTP) sample and the 7DdGTP and 2-aminodATP (2AdATP) sample, and slightly higher than the control sample (see Fig.11). The control sample and the 2AdATP-only sample had similar percentages of reads on-target. The samples with only 7DdGTP added had much higher deduplication depths than the control samples, while the samples with 2AdATP added (with or without 7DdGTP) had lower deduplication depths than the control samples. The percentage of bases within a 2-fold range was slightly higher for the samples with 7DdGTP added (with or without 2AdATP) than for the control samples and the 2AdATP-only sample. The percentage of group exonic regions greater than or equal to 1000X was much higher for the samples with 7DdATP than for the control samples, followed by the 2AdATP and 7DdATP samples. The percentage of group exonic regions greater than or equal to 1000X was slightly lower for the samples with only 2AdATP added compared to the control samples. The samples with only 7DdATP added had the same error rate percentage as the control samples, while the samples with 2AdATP added (with or without 7DdATP) had a higher error rate percentage compared to the control samples.

[0318] Compared with the samples with only 2AdATP added and the control samples, for the samples with 7DdTP added (with or without 2AdATP), the normalized position-deduplication coverage of the three high GC percentage targets in the no maximum Tm group was more uniform and close to the target normalized coverage of 1 (as indicated by the dotted line) ( Fig.12 ).

[0319] For samples captured using the no-maximum-Tm set, normalized position-deduplication coverage in high GC percentage targets was higher than for samples captured using the set designed using the current primer database from previous experiments; however, median normalized coverage levels were still below the ideal coverage of 1 (see Fig.13 ). After adding 7DdGTP in PCR1, the normalized coverage of the three targets was higher than 1.

[0320] Compared to all other samples with similar read-on-target percentages, the read-on-target percentage was greatly reduced by the addition of 10% DMSO (DMSO10) and slightly increased by the addition of betaine (see Fig.14 ). The deduplication depth was greatly reduced by the addition of DMSO10 compared to all other samples with similar deduplication depth. The percentage of bases within 2-fold range and the percentage of group exonic regions greater than or equal to 1000x were greatly reduced by the addition of DMSO10 compared to all other samples with similar performance. The error rate percentage was increased by the addition of DMSO10 compared to all other samples with similar error rate percentage.

[0321] Compared with the control samples, for the samples with added DMSO5 or betaine, the normalized position-deduplication coverage of the three high GC percentage targets in the no maximum Tm group was more uniform and close to the target normalized coverage of 1 (as indicated by the dotted line) (see Fig.15 ). The samples supplemented with DMSO10 had opposite profiles of CEBPB exon 1 and EGFR exon 1 compared to the control samples, as in the high coverage fractions of the control samples were low coverage in the DMSO10 samples and vice versa.

[0322] For samples captured using the no-maximum-Tm set, normalized position-deduplication coverage in high GC percentage targets was higher than for samples captured using the set designed using the current primer database from previous experiments; however, median normalized coverage levels were still below the ideal coverage of 1 (see Fig.16 ). After adding betaine in the capture extension, the normalized coverage of the three targets is closer to the ideal coverage of 1 compared to the sample without betaine (no maximum Tm group). After adding DMSO5 in the capture extension, the normalized coverage of the three targets is much higher than the ideal coverage of 1.

[0323] in conclusion

[0324] The addition of 7-deaza-dGTP in PCR1, betaine in capture extension, and 5% DMSO in capture extension all improved coverage of high GC percentage target regions. The addition of 7-deaza-dGTP also improved many sequencing QC metrics. The addition of betaine resulted in similar performance in sequencing QC metrics compared to no additive, while the addition of 5% DMSO showed a slight decrease in sequencing QC metric performance.

[0325] Example 6 - Titration of DMSO during capture extension

[0326] Purpose

[0327] The aim of this study was to titrate DMSO in capture extension using a large 1.7 Mb panel and evaluate the impact on sequencing QC metrics and coverage of high %GC targets.

[0328] Material

[0329] NA12891 cell line DNA

[0330] 1.7Mb group

[0331] DMSO

[0332] KAPA HyperPETE Kit

[0333] KAPA HyperCapture Bead Kit

[0334] KAPA HyperPlus Library Preparation Kit

[0335] KAPA UDI Primer Mix

[0336] KAPA Universal UMI Adapters

[0337] High Sensitivity D1000 Reagent

[0338] NextSeq500 / 550HO Reagents

[0339] method

[0340] Eighteen libraries were prepared with 10 ng of non-formalin damaged NA12891 cell line DNA according to the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 Chapter 4, with the exception that libraries were pooled after ligation cleanup and re-aliquoted in 20 μL volumes to limit differences in library preparation processing between samples. Enrichment was performed according to the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 Chapter 5, with some significant modifications to facilitate the use of a custom 1.7 Mb HyperPETE set that is beyond the size range of commercially available HyperPETE sets. The 1.7 Mb set was designed using an expanded primer database that includes %GC from 0-100 and Tm from 57-100°C.

