Improving next-generation target enrichment performance

Abstract

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

Description

[Technical Field]

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

[0002] Background to the disclosure Next-generation sequencing (NSS) is a massively parallel sequencing technology that offers high throughput, scalability, and speed. Given its sensitivity and specificity, NSS is a tool for investigating the molecular basis of disease (e.g., cancer).

[0003] Next-generation sequencing application data can result in poor performance, which can include low on-target rates, low genome equivalent recovery rates (deduplicated / intrinsic depth), and low coverage uniformity. Low uniformity can be observed in target enrichment applications where either higher GC content or higher AT content is enriched. This is commonly referred to as GC bias. Coverage uniformity is an important metric and affects the ability to uniformly enrich and sequence regions of interest. Low coverage uniformity results in low sequencing coverage in poorly performing regions, ultimately requiring more sequencing runs to achieve targeted sequencing depth in these poorly performing regions. Low on-target rates also result in the need for more sequencing runs and ultimately higher sequencing costs per sample.

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

[0005] A brief summary of the disclosure 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, particularly in reducing GC bias during sequencing of target-enriched samples. In particular, the applicant has unexpectedly discovered that the use of a composition comprising a primer with a high melting temperature and / or one or more enhancers improves coverage uniformity and / or reduces GC bias during one or more downstream sequencing operations. The applicant has also unexpectedly discovered that coverage uniformity can be improved by optimizing the temperature and time / duration parameters during capture extension. These and other improvements are described in further detail 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) in the range of about 57°C to about 95°C. In some embodiments, one or more primers have a Tm in the range of about 57°C to about 85°C. In some embodiments, one or more primers have a Tm in the range of about 57°C to about 75°C. In some embodiments, one or more primers have a Tm in the range of about 57°C to about 72°C. In some embodiments, one or more primers have a Tm in the range of about 57°C to about 69°C. In some embodiments, one or more primers have a Tm in the range of about 57°C to about 66°C. In some embodiments, one or more primers have a Tm in the range of 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., pre-amplification primers, capture primers, release primers, amplification primers, etc.).

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

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

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

[0014] In some embodiments, the one or more primers comprise pre-capture forward and reverse primers. In some embodiments, the one or more primers comprise a capture primer. In some embodiments, the capture primer comprises a capture moiety.

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

[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 do not include a limit on the percentage of guanine or cytosine bases, and at least one primer 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, up to 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, the at least one optional enhancer is selected from betaine, DMSO, a disaccharide, and a single-stranded DNA binding protein (SSB). In some embodiments, the at least one optional enhancer is betaine, and the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM.

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

[0019] 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. In some embodiments, the composition further comprises one or more polyols.

[0020] In some embodiments, the one or more primers comprise pre-capture forward and reverse primers. In some embodiments, the one or more primers are capture primers. In some embodiments, the composition further comprises one or more nucleic acid molecules.

[0021] In some embodiments, the one or more nucleic acid molecules comprise a first and a second adaptor. In some embodiments, the one or more primers comprise one or more capture primers, and the one or more capture primers can hybridize to 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 vessel (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 disclosed herein.

[0023] A fourth aspect of the present disclosure is the use of a composition, such as any of the compositions described in the first and second aspects of the present disclosure above, or as further described herein, in the amplification of 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), hi 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 in the range of about 57°C to about 95°C. In some embodiments, one or more primers have a Tm in the range of about 57°C to about 85°C. In some embodiments, one or more primers have a Tm in the range of about 57°C to about 75°C. In some embodiments, one or more primers have a Tm in the range of about 57°C to about 72°C. In some embodiments, one or more primers have a Tm in the range of about 57°C to about 69°C. In some embodiments, one or more primers have a Tm in the range of 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., pre-amplification primers, capture primers, release primers, amplification primers, etc.).

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

[0029] In some embodiments, the composition further comprises a divalent cation. In some embodiments, the divalent cation is Co 2+ , Mn 2+ , Mg 2+ , Cd 2+ , and Ca 2+ In some embodiments, the composition further comprises one or more buffers.

[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 a nucleic acid molecule comprising a target nucleic acid sequence and an extended capture primer hybridized to at least a portion of the target nucleic acid sequence.

[0031] A 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 do not include a limit on the percentage of guanine or cytosine bases, and at least one primer 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, up to 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, the one or more primers comprise one or more modified dNTPs. In some embodiments, the one or more modified dNTPs are selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate and 2-amino-2'deoxyadenosine-5'-triphosphate. In some embodiments, the at least one optional enhancer is selected from betaine, DMSO, a disaccharide, and a single-stranded DNA binding protein (SSB).

[0033] In some embodiments, the at least one optional enhancer is betaine, and 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 a nucleic acid molecule 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 vessel (or container, vial, reaction chamber, etc.) comprising the composition of any of the fifth or sixth aspects of the present disclosure described above or further herein.

[0036] An eighth aspect of the present disclosure is the use of the composition of any one of the fifth or sixth aspects of the present disclosure described above or herein for preferential enrichment of one or more target nucleic acid molecules in a library of nucleic acid molecules.

[0037] A ninth aspect of the present disclosure is a kit comprising (i) a set of capture primers, (ii) a set of release primers, (iii) a polymerase, (iv) dNTPs, and (v) betaine or a derivative or analog thereof. In some embodiments, the dNTPs comprise a mixture of unmodified and modified dNTPs. In some embodiments, the modified dNTPs comprise modified dGTP and modified dATP. In some embodiments, the set of capture and release primers comprises 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 set of capture primers and release primers has no limitation on melting temperature and / or GC content. In some embodiments, both the set of capture primers and the set of release primers have no limitation on melting temperature and / or GC content.

[0038] A tenth aspect of the present disclosure is a kit comprising: (i) a set of capture primers; (ii) a set of release primers; (iii) a polymerase; and (iv) dNTPs, wherein at least one primer in the set of capture primers has a melting temperature greater than about 65°C; and at least one primer in the set of release primers has a melting temperature greater than about 65°C. In some embodiments, at least one primer in the set of capture primers has a melting temperature greater than about 72°C; and at least one primer in the set of release primers has a melting temperature greater than about 72°C. In some embodiments, up to about 10% of the capture and / or release primers in the kit are high melting temperature and / or high GC content primers (e.g., pre-amplification primers, capture primers, release primers, amplification primers, etc.). In some embodiments, at least one primer in the set of capture primers comprises one or more modified dNTPs; and at least one primer in the set of release primers comprises one or more modified dNTPs. In some embodiments, 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.