[0341] The library input for primer extension target enrichment (PETE) was normalized to 1000ng library per sample. PETE workflow modifications included diluting the capture primers to 0.37x each primer concentration (relative to the standard 1x each primer concentration), thus keeping the total capture primer concentration at 2x the concentration of the largest group on the market (HyperPETE Pan-Cancer Group). Other modifications included cleaning up the concentrated Cot DNA using 3XHyperPure beads and adding twice the amount of human Cot DNA in the capture extension using 300μL capture beads (as opposed to 100μL in the standard protocol). In the capture extension step, the master mix was prepared to include 0-5% DMSO (Table 12). For the PCR amplification step, 13 cycles were used depending on the group size. The PETE libraries were sequenced on the NextSeq500 and analyzed using Roche internal channels, with secondary sampling at 60M total reads / sample.

[0342] Table 12 - Experimental setup.

[0343]

[0344] result

[0345] The on-target rate increases with increasing amount of DMSO in the capture extension (see Fig.17). The deduplication depth remained similar until 2% DMSO and then began to decline. For uniformity, the percentage of bases within a 2-fold range decreased with increasing DMSO amounts. The percentage of exon regions greater than or equal to 300x was optimal at 1% and 2% DMSO and began to decline with increasing DMSO amounts.

[0346] With increasing DMSO in the capture extension, normalized coverage of higher %GC targets improved (see Fig.18 ). At 2% DMSO, there was also minimal negative impact on coverage of low GC / high AT targets. Data for 1% and 3% are not shown, but the trend is the same.

[0347] in conclusion

[0348] Addition of 2% DMSO in the capture extension using the 1.7 Mb set improved coverage of high %GC targets while minimizing the negative impact on low %GC targets, and also improved or maintained similar enrichment performance based on sequencing quality metrics.

[0349] Additional Embodiments

[0350] Additional Example 1. A composition comprising: (a) a polymerase, (b) one or more primers, (c) unmodified dNTPs, and (d) at least one enhancer.

[0351] Additional Example 2. 4. The composition of additional embodiment 1, wherein the at least one enhancer is selected from the group consisting of betaine, dimethyl sulfoxide (DMSO), disaccharides, and single-stranded DNA binding proteins (SSBs).

[0352] Additional Example 3. The composition of any preceding additional embodiments wherein the at least one enhancer is betaine.

[0353] Additional Example 4. Embodiment 3: The composition according to additional embodiment 3, wherein the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM.

[0354] Additional Example 5. The composition according to additional embodiment 3, wherein the concentration of betaine in the composition ranges from about 0.3 mM to about 0.6 mM.

[0355] Additional Example 6. The composition according to additional embodiment 3, wherein the concentration of betaine in the composition is about 0.5 mM.

[0356] Additional Example 7.Embodiment 2 further includes providing a composition wherein the at least one enhancer is DMSO.

[0357] Additional Example 8. The composition according to additional embodiment 7, wherein the amount of DMSO in the composition ranges from about 1% (v / v) to about 10% (v / v).

[0358] Additional Example 9. The composition according to additional embodiment 7, wherein the amount of DMSO in the composition ranges from about 2% (v / v) to about 9% (v / v).

[0359] Additional Example 10. The composition of any preceding additional embodiments, wherein the one or more primers have a Tm ranging from about 57°C to about 95°C.

[0360] Additional Example 11. The composition of any preceding additional embodiments, wherein the one or more primers have a Tm ranging from about 57°C to about 85°C.

[0361] Additional Example 12. The composition of any preceding additional embodiments, wherein the one or more primers have a Tm ranging from about 57°C to about 75°C.

[0362] Additional Example 13. The composition of any preceding additional embodiments, wherein the one or more primers have a Tm ranging from about 57°C to about 72°C.

[0363] Additional Example 14. The composition of any preceding additional embodiments, wherein the one or more primers have a Tm ranging from about 57°C to about 69°C.

[0364] Additional Example 15. The composition of any preceding additional embodiments, wherein the one or more primers have a Tm ranging from about 57°C to about 66°C.

[0365] Additional Example 16. The composition of any preceding additional embodiments, wherein the one or more primers have a Tm ranging from about 57°C to about 63°C.

[0366] Additional Example 17. The composition of any preceding additional embodiments, wherein the one or more primers comprise one or more modified dNTPs.

[0367] Additional Example 18.The composition of additional embodiment 17, wherein the one or more modified dNTPs are selected from the group consisting of modified dGTP and modified dATP.

[0368] Additional Example 19. The composition of any preceding additional embodiments, further comprising one or more modified dNTPs.

[0369] Additional Example 20. The composition of additional embodiment 19, wherein the concentration of one or more modified dNTPs in the composition is about the same as the concentration of unmodified dNTPs in the composition.

[0370] Additional Example 21. 19. The composition of additional embodiment 19, wherein the concentration of one or more modified dNTPs in the composition ranges from about 0.1 mM to about 0.5 mM.

[0371] Additional Example 22. 19. The composition of additional embodiment 19, wherein the concentration of one or modified dNTP in the composition ranges from about 0.2 mM to about 0.4 mM.

[0372] Additional Example 23. The composition of additional embodiment 19, wherein the one or more modified dNTPs is a modified dGTP.

[0373] Additional Example 24. Embodiment 23. The composition of additional embodiment 23, wherein the modified dGTP comprises 7-deaza-2'-deoxyguanosine-5'-triphosphate.