[0039] An 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 library of nucleic acid molecules, wherein each nucleic acid molecule in the library has a first end comprising a first adapter and a second end comprising a second adapter; 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, a capture primer hybridized to a 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, e.g., DMF), a single-stranded DNA binding protein (e.g., a thermostable single-stranded DNA binding protein), and a disaccharide (e.g., trehalose). In some embodiments, the 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, the 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, the 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, a capture primer hybridized to a 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 dGTP comprises 7-deaza-2'-deoxyguanosine-5'-triphosphate. In some embodiments, the one or more modified dNTPs are modified dATPs. In some embodiments, the modified dATP comprises 2-amino-2'-deoxyadenosine-5'-triphosphate. In some embodiments, a capture primer hybridized to a 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 the target nucleic acid. In some embodiments, the release primer hybridizes to the target nucleic acid in a composition comprising at least one enhancer. In some embodiments, the at least one enhancer is betaine. In some embodiments, the release primer hybridizes to the 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] A 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 primer set, wherein at least one primer of the first primer set has a melting temperature greater than 63°C. In some embodiments, each nucleic acid molecule of the obtained plurality of nucleic acid molecules comprises a first and a second adapter. In some embodiments, the first composition further comprises betaine. In some embodiments, the concentration of betaine in the first composition is about 0.5 mM. In some embodiments, the method further comprises concentrating 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 primer set, wherein at least one primer of the second primer set 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 second composition is about 0.5 mM.In some embodiments, the obtained plurality of nucleic acid molecules is a target enriched library comprising a plurality of target nucleic acid molecules.In some embodiments, the method further comprises sequencing the amplified target enriched library.

[0045] A 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; and (ii) conducting a first amplification reaction in a first composition comprising the plurality of nucleic acid molecules, betaine, and a mixture of unmodified 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 of the first primer set has a high melting temperature and / or a high GC content. In some embodiments, each nucleic acid molecule of the obtained plurality of nucleic acid molecules comprises a first and a second adaptor, and 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 conducting a second amplification reaction in a second composition comprising one or more target nucleic acid molecules, betaine, and a mixture of unmodified and modified dNTPs. In some embodiments, the obtained plurality of nucleic acid molecules is a target-enriched library comprising a plurality of target nucleic acid molecules. In some embodiments, the method further comprises sequencing the amplified target-enriched library.

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

[0047] A fifteenth aspect of the present disclosure is a composition comprising: a library of nucleic acid molecules comprising at least one target nucleic acid, wherein each of the nucleic acid molecules in the library of nucleic acid molecules has a first end comprising a first adaptor, a second end comprising a second adaptor, and a region of interest intermediate the first adaptor and the second adaptor; an extended first oligonucleotide hybridized to the region of interest of the target nucleic acid, the extended first oligonucleotide comprising at least one capture moiety; a solid support bound to the at least one capture moiety; a second oligonucleotide hybridized to the target nucleic acid at a position 5' relative to 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 that do not have limitations 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 essentially consists of a polymerase, one or more primers that do not have limitations on melting temperature and / or GC content, a mixture of unmodified dNTPs and modified dNTPs, and at least one enhancer.In some embodiments, the at least one enhancer 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 that do not have limitations on melting temperature and / or GC content, a mixture of unmodified dNTPs and modified dNTPs, and at least one enhancer. In some embodiments, the at least one enhancer is betaine. In some embodiments, the composition further comprises one or more nucleic acid molecules.

[0051] A nineteenth aspect of the present disclosure is a reaction vessel comprising the composition of any one of the sixteenth, seventeenth, or eighteenth aspects of the present disclosure described above or herein.

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

[0053] A twenty-first aspect of the present disclosure is a method of amplifying one or more nucleic acid molecules, wherein the amplification is carried out in the presence of any one of the compositions of the sixteenth, seventeenth, or eighteenth aspects of the present disclosure described above or herein. In some embodiments, the thermal cycling step is carried out for a time ranging from about 25 minutes to about 35 minutes, e.g., about 34 minutes. [Brief explanation of the drawings]

[0054] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with 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, wherein like reference numerals are used throughout to identify identical elements.

[0056] [Figure 1]

[0013] Figure 1 provides a flow chart illustrating one embodiment of a method for enriching at least one target nucleic acid molecule in a library of nucleic acid molecules according to the present disclosure. The illustrated steps can be performed in the presence of any of the compositions described herein. [Figure 2A] This shows that primer availability in target areas with high %GC is limited when primer %GC is restricted to 20%-80% and primer Tm is restricted to 57°C-63°C. Primer coverage for the primary target is incomplete with these restrictions on primer %GC and primer Tm. [Figure 2B] We show that removing the restrictions on primer %GC content and primer Tm increases primer availability in target areas with high %GC content, improving primer coverage in these areas. [Figure 2C] It has been shown that increasing the maximum primer Tm allowed during primer design reduces the number of target bases that are not covered by the primer. [Figure 3A] Samples enriched with primer test panels designed with primer Tms ranging from approximately 57°C to approximately 63°C (current primer database), approximately 57°C to approximately 66°C (maximum Tm 66), approximately 57°C to approximately 69°C (maximum Tm 69), approximately 57°C to approximately 62°C (maximum Tm 72°C), and approximately 57°C to "no Tm limit" (no maximum Tm). Samples enriched with the "no maximum Tm" test panel had the highest percentage of bases within a 2-fold range and a panel exon region percentage of 1000X or greater, a measure of sequencing coverage uniformity for the target. [Figure 3B]Samples enriched with primer test panels designed with primer Tms between approximately 57°C and approximately 63°C (current primer database), between approximately 57°C and approximately 66°C (max Tm 66), between approximately 57°C and approximately 69°C (max Tm 69), between approximately 57°C and approximately 62°C (max Tm 72°C), and between approximately 57°C and no limit (max Tm no) are shown. Normalized sequencing coverage at high %GC targets (65%, 75%, and 77%) is most improved for samples enriched with the no max Tm test panel compared to samples enriched with the current primer database. [Figure 4] Samples prepared into libraries with or without betaine (PCR1+B, PCR1-B) and with or without 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 yield of PCR1+B, which contained the PCR1-7 pre-capture input library, was higher than that of PCR1-B, which contained the PCR1-7 pre-capture input library. The yield of the PCR1+7 pre-capture input library was relatively low. [Figure 5] Samples prepared into libraries with or without betaine (PCR1+B, PCR1-B) and with or without 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). Compared to the PCR1-B sample, the PCR1+B and PCR1-7 samples had higher deduplication depths and average fragment lengths. Compared to the CapExt-B sample, the PCR1-7 containing CapExt+B sample had a 2-fold higher base percentage and over 1000x higher panel exon region percentage. The PCR1+7 sample had a 2-fold higher base percentage and over 1000x higher panel exon region percentage, regardless of the use of betaine in PCR1 or capture extension. [Figure 6A]Shown are samples prepared into libraries with and without 7-deaza-dGTP (PCR1 + 7-deaza-dGTP, PCR1 - 7-deaza-dGTP). The libraries were then used as input to HyperPETE with and without betaine in the capture extension (capture extension + betaine, capture extension - betaine). Compared to the PCR1-7 sample, the normalized coverage across the three high GC targets is more uniform for the PCR1+7 sample. [Figure 6B] Samples prepared into libraries with and without 7-deaza-dGTP (PCR1 + 7-deaza-dGTP, PCR1 - 7-deaza-dGTP) are shown. The libraries were then used as input to HyperPETE with and without betaine in the capture extension (capture extension + betaine, capture extension - betaine). For the PCR1 - 7-deaza-dGTP sample, the normalized coverage across the three high GC targets is more uniform for the capture extension + betaine sample than for the capture extension - betaine sample. [Figure 7] Samples were prepared into libraries with and without betaine (PCR1 + betaine, PCR1 - betaine) and with and without 7-deaza dGTP (PCR1 + 7-deaza dGTP, PCR1 - 7-deaza dGTP). The libraries were then used as input to HyperPETE with and without betaine in the capture extension (capture extension + betaine, capture extension - betaine). The PCR1 + 7-deaza dGTP sample had the highest normalized coverage for high GC targets (75%, 80%), followed by the PCR1 - 7-deaza dGTP + capture extension + betaine sample. The PCR1 + betaine sample had higher coverage for low GC targets (30%) compared to all other sample groups. [Figure 8]Samples enriched using the variable capture extension test programs and the control capture extension program with HyperPETE instructions for use (IFU) are shown in Tables 3-6 herein. All samples enriched with the test programs (Programs 1-4) had higher on-target read percentages and deduplication depths compared to the IFU programs. Average fragment lengths were much higher for the IFU samples compared to the test program samples. Program 3 samples had the highest percentage of bases within 2-fold range and the highest percentage of panel exon regions over 1000x. [Figure 9] Samples enriched using the variable capture extension test programs and the control capture extension program with HyperPETE instructions for use (IFU) are shown in Tables 3-6 herein. Samples enriched using the test programs (Programs 1-4) had higher normalized coverage in the high %GC targets (75%, 80%) compared to the IFU samples with Program 3 samples, which had the highest coverage. [Figure 10] Of all conditions tested to improve coverage in high % GC targets, samples generated from libraries enriched with a panel designed with 7-deaza dGTP and no primer % GC or maximum Tm limitations (no maximum Tm + PCR1 w / 7-deaza dGTP) showed the highest normalized coverage in the three high % GC targets (65%, 75%, 77%), exceeding 90% for all three targets. [Figure 11] FIG. 1 shows sequencing quality control metrics. [Figure 12] Normalized positional deduplication coverage over high GC percentage regions of interest is shown. [Figure 13] Normalized positional deduplication coverage by region of interest is shown. [Figure 14] Sequencing quality control metrics are shown. [Figure 15] Normalized positional deduplication coverage over high GC percentage regions of interest is shown. [Figure 16]Normalized positional deduplication coverage by region of interest is shown. [Figure 17] Provides sequencing quality control metrics for DMSO titration using a 1.7 Mb panel. [Figure 18] DMSO titration with a 1.7 Mb panel is shown, demonstrating normalized positional deduplication coverage and high GC percentage. DETAILED DESCRIPTION OF THE INVENTION

[0057] Detailed Description It is also to be understood that, unless expressly stated to the contrary, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0058] definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "comprising" is defined inclusively, such that "including A or B" means including A, B, or A and B.

[0059] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as inclusive, e.g., including at least one of a number or list of elements, but including a plurality, and optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," shall refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (e.g., "one or the other, but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0060] Terms such as "comprising," "including," and "having" are used interchangeably and have the same meaning. Similarly, "comprises," "includes," "has," and the like are used interchangeably and have the same meaning. Specifically, each term is defined consistent with the general U.S. patent law definition of "comprising," and therefore is to be interpreted as open term meaning "at least the following" and not excluding additional features, limitations, aspects, etc. Thus, 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 including steps a, b, and c" means that the method includes at least steps a, b, and c. Additionally, although steps and processes may be outlined herein in a particular order, those skilled in the art will recognize that the ordering of steps and processes may vary.

[0061] As used in the specification and claims herein, the phrase "at least one" in connection with a list of one or more elements should be understood to mean at least one element selected from any one or more elements of the list of elements, but may not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for elements other than those specifically identified in the list of elements to which the phrase "at least one" refers, whether related to those elements specifically identified or not. 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 in one embodiment to at least one, optionally more than one A, and no B (and optionally including elements other than B); in another embodiment to at least one, optionally more than one B, and no A (and optionally including elements other than A); in yet another embodiment to at least one, optionally more than one A, and at least one, optionally more than one B (and optionally including other elements); and so forth.

[0062] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0063] The term "adapter" refers to a nucleotide sequence that can be added to another sequence to confer additional properties to the sequence. Adapters can be single-stranded or double-stranded, or can have both single-stranded and double-stranded portions.

[0064] As used herein, "amplification" refers to the process of increasing copy number. Amplification can be a process in which replication occurs repeatedly over time to form multiple copies of a template. Amplification can result in an exponential or linear increase in copy number as amplification progresses. Exemplary amplification strategies include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), rolling circle replication (RCA), cascade RCA, and nucleic acid-based amplification (NASBA). Amplification can also utilize linear or circular templates. Amplification can be performed under any suitable temperature conditions, such as using thermal cycling or isothermal amplification. Furthermore, amplification can be performed in an amplification mixture (or reagent mixture), which is any composition capable of amplifying a nucleic acid target, if present in the mixture. PCR amplification relies on repeated cycles of heating and cooling (i.e., thermal cycling) to achieve successive rounds of replication. PCR can be carried out by thermal cycling between two or more temperature setpoints, such as a higher denaturation temperature and a lower annealing / extension temperature, or between three or more temperature setpoints, such as a higher denaturation temperature, a lower annealing temperature, and an intermediate extension temperature. PCR can be carried out using a thermostable polymerase such as Taq DNA polymerase. PCR generally results in an exponential increase in the amount of product amplicon over successive cycles. PCR is described in, for example, U.S. Patent No. 4,683,202, U.S. Patent No. 4,683,195, U.S. Patent No. 4,000,159, U.S. Patent No. 4,965,188, and U.S. Patent No. 5,176,995, the disclosures of which are incorporated herein by reference in their entirety.

[0065] As used herein, the term "complementary" generally refers to the ability to form precise 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 ionic buffer conditions. Complementarity is achieved through the differential interactions between the nucleobases adenine, thymine (uracil in RNA), guanine, and cytosine, where adenine pairs with thymine or uracil, and guanine pairs with cytosine. For example, if a nucleotide at a given position in a nucleic acid can hydrogen bond with a nucleotide in another nucleic acid, the two nucleic acids are considered complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules can be "partial," in which only a portion of the nucleotides bind, or complete, in which total complementarity exists between the single-stranded molecules. A first nucleotide sequence can be said to be the "complement" of a second sequence if the first nucleotide sequence is complementary to the second nucleotide sequence. A first nucleotide sequence can be said to be the "reverse complement" of a second sequence if the first nucleotide sequence is complementary to a sequence that is the reverse of the second sequence (i.e., the order of nucleotides is reversed).

[0066] As used herein, the term "enrichment" refers to a process that increases 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 before processing. Thus, an enrichment step provides a percentage or fractional increase rather than a direct increase in the copy number of a nucleic acid sequence of interest, as does, for example, amplification methods such as the polymerase chain reaction.

[0067] As used herein, the term "hybridize" refers to base pairing between different nucleic acid molecules that match their nucleotide sequences.