[0374] Additional Example 25. The composition of additional embodiment 19, wherein the one or more modified dNTPs is a modified dATP.

[0375] Additional Example 26. Embodiment 25. The composition of additional embodiment 25, wherein the modified dATP comprises 2-amino-2'deoxyadenosine-5'-triphosphate.

[0376] Additional Example 27. Embodiment 26 further comprises a divalent cation.

[0377] Additional Example 28. Embodiment 27: The composition of claim 27, wherein the divalent cation is selected from the group consisting of: Co 2+ , Mn 2+ Mg 2+ 、Cd 2+ and Ca2+ .

[0378] Additional Example 29. The composition of any preceding additional embodiments, wherein the composition further comprises one or more buffering agents.

[0379] Additional Example 30. The composition of any preceding additional embodiments, wherein the composition further comprises one or more polyols.

[0380] Additional Example 31. The composition of any preceding additional embodiments, wherein the one or more primers comprises a pre-capture forward primer and a pre-capture reverse primer.

[0381] Additional Example 32. The composition of any preceding additional embodiments, wherein the one or more primers comprises a capture primer.

[0382] Additional Example 33. The composition of any preceding additional embodiments, wherein the capture primer comprises a capture moiety.

[0383] Additional Example 34. The composition of any preceding additional embodiments, wherein the composition further comprises an input nucleic acid molecule.

[0384] Additional Example 35. The composition of additional embodiment 34, wherein the input nucleic acid molecule comprises a library of nucleic acid molecules, wherein each nucleic acid molecule in the library of nucleic acid molecules comprises a first adaptor and a second adaptor.

[0385] Additional Example 36. The composition of additional embodiment 34, wherein the one or more primers comprise one or more capture primers, and wherein the one or more capture primers are capable of hybridizing to a target nucleic acid sequence within the library of nucleic acid molecules.

[0386] Additional Example 37. A composition comprising: (a) a polymerase, (b) one or more primers, (c) dNTPs, and (d) optionally at least one enhancer, wherein the one or more primers are not limited in terms of the percentage of guanine or cytosine bases, and wherein at least one of the one or more primers has a melting temperature greater than 63°C.

[0387] Additional Example 38. Embodiment 37. The composition of additional embodiment 37, wherein the at least one primer has a melting temperature greater than about 69°C.

[0388] Additional Example 39.Embodiment 37. The composition of additional embodiment 37, wherein the at least one primer has a melting temperature greater than about 75°C.

[0389] Additional Example 40. Embodiment 37. The composition of additional embodiment 37, wherein the at least one primer has a melting temperature greater than about 85°C.

[0390] Additional Example 41. Embodiment 37. The composition of additional embodiment 37, wherein the at least one primer has a melting temperature greater than about 95°C.

[0391] Additional Example 42. 44. The composition of any one of additional embodiments 37 to 41, wherein the at least one optional enhancer is selected from the group consisting of betaine, DMSO, a disaccharide, and a single-stranded DNA binding protein (SSB).

[0392] Additional Example 43. 46. ​​The composition of additional embodiment 42, wherein the at least one optional enhancer is betaine, and wherein the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM.

[0393] Additional Example 44. The composition of any one of additional embodiments 37 to 43, wherein the dNTPs comprise a mixture of unmodified dNTPs and modified dNTPs.

[0394] Additional Example 45. Embodiment 44. The composition of additional embodiment 44, wherein the one or more modified dNTPs comprise 7-deaza-2'-deoxyguanosine-5'-triphosphate.

[0395] Additional Example 46. The composition of additional embodiment 44, wherein the one or more modified dNTPs comprises 2-amino-2'deoxyadenosine-5'-triphosphate.

[0396] Additional Example 47. The composition of any one of additional embodiments 44 to 46, wherein the concentration of the one or more modified dNTPs in the composition ranges from about 0.2 mM to about 0.8 mM.

[0397] Additional Example 48. The composition of any one of additional embodiments 37 to 41, wherein the one or more primers comprise one or more modified dNTPs.

[0398] Additional Example 49.48. The composition of additional embodiment 48, wherein the one or more modified dNTPs are selected from the group consisting of 7-deaza-2'-deoxyguanosine-5'-triphosphate and 2-amino-2'deoxyadenosine-5'-triphosphate.

[0399] Additional Example 50. Embodiment 49. The composition of any one of additional embodiments 37 to 49, wherein the composition further comprises a divalent cation.

[0400] Additional Example 51. The composition according to additional embodiment 50, wherein the divalent cation is selected from the group consisting of: Co2+, Mn2+, Mg2+, Cd2+ and Ca2+.

[0401] Additional Example 52. The composition according to any one of additional embodiments 37 to 51, wherein the composition further comprises one or more buffering agents.

[0402] Additional Example 53. The composition according to any of additional embodiments 37 to 52, wherein the composition further comprises one or more polyols.

[0403] Additional Example 54. The composition of any one of additional embodiments 37 to 53, wherein the one or more primers comprises a pre-capture forward primer and a pre-capture reverse primer.

[0404] Additional Example 55. Embodiment 54. The composition of any one of additional embodiments 37 to 53, wherein the one or more primers are capture primers.