[0068] 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 modified nucleotides must be capable of the same or similar base pairing as the unmodified dNTPs they replace. Modified nucleotides or modified dNTPs may have a chemical structure similar to that of the corresponding nucleotide or dNTP, but may differ from the nucleotide or dNTP in at least one atom or at least one bond type.

[0069] As used herein, the terms "nucleic acid" or "polynucleotide" (used interchangeably herein) refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogs. Polynucleotides may have any three-dimensional structure and may perform any function, known or unknown. Unless specifically limited, the term encompasses nucleic acids or polynucleotides containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, locus(s) defined from phylogenetic 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.

[0070] A polynucleotide may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified, such as by conjugation with a labeling component. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base 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)).

[0071] As used herein, the term "oligonucleotide" refers to an oligomer of nucleotide or nucleoside monomeric units, where the oligomer optionally contains non-nucleotide monomeric units and / or other chemical groups attached to internal and / or external positions of the oligomer. The oligomer can be natural or synthetic and can include naturally occurring oligonucleotides or oligomers containing nucleosides with non-naturally occurring (or modified) bases, sugar moieties, phosphodiester-analog linkages, and / or alternative monomeric unit chirality and isomeric structures (e.g., 5'->2'-linkages, L-nucleosides, α-anomeric nucleosides, β-anomeric nucleosides, locked nucleic acids (LNA), peptide nucleic acids (PNA)). By way of example, oligonucleotides can be 10-20, 11-30, 31-40, 41-50, 51-60, 61-70, 71-80, 80-100, 100-150 or 150-200 nucleotides in length.

[0072] As used herein, the term "polymerase" refers to an enzyme that performs template-directed synthesis of polynucleotides. DNA polymerases can add free nucleotides only to the 3' end of a newly formed chain, thereby extending the newly formed chain in the 5'-3' direction. Known DNA polymerases cannot initiate new chains (de novo). DNA polymerases can only add nucleotides to an existing 3'-OH group and therefore require a primer to which the first nucleotide can be added.

[0073] As used herein, the term "primer" refers to an oligonucleotide that binds to a specific region of a single-stranded template nucleic acid molecule, extends from the 3' end of the primer, and initiates nucleic acid synthesis via a polymerase-mediated enzymatic reaction complementary to the sequence of the template molecule. PCR amplification primers can be referred to as "forward" and "reverse" primers, one of which is complementary to a nucleic acid strand and the other to the complement of that strand. Typically, primers contain fewer than about 100 nucleotides, preferably fewer than about 50 nucleotides. Exemplary primers range from about 5 to about 25 nucleotides. Primers can contain, for example, RNA and / or DNA bases, as well as non-naturally occurring bases.

[0074] As used herein, the term "unmodified dNTP" or "unmodified nucleoside triphosphate" refers to the four deoxyribonucleotide triphosphates commonly used as building blocks in the synthesis of DNA: dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate), and dTTP (deoxythymidine triphosphate).

[0075] As used herein, the term "sequence," when used in reference to a nucleic acid molecule, refers to the order of nucleotides (or bases) in the nucleic acid molecule. When different types of nucleotides are present in a nucleic acid molecule, the sequence includes the identity of the type of nucleotide (or base) at each position in the nucleic acid molecule. A sequence is a property of all or a portion of a nucleic acid molecule. The term can similarly be used to describe the order and positional identity of monomer units in other polymers, such as the amino acid monomer units of a protein polymer.

[0076] As used herein, the term "sequencing" refers to determining the order and position of bases in nucleic acid molecules.More specifically, the term "sequencing" refers to a biochemical method for determining the order of nucleotide bases, adenine, guanine, cytosine and thymine, in DNA oligonucleotides.As used herein, sequencing can 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 any other modern automated DNA sequencing device.

[0077] As used herein, the term "target" or "target sequence" refers to a nucleic acid molecule sequence of interest, e.g., a nucleic acid molecule sequence that hybridizes to an oligonucleotide probe.

[0078] As used herein, the term "universal primer" refers to a primer that can hybridize to and support the amplification of a target polynucleotide having a shared, complementary universal primer binding site. Similarly, the term "universal primer pair" refers to a forward and reverse primer pair that can hybridize to and support the PCR amplification of a target polynucleotide having a shared, complementary forward and reverse universal primer binding site. Such universal primer(s) and universal primer binding site(s) can enable single- or dual-primer-mediated universal amplification of a target polynucleotide region of interest (e.g., universal PCR). The headings provided herein are for convenience only and do not interpret the scope or meaning of the disclosed embodiments.

[0079] overview The present disclosure relates to compositions comprising one or more components that facilitate improved coverage uniformity and / or reduced GC bias during one or more downstream sequencing operations. In some embodiments, the compositions described herein are useful for preparing target-enriched samples and / or amplifying such target-enriched samples. Methods for preparing target-enriched samples and / or amplifying target-enriched samples are disclosed in U.S. Patent Application Publication Nos. 2018 / 0016630 and 2020 / 0032244 and International Application Nos. PCT / US2017 / 041748 and PCT / EP2018 / 08527, the disclosures of which are incorporated herein in their entireties. In some embodiments, the compositions of the present disclosure can be used in one or more steps of preparing target-enriched samples, including during pre-capture amplification, capture extension, and release primer hybridization and / or extension, and subsequent post-capture amplification of the prepared target-enriched samples.

[0080] The present disclosure also relates to methods for preparing target-enriched samples and for amplifying one or more targets in the prepared target-enriched samples. In some embodiments, the pre-capture, capture extension, release primer hybridization and extension, and / or post-capture amplification steps are performed in the presence of one or more components that improve coverage uniformity and / or reduce GC bias during one or more downstream sequencing operations. In some embodiments, the 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, the one or more components that increase coverage uniformity and / or reduce GC bias during sequencing include high-melting temperature primers or primers with a high GC content. In yet other embodiments, the one or more components that increase coverage uniformity and / or reduce GC bias are a dNTP mixture containing unmodified dNTPs and one or more modified dNTPs; or a primer containing one or more modified dNTPs. Of course, any combination of any of these components may be used in any hybridization and / or extension reaction, as described in more detail herein.

[0081] The present disclosure also provides methods for improving coverage uniformity and / or reducing GC bias during sequencing by optimizing specific parameters of specific incubation steps utilized during preparation of target-enriched samples. For example, the present disclosure provides methods for incubating samples at higher temperatures and / or for longer durations compared to a control incubation program. These and other embodiments are further described herein.

[0082] composition As described above, the present disclosure relates to a composition for use in hybridization and / or extension reactions, the composition comprising one or more components that facilitate improved coverage uniformity and / or reduced GC bias during sequencing. In some embodiments, the one or more components that facilitate improved coverage uniformity during sequencing comprise: (i) a primer with a high GC content and / or a high melting temperature; (ii) a primer containing one or more modified dNTPs; (iii) an enhancer; and / or (iv) a dNTP mixture containing unmodified and modified dNTPs. In some embodiments, the enhancer includes, but is not limited to, betaine, DMSO, single-stranded DNA binding proteins, disaccharides (e.g., trehalose), and combinations thereof. Each of these components is described herein.

[0083] In some embodiments, the composition of the present disclosure comprises both (a) a primer with a high GC content and / or a high melting temperature; and (b) one or more enhancers. In other embodiments, the composition of the present disclosure comprises both (a) a primer with a high GC content and / or a high melting temperature; and (b) a primer containing one or more modified dNTPs. In still other embodiments, the composition of the present disclosure comprises both (a) a primer containing one or more modified dNTPs; and (b) one or more enhancers. In further embodiments, the composition of the present disclosure comprises both (a) one or more enhancers; and (b) a mixture of modified and unmodified dNTPs. In still further embodiments, the composition of the present disclosure comprises (a) a primer with a high GC content and / or a high melting temperature, (b) a primer containing one or more modified dNTPs, and (c) one or more enhancers. Furthermore, in still further embodiments, the compositions of the present disclosure comprise (a) primers with high GC content and / or high melting temperature, (b) primers comprising one or modified dNTPs, and (c) a mixture of unmodified and modified dNTPs.

[0084] In some embodiments, the composition comprises one or more primers; one or more polymerases; deoxynucleoside triphosphates (dNTPs) (e.g., unmodified dNTPs or a mixture of unmodified and modified dNTPs); and at least one enhancer. In other embodiments, the composition comprises one or more primers, at least one of which has a high melting temperature (or high GC content) and / or comprises one or more modified dNTPs; one or more polymerases; dNTPs (e.g., unmodified dNTPs or a mixture of unmodified and modified dNTPs); and optionally, at least one enhancer. In some embodiments, the composition further comprises 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.

[0085] In some embodiments, the composition comprises one or more primers; one or more nucleic acid molecules; and at least one enhancer. In other embodiments, the composition comprises one or more primers, and at least one primer of the one or more primers has a high melting temperature (or high GC content) and / or comprises one or more modified dNTPs (for example, unmodified dNTPs or a mixture of unmodified and modified dNTPs); one or more nucleic acid molecules; and optionally, one or more enhancers. In some embodiments, the composition further comprises one or more polymerases; dNTPs; one or more buffers; one or more divalent cations; one or more cofactors; and / or one or more polyols.

[0086] Primer Primers comprising any of the pre-capture primers (e.g., pre-capture forward and pre-capture reverse primers), capture primers, release primers, and / or post-capture amplification primers described herein can be synthesized to contain at least one modified dNTP, including any of the modified dNTPs described herein.Similarly, primers comprising any of the pre-capture primers (e.g., pre-capture forward and reverse primers), capture primers, release primers, and / or post-capture amplification primers described herein can be synthesized to contain a high GC content and / or have a high melting temperature.In addition, primers comprising any of the pre-capture primers (e.g., pre-capture forward and reverse primers), capture primers, release primers, and / or post-capture amplification primers described herein can be synthesized to contain (i) a high GC content and / or a high melting temperature; and (ii) one or more modified dNTPs.

[0087] In some embodiments, the compositions of the present disclosure comprise one or more primers, wherein at least one of the one or more primers has a high GC content and / or a high melting temperature. "High GC content" means that at least one of the one or more primers has a GC content of greater than about 70%. For example, a high GC content primer can have a GC content of at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. In some embodiments, a high GC content primer does not include a limitation on GC content.

[0088] By "high melting temperature" it is meant that at least one primer of the one or more primers has a melting temperature above 63°C. In some embodiments, at least one primer 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, about 91°C, at least 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, etc.

[0089] In some embodiments, the primers included in any primer set may be at a temperature of about 57°C to about 63°C, 57°C to about 64°C, 57°C to about 65°C, 57°C to about 66°C, 57°C to about 67°C, 57°C to about 68°C, 57°C to about 69°C, 57°C to about 70°C, 57°C to about 71°C, 57°C to about 72°C, 57°C to about 73°C, 57°C to about 74°C, 57°C to about 75°C, 57°C to about 76°C, 57°C to about 77°C, 57°C to about 78°C, 57°C to about 79°C, or 57°C to about 80°C. The melting temperature may be in the range of 63°C, 57°C to about 81°C, 57°C to about 82°C, 57°C to about 83°C, 57°C to about 84°C, 57°C to about 85°C, 57°C to about 86°C, 57°C to about 87°C, 57°C to about 88°C, 57°C to about 89°C, 57°C to about 90°C, 57°C to about 91°C, 57°C to about 92°C, 57°C to about 93°C, 57°C to about 94°C, 57°C to about 95°C, 57°C to about 96°C, 57°C to about 97°C, 57°C to about 98°C, 57°C to about 99°C, or 57°C to about 100°C. In some embodiments, at least one primer in a primer set having any of the above temperature ranges has a melting temperature greater than 63°C. Of course, one of skill in the art will understand that any primer in a primer set (including at least one primer with a melting temperature greater than 63°C) may contain one or more modified dNTPs, such as one or more modified dGTPs or dATPs.

[0090] In some embodiments, up to about 1% of the primers in any primer set (in any composition) have a high GC content and / or a high melting temperature. In some embodiments, up to about 2% of the primers in any primer set have a high GC content and / or a high melting temperature. In some embodiments, up to about 3% of the primers in any primer set have a high GC content and / or a high melting temperature. In some embodiments, up to about 4% of the primers in any primer set have a high GC content and / or a high melting temperature. In some embodiments, up to about 5% of the primers in any primer set have a high GC content and / or a high melting temperature. In some embodiments, up to about 6% of the primers in any primer set have a high GC content and / or a high melting temperature. In some embodiments, up to about 7% of the primers in any primer set have a high GC content and / or a high melting temperature. In some embodiments, up to about 8% of the primers in any primer set have a high GC content and / or a high melting temperature. In some embodiments, up to about 9% of the primers in any primer set have a high GC content and / or a high melting temperature. In some embodiments, up to about 10% of the primers in any primer set have a high GC content and / or a high melting temperature. In some embodiments, up to about 15% of the primers in any primer set have a high GC content and / or a high melting temperature. In some embodiments, up to about 20% of the primers in any primer set have a high GC content and / or a high melting temperature. In some embodiments, up to about 25% of the primers in any primer set have a high GC content and / or a high melting temperature. In some embodiments, up to about 30% of the primers in any primer set have a high GC content and / or a high melting temperature.

[0091] In some embodiments, two or more modified dNTPs can be used in any of the primers in any of the compositions described herein. In some embodiments, a primer can contain two or more of the same modified dNTPs. In other embodiments, a primer can contain two or more different modified dNTPs. In some embodiments, the forward primer and the reverse primer can contain the same or different dNTP analogs. In some embodiments, up to about 5% of the nucleoside triphosphates contained in any primer are modified dNTPs. In some embodiments, up to about 10% of the nucleoside triphosphates contained in any primer are modified dNTPs. In other embodiments, up to about 15% of the nucleoside triphosphates contained in any primer are modified dNTPs. In still other embodiments, up to about 20% of the nucleoside triphosphates contained in any primer are modified dNTPs. In a further embodiment, up to about 25% of the nucleoside triphosphates contained in any primer are modified dNTPs. In yet a further embodiment, up to about 30% of the nucleoside triphosphates contained in any primer are modified dNTPs. Additionally, in still further embodiments, up to about 35% of the nucleoside triphosphates contained within any primer are modified dNTPs.