[0405] Additional Example 56. The composition according to any one of additional embodiments 37 to 55, wherein the composition further comprises an input nucleic acid molecule.

[0406] Additional Example 57. Embodiment 56: The composition of additional embodiment 56, wherein the input nucleic acid molecule comprises a first adaptor and a second adaptor.

[0407] Additional Example 58.

[00136] The composition of additional embodiment 56, wherein the one or more primers comprise one or more capture primers, and wherein the one or more capture primers are capable of hybridizing to a target nucleic acid sequence of the nucleic acid molecule.

[0408] Additional Example 59. The composition of additional embodiment 56, wherein the input nucleic acid molecule comprises DNA.

[0409] Additional Example 60.A reaction tube comprising the composition of any one of Additional Embodiments 1 to 59.

[0410] Additional Example 61. Use of the composition according to any one of additional embodiments 1 to 33 and 37 to 55 in amplifying an input nucleic acid molecule.

[0411] Additional Example 62. A composition comprising: (a) one or more primers, (b) an input nucleic acid molecule, and (c) at least one enhancer selected from the group consisting of: betaine, DMSO, a disaccharide, and a single-stranded DNA binding protein (SSB).

[0412] Additional Example 63. Embodiment 63. The composition of additional embodiment 62, wherein the at least one enhancer is betaine.

[0413] Additional Example 64. 64. The composition according to additional embodiment 63, wherein the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM.

[0414] Additional Example 65. 64. The composition according to additional embodiment 63, wherein the concentration of betaine in the composition ranges from about 0.3 mM to about 0.6 mM.

[0415] Additional Example 66. Embodiment 63. The composition of additional embodiment 62, wherein the at least one enhancer is DMSO.

[0416] Additional Example 67. Embodiment 66: The composition of additional embodiment 66, wherein the amount of DMSO in the composition ranges from about 1% (v / v) to about 10% (v / v).

[0417] Additional Example 68. Embodiment 66: The composition of additional embodiment 66, wherein the amount of DMSO in the composition ranges from about 2% (v / v) to about 8% (v / v).

[0418] Additional Example 69. 64. The composition of additional embodiment 62, wherein the one or more primers have a Tm ranging from about 57°C to about 95°C.

[0419] Additional Example 70. 64. The composition of additional embodiment 62, wherein the one or more primers have a Tm ranging from about 57°C to about 85°C.

[0420] Additional Example 71.64. The composition of additional embodiment 62, wherein the one or more primers have a Tm ranging from about 57°C to about 75°C.

[0421] Additional Example 72. 64. The composition of additional embodiment 62, wherein the one or more primers have a Tm ranging from about 57°C to about 72°C.

[0422] Additional Example 73. 64. The composition of additional embodiment 62, wherein the one or more primers have a Tm ranging from about 57°C to about 69°C.

[0423] Additional Example 74. 64. The composition of additional embodiment 62, wherein the one or more primers have a Tm ranging from about 57°C to about 66°C.

[0424] Additional Example 75. The composition of additional embodiment 62, wherein the one or more primers comprise one or more modified dNTPs.

[0425] Additional Example 76. The composition of additional embodiment 75, wherein the one or more modified dNTPs are selected from the group consisting of modified dGTP and modified dATP.

[0426] Additional Example 77. The composition according to any one of additional embodiments 62-76, wherein the composition further comprises a divalent cation.

[0427] Additional Example 78. The composition according to additional embodiment 77, wherein the divalent cation is selected from the group consisting of: Co 2+ , Mn 2+ Mg 2+ 、Cd 2+ and Ca 2+ .

[0428] Additional Example 79. The composition according to any one of additional embodiments 62 to 78, wherein the composition further comprises one or more buffering agents.

[0429] Additional Example 80. The composition of any one of additional embodiments 62 to 79, wherein the input nucleic acid molecules comprise a prepared nucleic acid library.

[0430] Additional Example 81. The composition of any one of additional embodiments 62 to 79, wherein the input nucleic acid molecules comprise captured nucleic acid molecules.

[0431] Additional Example 82. The composition of additional embodiment 81, wherein the captured nucleic acid molecule comprises a complex of nucleic acid molecules comprising a target nucleic acid sequence and an extended capture primer hybridized to at least a portion of the target nucleic acid sequence.

[0432] Additional Example 83. A composition comprising: (a) an input nucleic acid molecule, and (b) one or more primers, and optionally (c) at least one enhancer, wherein the one or more primers are not restricted in terms of the percentage of guanine or cytosine bases, and wherein at least one of the one or more primers has a melting temperature greater than 63°C.

[0433] Additional Example 84. Embodiment 83. The composition of additional embodiment 83, wherein the at least one primer has a melting temperature greater than about 69°C.

[0434] Additional Example 85. Embodiment 83. The composition of additional embodiment 83, wherein the at least one primer has a melting temperature greater than about 75°C.

[0435] Additional Example 86. Embodiment 83. The composition of additional embodiment 83, wherein the at least one primer has a melting temperature greater than about 85°C.

[0436] Additional Example 87. Embodiment 83. The composition of additional embodiment 83, wherein the at least one primer has a melting temperature greater than about 95°C.

[0437] Additional Example 88. The composition of any one of additional embodiments 83 to 87, wherein the one or more primers comprise one or more modified dNTPs.