[0092] In some embodiments, at least one primer in any composition may (i) have both a high GC content and / or a high melting temperature; (ii) comprise one or more modified dNTPs, e.g., 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. In other embodiments, multiple primers included within any composition may (i) have both a high GC content and / or a high melting temperature; (ii) comprise one or more modified dNTPs, e.g., 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.

[0093] In some embodiments, the primer is a target-specific primer having a sequence complementary to the sequence of the target nucleic acid. An example of a target-specific primer is a gene-specific primer designed to hybridize to or near (e.g., upstream of, or 5' from) a gene of interest (e.g., cDNA, genomic DNA). The target nucleic acid can be RNA, DNA, or a combination thereof.

[0094] dNTP In some embodiments, the composition of the present disclosure comprises one or more dNTPs, including unmodified dNTPs and / or modified dNTPs.In some embodiments, the 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 instead of or in addition to unmodified dNTPs.

[0095] 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 (thio-T TP), 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-thiol) Modified dNTPs include 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.

[0096] In some embodiments, the composition of the present disclosure is a mixture of unmodified dNTPs and modified dNTPs. In some embodiments, the ratio of modified dNTPs to corresponding unmodified dNTPs can be in the range of 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.

[0097] In some embodiments, the composition may comprise about 0.1 mM to about 0.7 mM unmodified dNTPs; and may further comprise one or more modified dNTPs. In other embodiments, the composition may comprise about 0.1 mM to about 0.6 mM unmodified dNTPs; and may further comprise one or more modified dNTPs. In yet other embodiments, the composition may comprise about 0.2 mM to about 0.5 mM unmodified dNTPs; and may further comprise one or more modified dNTPs.

[0098] In some embodiments, the composition may contain about 0.1 mM to about 0.7 mM unmodified dNTPs and may further contain about 0.1 mM to about 0.7 mM of one or more modified dNTPs. In other embodiments, the composition may contain about 0.1 mM to about 0.6 mM unmodified dNTPs and may further contain about 0.1 mM to about 0.6 mM of one or more modified dNTPs. In still other embodiments, the composition may contain about 0.2 mM to about 0.5 mM unmodified dNTPs and may further contain about 0.2 mM to about 0.5 mM of one or more modified dNTPs. In further embodiments, the composition may contain about 0.2 mM to about 0.4 mM unmodified dNTPs and may further contain about 0.2 mM to about 0.4 mM of one or more modified dNTPs. In still further embodiments, the composition may contain about 0.3 mM unmodified dNTPs and may further contain about 0.2 mM to about 0.5 mM of one or more modified dNTPs. In still further embodiments, the composition may comprise about 0.3 mM unmodified dNTPs; and may further comprise about 0.3 mM of one or more modified dNTPs.

[0099] In some embodiments, the composition may comprise about 0.1 mM to about 0.7 mM unmodified dNTPs and may further comprise about 0.1 mM to about 0.8 mM of one or more modified dNTPs. In other embodiments, the composition may comprise about 0.1 mM to about 0.6 mM unmodified dNTPs and may further comprise about 0.2 mM to about 0.7 mM of one or more modified dNTPs. In yet other embodiments, the composition may comprise about 0.2 mM to about 0.5 mM unmodified dNTPs and may further comprise about 0.2 mM to about 0.6 mM of one or more modified dNTPs.

[0100] Enhancer 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 having similar chemical and / or physical properties, e.g., 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.

[0101] In some embodiments, the enhancer is betaine (or a derivative or analog thereof), and the concentration of betaine in any of the compositions ranges from about 0.05 mM to about 1 mM. In other embodiments, the enhancer is betaine, and the concentration of betaine in any of the compositions ranges from about 0.1 mM to about 0.9 mM. In other embodiments, the enhancer is betaine, and the concentration of betaine in any of the compositions ranges from about 0.2 mM to about 0.8 mM. In other embodiments, the enhancer is betaine, and the concentration of betaine in any of the compositions ranges from about 0.3 mM to about 0.7 mM. In other embodiments, the enhancer is betaine, and the concentration of betaine in any of the compositions ranges from 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.

[0102] In some embodiments, the enhancer is DMSO, and the 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 the 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 the 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 the 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 the 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 the 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 the 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 of up to about 3% (v / v). In some embodiments, the enhancer is DMSO, and DMSO may be present in any composition in an amount of up to about 2% (v / v). In some embodiments, the enhancer is DMSO, and DMSO may be present in any composition in an amount of up to about 1% (v / v).

[0103] 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.

[0104] In some embodiments, the single-stranded DNA binding protein is a thermostable binding protein, such as one derived from a hyperthermophilic microorganism (e.g., ET SSB available from New England BioLabs, Inc.). In other embodiments, the single-stranded DNA binding protein is derived from E. coli, Drosophila, or Xenopus, the 32, 41, 44, 45, or 61 gene proteins from T4 bacteriophage, or the eukaryotic RPA protein. Non-limiting examples of single-stranded DNA binding proteins include the gene 2.5 protein of bacteriophage T7 and equivalent T7-type phage proteins or other proteins. Examples of T7-type phages include T7, T3, φI, φII, H, W31, gh-1, Y, A1122, and SP6. In some embodiments, the single-stranded DNA binding protein may be present in an amount ranging from 0 ng / μL to about 50 ng / μL. In some embodiments, the single-stranded DNA binding protein may be present in an amount ranging from 1 ng / μL to about 50 ng / μL. In some embodiments, the single-stranded DNA binding protein may be present in an amount ranging from 5 ng / μL to about 50 ng / μL. In some embodiments, the single-stranded DNA binding protein may be present in an amount ranging from 10 ng / μL to about 50 ng / μL. In some embodiments, the single-stranded DNA binding protein may be present in an amount ranging from 5 ng / μL to about 40 ng / μL.

[0105] In some embodiments, the compositions of the present disclosure include a disaccharide, such as trehalose. In some embodiments, the disaccharide is present at a concentration ranging from about 0 M to about 1 M. In some embodiments, the disaccharide is present at a concentration ranging from about 0.1 M to about 1 M. In some embodiments, the disaccharide is present at a concentration ranging from about 0.1 M to about 0.8 M. In some embodiments, the disaccharide is present at a concentration ranging from about 0.2 M to about 0.8 M.

[0106] polymerase In some embodiments, the composition of the present disclosure comprises 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, telomerase, reverse transcriptase, and RNA polymerase. Reverse transcriptase is an RNA-dependent DNA polymerase that synthesizes DNA from an RNA template. The reverse transcriptase family contains both DNA polymerase functionality and RNase H functionality, which degrades RNA base-paired with DNA. RNA polymerase is an enzyme that synthesizes RNA using DNA as a template in the process of gene transcription. RNA polymerase polymerizes ribonucleotides at the 3' end of RNA transcripts.

[0107] In some embodiments, polymerases from the following can be used for polymerase-mediated primer extension, end modification (e.g., terminal transferase, degradation, or polishing), 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 sp. strain KOD (Pfx, GenBank: AAE68738)), Thermococcus gorgonarius (Tgo, Pdb: 4699806), Sulfolobus solataricus (GenBank:NC002754, P26811), Aeropyrum pernix (GenBank:BAA81109), Archaeglobus fulgidus (GenBank:029753), Pyrobaculum aerophilum (GenBank:AAL63952), Pyrodictium occultum (GenBank:BAA07579, BAA07580), Thermococcus 9 degree Nm (GenBank:AAA88769, Q56366), Thermococcus fumicolans (GenBank:CAA93738, P74918), Thermococcus hydrothermalis (GenBank:CAC18555), Thermococcus sp.GE8 (GenBank:CAC12850), Thermococcus sp.JDF-3 (GenBank:AX135456;WO0132887), Thermococcus sp.TY(GenBank:CAA73475)、Pyrococcus abyssi(GenBank:P77916)、Pyrococcus glycovorans(GenBank:CAC12849)、Pyrococcus horikoshii(GenBank:NP143776)、Pyrococcus sp.GE23(GenBank:CAA90887)、Pyrococcus sp.ST700(GenBank:CAC12847)、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) Thermococcus thioreducens, Thermococcus onnurineus NA1, Sulfolobus acidocaldarium, Sulfolobus tokodaii, Pyrobaculum calidifontis, Pyrobaculum islandicum(GenBank:AAF27815), Methanococcus jannaschii (GenBank:Q58295) , Desulforococcus , TOK , Desulforococcus , Pyrolobus , Pyrodictium , Staphylothermus, Vulcanisaetta, Methanococcus (GenBank:P52025) and a number of strain B strains from GenBank AAC62712, P956901, BAAA07579)) Manufacturer Thermus (flavus, ruber, thermophilus, lacteus, rubens, aquaticus), Bacillus stearothermophilus、Thermotoga maritima、Methanothermus fervidus、KODメメラー、TNA1ポメーー、Thermococcus sp.9 degrees N-7, T4, T7, phi29, Pyrococcus furiosus, P. abyssi, T. gorgonarius, T. litoralis, T. zilligii, T. sp.GT, P. sp.GB-D, CODE, Pfu, T. gorgonarius, T. zilligii, T. litoralis, etc. Thermococcus sp.9N-7protein.

[0108] In some embodiments, the composition of the present disclosure comprises one or more thermostable polymerases. As used herein, the term "thermostable polymerase" refers to an enzyme that is heat-stable and thermotolerant, and that retains sufficient activity to subsequently carry out a polynucleotide extension reaction when exposed to high temperatures for the time required to denature double-stranded nucleic acids, and is not irreversibly denatured (inactivated). The heating conditions required for nucleic acid denaturation are known in the art and are exemplified 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 terminal modification (e.g., terminal transferase, degradation, or polishing) reactions. For purposes of this specification, irreversible denaturation refers to the permanent and complete loss of enzyme activity. For thermostable polymerases, enzymatic activity refers to the catalysis of the combination of nucleotides in the appropriate manner to form polynucleotide extension products that are complementary to a template nucleic acid strand. Thermostable DNA polymerases include those from Thermotoga maritima, Thermus aquaticus, Thermus thermophilus, Thermus flavus, Thermus filiformis, Thermus sp. sp17, Thermus sp. Z05, Thermus caldophilus, Bacillus caldotenax, Thermotoga neopolitana, Thermosipho africanus, and other thermostable DNA polymerases disclosed above.

[0109] An example of a polymerase is Taq or a Taq-derived polymerase (e.g., KAPA 2G polymerase from KAPA BIOSYSTEMS). Another exemplary polymerase is a B family DNA polymerase (e.g., KAPA HIFI polymerase from KAPA BIOSYSTEMS).

[0110] Input nucleic acid molecule In some embodiments, the composition of the present disclosure includes an input nucleic acid molecule or a library of input nucleic acid molecules. In some embodiments, the nucleic acid molecules in the resulting nucleic acid library are selected from DNA molecules, RNA molecules, genomic DNA molecules, cDNA molecules, mRNA molecules, rRNA molecules, mtDNA, siRNA molecules, or any combination thereof. In some embodiments, the plurality of nucleic acid molecules comprises single-stranded polynucleotides. In some embodiments, the plurality of nucleic acid molecules is derived from a tissue sample, such as a tissue sample derived from a mammalian subject. In other embodiments, the plurality of nucleic acid molecules is derived from a formalin-fixed, paraffin-embedded sample or a cytology sample, such as a cytology sample derived from a mammalian subject.

[0111] In some embodiments, the resulting nucleic acid library comprises a plurality of target nucleic acid molecules and / or a plurality of non-target nucleic acid molecules. In some embodiments, the non-target nucleic acid molecules are highly abundant relative to the target nucleic acid molecules in the nucleic acid library. In some embodiments, the non-target nucleic acid molecules represent at least 70% of the nucleic acid molecules in the resulting nucleic acid library. In other embodiments, the non-target nucleic acid molecules represent at least 75% of the nucleic acid molecules in the resulting nucleic acid library. In still other embodiments, the non-target nucleic acid molecules represent at least 80% of the nucleic acid molecules in the resulting nucleic acid library. In further embodiments, the non-target nucleic acid molecules represent at least 85% of the nucleic acid molecules in the resulting nucleic acid library. In still further embodiments, the non-target nucleic acid molecules represent at least 90% of the nucleic acid molecules in the resulting nucleic acid library. In still further embodiments, the non-target nucleic acid molecules represent at least 95% of the nucleic acid molecules in the resulting nucleic acid library. In some embodiments, the non-target nucleic acid molecules represent at least 96% of the nucleic acid molecules in the resulting nucleic acid library. In some embodiments, the non-target nucleic acid molecules represent at least 97% of the nucleic acid molecules in the resulting nucleic acid library. In some embodiments, the non-target nucleic acid molecules represent at least 98% of the nucleic acid molecules in the resulting nucleic acid library. In some embodiments, the non-target nucleic acid molecules represent at least 99% of the nucleic acid molecules in the resulting nucleic acid library.

[0112] In some embodiments, nucleic acid molecules in any library contain ligated first and second adaptors. For example, in some embodiments, nucleic acid fragments are first prepared from a biological sample, such as a tissue sample and / or a cytology sample. DNA sequencing libraries can be constructed from genomic DNA for genome analysis or cDNA prepared from RNA or mRNA for transcriptome analysis, and can be constructed from DNA or cDNA from any organism from which these nucleic acids can be extracted. In some embodiments, the resulting sample is sheared into fragments to obtain a population of nucleic acid fragments. In some embodiments, shearing of the resulting genomic sample is achieved using mechanical fragmentation (e.g., nebulization or sonication) and / or enzymatic fragmentation (e.g., restriction endonucleases). In some embodiments, the generated nucleic acid fragments are randomly sized. In some embodiments, the generated nucleic acid fragments have lengths less than about 1000 base pairs. In other embodiments, the generated nucleic acid fragments include sequence fragments having sequence sizes ranging from about 100 to about 1000 base pairs in length. In yet another embodiment, the generated nucleic acid fragments include sequence fragments having sequence sizes ranging from about 500 to about 750 base pairs in length.