[0438] Additional Example 89. Embodiment 87. The composition of additional embodiment 88, wherein the one or more modified dNTPs are selected from the group consisting of 7-deaza-2'-deoxyguanosine-5'-triphosphate and 2-amino-2'deoxyadenosine-5'-triphosphate.

[0439] Additional Example 90. The composition according to any one of additional embodiments 83 to 89, wherein the at least one optional enhancer is selected from the group consisting of betaine, DMSO, a disaccharide, and a single-stranded DNA binding protein (SSB).

[0440] Additional Example 91. 93. The composition of additional embodiment 90, wherein the at least one optional enhancer is betaine, and wherein the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM.

[0441] Additional Example 92. The composition according to any one of additional embodiments 83 to 91, wherein the composition further comprises a divalent cation.

[0442] Additional Example 93. The composition according to additional embodiment 92, wherein the divalent cation is selected from the group consisting of: Co2+, Mn2+, Mg2+, Cd2+ and Ca2+.

[0443] Additional Example 94. The composition according to any one of additional embodiments 83 to 93, wherein the composition further comprises one or more buffering agents.

[0444] Additional Example 95. The composition of any one of additional embodiments 83 to 94, wherein the input nucleic acid molecules comprise a prepared nucleic acid library.

[0445] Additional Example 96. The composition of any one of additional embodiments 83 to 94, wherein the input nucleic acid molecules comprise captured DNA.

[0446] Additional Example 97. The composition of additional embodiment 96, wherein the captured nucleic acid molecule comprises a complex of nucleic acid molecules comprising a target nucleic acid sequence and an extended capture primer hybridized to at least a portion of the target nucleic acid sequence.

[0447] Additional Example 98. A reaction tube comprising the composition of any one of Additional Embodiments 62 to 97.

[0448] Additional Example 99. Use of the composition according to any one of additional embodiments 62 to 79 and 83 to 94 for preferentially enriching one or more target nucleic acid molecules in a nucleic acid molecule library.

[0449] Additional Example 100. A kit comprising: (a) a capture primer set; (b) a release primer set; (c) a polymerase; (d) dNTPs; and (e) betaine or a derivative or analog thereof.

[0450] Additional Example 101. The kit according to additional embodiment 100, wherein the dNTPs comprise a mixture of unmodified dNTPs and modified dNTPs.

[0451] Additional Example 102. The kit of additional embodiment 101, wherein the modified dNTPs comprise modified dGTP and modified dATP.

[0452] Additional Example 103. The kit according to any one of additional embodiments 100 to 102, wherein the capture primer set and the release primer set comprise one or more modified dNTPs.

[0453] Additional Example 104. The kit according to additional embodiment 100, further comprising instructions for preparing a capture extension master mix, wherein the prepared capture extension master mix comprises betaine at a concentration of about 0.5 mM.

[0454] Additional Example 105. 104. The kit according to any one of additional embodiments 100 to 104, further comprising one or more buffers.

[0455] Additional Example 106. Embodiment 105 further comprising one or more divalent cations.

[0456] Additional Example 107. Embodiment 106 further comprising one or more polyols.

[0457] Additional Example 108. The kit according to any one of additional embodiments 100 to 107, wherein at least one of the capture primer set and the release primer set has no restrictions on melting temperature and / or GC content.

[0458] Additional Example 109. The kit according to any one of additional embodiments 100 to 108, wherein both the capture primer set and the release primer set have no restrictions on melting temperature and / or GC content.

[0459] Additional Example 110. A kit comprising: (a) a capture primer set; (b) a release primer set; (c) a polymerase; and (d) dNTPs; wherein at least one primer in the capture primer set has a melting temperature greater than about 65°C; and wherein at least one primer in the release primer set has a melting temperature greater than about 65°C.

[0460] Additional Example 111. The kit of additional embodiment 110, wherein at least one primer in the capture primer set has a melting temperature greater than about 72°C; and wherein at least one primer in the release primer set has a melting temperature greater than about 72°C.

[0461] Additional Example 112.The kit of any one of additional embodiments 110 to 111, wherein at least one primer in the capture primer set comprises one or more modified dNTPs; and wherein at least one primer in the release primer set comprises one or more modified dNTPs.

[0462] Additional Example 113. The kit according to any one of additional embodiments 110 to 112, wherein the kit further comprises at least one enhancer selected from the group consisting of betaine or a derivative or analog thereof, DMSO, a single-stranded DNA binding protein, or a disaccharide.

[0463] Additional Example 114. A method for producing a capture primer extension complex comprising a target nucleic acid molecule and a capture primer, the method comprising: (a) hybridizing a capture primer with a portion of the target nucleic acid molecules in a nucleic acid molecule library, wherein each of the nucleic acid molecules in the nucleic acid molecule library has a first end comprising a first linker and a second end comprising a second linker; and (b) extending the hybridized capture primer with a first polymerase to produce a capture primer extension complex; wherein the capture primer has a melting temperature greater than 63°C.

[0464] Additional Example 115. The method according to additional embodiment 114, wherein the melting temperature of the capture primer is greater than 66°C.

[0465] Additional Example 116. The method according to additional embodiment 114, wherein the melting temperature of the capture primer is greater than 72°C.

[0466] Additional Example 117. The method according to additional embodiment 114, wherein the melting temperature of the capture primer is greater than 75°C.

[0467] Additional Example 118. According to the method described in any of the additional embodiments 114 to 117, wherein the capture primer hybridized to the target nucleic acid molecule is extended in a composition comprising at least one enhancer, wherein the at least one enhancer is selected from the group consisting of: betaine or its derivative or analogue, DMSO, a single-stranded DNA binding protein or a disaccharide.

[0468] Additional Example 119. The method according to additional embodiment 118, wherein the at least one enhancer is betaine, and wherein the concentration of betaine ranges from about 0.2 mM to about 0.8 mM.

[0469] Additional Example 120. The method according to additional embodiment 118, wherein the at least one enhancer is betaine, and wherein the concentration of betaine ranges from about 0.3 mM to about 0.7 mM.

[0470] Additional Example 121. The method according to additional embodiment 118, wherein the at least one enhancer is betaine, and wherein the concentration of betaine ranges from about 0.4 mM to about 0.6 mM.

[0471] Additional Example 122. The method of any one of additional embodiments 114 to 117, wherein the capture primer hybridized to the target nucleic acid molecule is extended in a composition comprising one or more modified dNTPs.

[0472] Additional Example 123. The method according to Additional Embodiment 122, wherein the one or more modified dNTPs is a modified dGTP.

[0473] Additional Example 124. The method according to additional embodiment 123, wherein the modified dGTP comprises 7-deaza-2'-deoxyguanosine-5'-triphosphate.

[0474] Additional Example 125. The method according to additional embodiment 122, wherein the one or more modified dNTPs is modified dATP.

[0475] Additional Example 126. The method according to additional embodiment 125, wherein the modified dATP comprises 2-amino-2'deoxyadenosine-5'-triphosphate.

[0476] Additional Example 127. The method of any one of additional embodiments 114 to 117, wherein the capture primer hybridized to the target nucleic acid molecule is extended in a composition comprising betaine and one or more modified dNTPs.

[0477] Additional Example 128. The method according to additional embodiment 127, wherein the concentration of betaine in the composition is about 0.5 mM.

[0478] Additional Example 129. Embodiment 127. The method of any one of additional embodiments 114 to 128, further comprising capturing the capture primer-extension complex.

[0479] Additional Example 130. Embodiment 129 further comprising hybridizing a release primer to the target nucleic acid.

[0480] Additional Example 131. The method according to additional embodiment 130, wherein the release primer is hybridized to the target nucleic acid in a composition comprising at least one enhancer.

[0481] Additional Example 132. The method according to additional embodiment 131, wherein the at least one enhancer is betaine.

[0482] Additional Example 133. The method according to additional embodiment 130, wherein the release primer is hybridized to the target nucleic acid in a composition comprising one or more dNTPs.

[0483] Additional Example 134. The method according to additional embodiment 130, wherein the release primer has a melting temperature greater than 63°C.

[0484] Additional Example 135. The method according to additional embodiment 134, wherein the melting temperature is greater than 65°C.

[0485] Additional Example 136. The method according to additional embodiment 134, wherein the melting temperature is greater than 72°C.

[0486] Additional Example 137. A method according to additional embodiment 130, further comprising extending the release primer hybridized to the target nucleic acid with a second polymerase.

[0487] Additional Example 138. A method for amplifying one or more nucleic acid molecules, the method comprising: (a) obtaining a plurality of nucleic acid molecules; and (b) performing a first amplification reaction in a first composition comprising the obtained plurality of nucleic acid molecules and a first set of primers, wherein at least one primer in the first set of primers has a melting temperature greater than 63°C.

[0488] Additional Example 139. A method according to additional embodiment 138, wherein each nucleic acid molecule in the obtained plurality of nucleic acid molecules comprises a first adaptor and a second adaptor.

[0489] Additional Example 140. The method according to any one of additional embodiments 138-139, wherein the first composition further comprises betaine.

[0490] Additional Example 141. The method according to additional embodiment 140, wherein the concentration of betaine in the first composition is about 0.5 mM.

[0491] Additional Example 142. According to the method described in additional embodiment 139, it further comprises enriching the obtained multiple nucleic acid molecules for one or more target nucleic acid molecules.

[0492] Additional Example 143.A method according to additional embodiment 142, further comprising performing a second amplification reaction in a second composition comprising one or more target nucleic acid molecules and a second set of primers, wherein at least one primer in the second set of primers has a melting temperature greater than 63°C.

[0493] Additional Example 144. The method according to additional embodiment 143, wherein the second composition further comprises betaine.

[0494] Additional Example 145. The method according to additional embodiment 144, wherein the concentration of betaine in the second composition is about 0.5 mM.

[0495] Additional Example 146. A method according to additional embodiment 138, wherein the obtained plurality of nucleic acid molecules is a target enrichment library comprising a plurality of target nucleic acid molecules.

[0496] Additional Example 147. The method according to additional embodiment 146, further comprising sequencing the amplified target enriched library.

[0497] Additional Example 148. A method for amplifying one or more nucleic acid molecules, the method comprising: (a) obtaining a plurality of nucleic acid molecules; and (b) performing a first amplification reaction in a first composition comprising the plurality of nucleic acid molecules, betaine, and a mixture of unmodified dNTPs and modified dNTPs.

[0498] Additional Example 149. The method according to additional embodiment 148, wherein the concentration of betaine in the first composition is about 0.5 mM.

[0499] Additional Example 150. The method of any one of additional embodiments 148 to 149, wherein at least one primer in the first set of primers has a high melting temperature and / or a high GC content.

[0500] Additional Example 151. The method according to additional embodiment 148, wherein each nucleic acid molecule in the obtained plurality of nucleic acid molecules comprises a first adaptor and a second adaptor, and wherein the method further comprises enriching the obtained plurality of nucleic acid molecules for one or more target nucleic acid molecules.

[0501] Additional Example 152. The method according to additional embodiment 151, further comprising performing a second amplification reaction in a second composition comprising one or more target nucleic acid molecules, betaine, and a mixture of unmodified dNTPs and modified dNTPs.

[0502] Additional Example 153.A method according to additional embodiment 148, wherein the obtained plurality of nucleic acid molecules is a target enrichment library comprising a plurality of target nucleic acid molecules.

[0503] Additional Example 154. The method according to additional embodiment 153 further comprises sequencing the amplified target enriched library.

[0504] Additional Example 155. A kit for enriching at least one target nucleic acid molecule in a nucleic acid molecule library, the kit comprising: (a) a first oligonucleotide complementary to a target nucleic acid in the nucleic acid molecule library, each of the nucleic acid molecules in the nucleic acid molecule library having a first end comprising a first adaptor and a second end comprising a second adaptor; (b) a second oligonucleotide complementary to the target nucleic acid; a first amplification primer; and (c) a second amplification primer; and wherein at least one of the first oligonucleotide or the second oligonucleotide has a high melting temperature and / or a high GC content.

[0505] Additional Example 156. The kit according to additional embodiment 155, wherein the first oligonucleotide and the second oligonucleotide both have a high melting temperature and / or a high GC content.

[0506] Additional Example 157. A composition comprising: (a) a library of nucleic acid molecules comprising at least one target nucleic acid, each of the nucleic acid molecules in the library having a first end comprising a first adaptor, a second end comprising a second adaptor, and a target region between the first adaptor and the second adaptor; (b) an extended first oligonucleotide hybridized to the target region of the target nucleic acid, the extended first oligonucleotide comprising at least one capture portion; a solid support bound to the at least one capture portion; (c) a second oligonucleotide hybridized to the target nucleic acid at the 5' position of the first extended oligonucleotide; and (d) a polymerase associated with the 3' end of the second oligonucleotide; wherein at least one of the first oligonucleotide or the second oligonucleotide has a high melting temperature and / or a high GC content.

[0507] Additional Example 158. A composition comprising: (a) a polymerase, (b) one or more primers that are not limited in terms of melting temperature and / or GC content, (c) a mixture of unmodified dNTPs and modified dNTPs, and (d) at least one enhancer.

[0508] Additional Example 159. Embodiment 159. The composition of additional embodiment 158, wherein the at least one enhancer is betaine.

[0509] Additional Example 160.Embodiment 157. The composition of any one of additional embodiments 158-159, further comprising one or more nucleic acid molecules.

[0510] Additional Example 161. A composition consisting essentially of a polymerase, one or more primers with no limitations on melting temperature and / or GC content, a mixture of unmodified dNTPs and modified dNTPs, and at least one enhancer.

[0511] Additional Example 162. Embodiment 164: The composition of additional embodiment 161, wherein the at least one enhancer is betaine.

[0512] Additional Example 163. The composition of any one of additional embodiments 161 to 162, further comprising one or more nucleic acid molecules.

[0513] Additional Example 164. A composition consisting of a polymerase, one or more primers with no restrictions on melting temperature and / or GC content, a mixture of unmodified dNTPs and modified dNTPs, and at least one enhancer.

[0514] Additional Example 165. Embodiment 164: The composition of additional embodiment 164, wherein the at least one enhancer is betaine.

[0515] Additional Example 166. The composition according to any of additional embodiments 164-165, further comprising one or more nucleic acid molecules.

[0516] Additional Example 167. A reaction tube comprising the composition of any one of Additional Embodiments 158 to 166.

[0517] Additional Example 168. Use of the composition of any one of additional embodiments 158, 159, 161, 162, 164 and 165 in amplifying an input nucleic acid molecule.

[0518] Additional Example 169. A method of amplifying one or more nucleic acid molecules, wherein the amplification is performed in the presence of any of the compositions described in additional embodiments 158, 159, 161, 162, 164 and 165.

[0519] Additional Example 170. The method according to additional embodiment 169, wherein the thermal cycling step is performed for about 34 minutes.

Claims

1. A composition comprising: (a) a polymerase, (b) one or more primers, (c) unmodified dNTPs, (d) at least one enhancer selected from the group consisting of betaine, dimethyl sulfoxide (DMSO), trehalose, and a single-stranded DNA binding protein; and (e) optionally one or more modified dNTPs.

2. The composition of claim 1, wherein the at least one enhancing agent is DMSO, and wherein the amount of DMSO in the composition ranges from about 1% (v / v) to about 3% (v / v).

3. The composition of claim 1, wherein the at least one enhancer is betaine, and wherein the concentration of betaine in the composition ranges from about 0.4 mM to about 0.6 mM.

4. The composition of claim 1, wherein the at least one enhancer is trehalose, and wherein the concentration of trehalose in the composition is about 0.1 M.

5. The composition of claim 1, wherein the at least one enhancing agent is a single-stranded binding protein, and wherein the concentration of the single-stranded binding protein in the composition ranges from about 1 ng / mL to about 9 ng / mL.

6. The composition of any of the preceding claims, wherein the one or more primers are capture primers, and wherein the capture primers comprise a capture moiety.

7. The method according to any one of claims 1 to 6, wherein at least two enhancers are present in the composition.

8. A composition comprising: (a) a polymerase; (b) one or more primers; (c) a dNTP mixture comprising (i) unmodified dNTPs, and (ii) modified dNTPs, wherein the modified dNTPs are selected from the group consisting of: (i) 7-deaza dGTP, (ii) 2-amino dATP, and (iii) 7-deaza dGTP and 2-amino dATP; and (d) optionally, at least one enhancer selected from the group consisting of: betaine or a derivative or analog thereof, DMSO, a single-stranded DNA binding protein, and a disaccharide, wherein the one or more primers include no restriction on the percentage of guanine or cytosine bases, and wherein at least one of the one or more primers has a melting temperature greater than 63°C.

9. The composition of claim 8, wherein the modified dNTPs comprise both 7-deaza dGTP and 2-amino dATP, and wherein the 7-deaza dGTP and 2-amino dATP are each present at a concentration ranging between about 0.2 mM to about 0.4 mM.

10. A method for producing a capture primer extension complex comprising a target nucleic acid molecule and a capture primer, the method comprising: (a) hybridizing a capture primer to a portion of the target nucleic acid molecule in a nucleic acid molecule library, wherein each of the nucleic acid molecules in the nucleic acid molecule library has a first end comprising a first linker and a second end comprising a second linker; and (b) extending the hybridized capture primer with a first polymerase to produce the capture primer extension complex, wherein the capture primer hybridized to the target nucleic acid molecule is extended in a composition comprising at least one enhancer, wherein the at least one enhancer is selected from the group consisting of: betaine or a derivative or analog thereof, DMSO, a single-stranded DNA binding protein and a disaccharide.

11. The composition of claim 10, wherein the at least one enhancing agent is DMSO, and wherein the amount of DMSO in the composition ranges from about 1% (v / v) to about 3% (v / v).

12. The composition of claim 10, wherein the at least one enhancer is betaine, and wherein the concentration of betaine in the composition ranges from about 0.4 mM to about 0.6 mM.

13. The composition of claim 10, wherein the at least one enhancer is trehalose, and wherein the concentration of trehalose in the composition is about 0.1 M.

14. The composition of claim 10, wherein the at least one enhancing agent is a single-stranded binding protein, and wherein the concentration of the single-stranded binding protein in the composition ranges from about 21 ng / mL to about 9 ng / mL.

15. The method according to any one of claims 10 to 14, wherein the composition comprises at least two enhancers.

16. A method for amplifying one or more nucleic acid molecules, the method comprising: (a) obtaining a plurality of nucleic acid molecules; (b) performing a first amplification reaction to amplify the obtained plurality of nucleic acid molecules, wherein the first amplification is performed using a first composition comprising: (i) polymerase; (ii) one or more primers; (iii) a dNTP mixture comprising: (a) unmodified dNTPs, and (b) a modified dNTP, wherein the modified dNTP is selected from the group consisting of: (i) 7-deaza-dGTP, (ii) 2-aminodATP, and (iii) 7-deaza-dGTP and 2-amino-dATP; and (iv) optionally, at least one enhancer selected from the group consisting of betaine or a derivative or analog thereof, DMSO, a single-stranded DNA binding protein, and a disaccharide; Wherein the one or more primers include no restriction on the percentage of guanine or cytosine bases, and wherein at least one primer of the one or more primers has a melting temperature greater than 63°C.

17. The composition of claim 16, wherein the modified dNTPs comprise both 7-deaza dGTP and 2-amino dATP, and wherein the 7-deaza dGTP and 2-amino dATP are each present at a concentration ranging between about 0.2 mM to about 0.4 mM.

18. The method according to any one of claims 16 to 17, further comprising enriching the obtained plurality of nucleic acid molecules for one or more target nucleic acid molecules before performing the first amplification reaction.

19. The method according to any one of claims 16 to 18, further comprising performing a second amplification reaction, wherein the second amplification is performed using a second composition comprising: (i) polymerase; (ii) one or more primers; (iii) a dNTP mixture comprising: (a) unmodified dNTPs, and (b) a modified dNTP, wherein the modified dNTP is selected from the group consisting of: (i) 7-deaza-dGTP, (ii) 2-aminodATP, and (iii) 7-deaza-dGTP and 2-amino-dATP; and (iv) optionally, at least one enhancer selected from the group consisting of betaine or a derivative or analog thereof, DMSO, a single-stranded DNA binding protein and a disaccharide.

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