[0113] In some embodiments, adapters are then added to the collection of nucleic acid molecules through ligation reaction.In some embodiments, the adapters comprise one or more barcode sequences.Methods for ligating adapters to nucleic acid molecules are described in U.S. Patent Application Publication Nos. 2017 / 0037459, 2018 / 0334709, 2018 / 0016630 and PCT Publication No. WO2017021449, the disclosures of which are incorporated herein by reference in their entirety.

[0114] In some embodiments, the input nucleic acid molecule comprises a complex containing a target nucleic acid molecule to which at least one primer is hybridized. For example, the input nucleic acid molecule may comprise a complex of a target nucleic acid to which a capture primer is hybridized. In some embodiments, the capture primer of the complex may be extended.

[0115] In other embodiments, the input nucleic acid molecule comprises a complex containing a target nucleic acid molecule to which a first and second primer hybridize (including a primer with a high melting temperature and / or a primer without GC content restrictions). For example, the input nucleic acid molecule may comprise a complex of a target nucleic acid molecule and an extended capture primer; the complex further comprises a release primer.

[0116] In yet other embodiments, the input nucleic acid molecule may comprise a complex of the target nucleic acid molecule and the extended-release primer.

[0117] In some embodiments, the input nucleic acid molecules are target-enriched, i.e., comprise a plurality of target nucleic acid molecules and are devoid or substantially devoid of non-target nucleic acid molecules.

[0118] buffer solution The compositions of the present disclosure may contain one or more buffers. Non-limiting examples of buffers include citric acid, potassium dihydrogen phosphate, boric acid, diethylbarbituric acid, piperazine-N,N'-bis(2-ethanesulfonic acid), dimethylarsinic 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 combinations thereof. Additional wash solutions, transfer solutions, acidic 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.

[0119] divalent cations In some embodiments, the compositions of the present disclosure include one or more divalent cations. In some embodiments, the divalent cations are Co 2+ , Mn 2+ , Mg 2+ , Cd 2+ , and Ca 2+ Selected from 。

[0120] In some embodiments, any of the compositions of the present disclosure may comprise a concentration of divalent cations that is 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 may have a concentration of CoCl 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 may have a concentration of MnCl that is 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 may have a concentration of MgCl that is 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 may have a concentration of CdCl that is 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 may have a concentration of CaCl that is 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.

[0121] polyol In some embodiments, any of the compositions of the present disclosure may include one or more polyols. Suitable polyols include 1,2-ethanediol, 1,2-propanediol, 1,3-propanediol, 2-methyl-1,3-propanediol, 2-methyl-1,2-propanediol, 2,2-dimethyl-1,3-propanediol, 2,2-diethyl-1,3-propanediol, 2-methyl-2-propyl-1,3-propanediol, 2-butyl-2-ethyl-1,3-propanediol, dihydroxyacetone, 2,2-dibutyl-1,3-propanediol, 3-methoxyacetone, 3-methoxy-2-propyl-1,3-propanediol ... 1,3-propanediol, 3-methoxy-1,2-propanediol, 3-methoxy-2,3-propanediol, 2-methoxymethyl-1,3-propanediol, 3-ethoxy-1,3-propanediol, 3-ethoxy-1,2-propanediol, 3-ethoxy-2,3-propanediol, 3-allyloxy-1,2-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, 2,3-dimethyl-2,3-butanediol hexanediol, 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, 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-nonadiol, 1,9-Nonadiol, 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, ethylene 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- 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-cycloheptanetriol, 1,2,4-cycloheptanetriol, 1,2,3-propanetriol, 3-ethyl-2-hydroxymethyl-1,3-propanediol, 2-hydroxymethyl-2-methyl-1,3-propanediol, 2-hydroxymethyl- Methyl-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-octanetriol, 1,2,8-octanetriol, 1,2,3-nonatriol, 1,2,9-nonatriol, 1,2,3-decanetriol, 1,2,10-decanetriol, 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-butanetetraol, 1,2,3,4-pentanetetraol, 1,2,3,5-pentanetriol, Examples of suitable ethanol include 1,2,3,4-hexanetetraol, 1,2,3,6-hexanetetraol, 1,2,3,4-heptanetetraol, 1,2,3,7-heptanetetraol, 1,2,3,4-octanetetraol, 1,2,3,8-octanetetraol, 1,2,3,4-nonanetetraol, 1,2,3,9-nonanetetraol, 1,2,3,4-decanetetraol, 1,2,3,10-decanetetraol, trimethylolpropanol, pentaerythritol, mannitol, sorbitol, or sugar alcohols such as arabitol, hexanehexol, 1,2,3,4,5-pentanepentol, and 1,2,3,4,5,6-hexanehexaol.

[0122] In some embodiments, the polyol is 1,2,3-propanetriol (also called glycerol).

[0123] In some embodiments, the polyol may be present in an amount ranging from about 1% to about 10% of the total volume of the composition; from about 2% to about 8% of the total volume of the composition; from about 3% to about 7% of the total volume of the composition; or from about 4% to about 6% of the total volume of the composition.

[0124] Composition Examples An example composition includes a primer set; a polymerase; and betaine. In some embodiments, the betaine is present at a concentration ranging from about 0.3 mM to about 0.7 mM. In some embodiments, the concentration of betaine is about 0.5 mM. 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 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 primer in the primer set. In some embodiments, up to 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 and unmodified 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 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 is useful for amplifying nucleic acid molecules, for example, nucleic acid molecules present in a nucleic acid library. In some embodiments, the composition is useful for pre-capture amplification. In some embodiments, the composition is useful in one or more steps of target enrichment, such as during capture primer hybridization and extension (as further described herein).

[0125] Another example of a composition comprises a primer set; a polymerase; and optionally 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 of the primer set has a melting temperature of 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, about 91°C, at least 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, etc. 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 primer in the primer set. In some embodiments, up to 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 at a concentration ranging from about 0.3 mM to about 0.7 mM. In some embodiments, the composition further comprises a mixture of modified and unmodified 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 is useful for amplifying nucleic acid molecules, e.g., nucleic acid molecules present in a nucleic acid library. In some embodiments, the composition is useful for capture pre-amplification. In some embodiments, the compositions are useful in one or more steps of target enrichment, such as during capture primer hybridization and extension (as further described herein).

[0126] method The present disclosure also relates to methods for concentrating a plurality of target nucleic acid molecules in a sample, for example, about 1 to about 10,000 target nucleic acid molecules, or about 1 to about 5,000 target nucleic acid molecules, or about 1 to about 1,000 target nucleic acid molecules.

[0127] The present disclosure also relates to a method for amplifying a target-enriched sample, such as a target-enriched sample prepared using any one of the methods described herein. Furthermore, the present disclosure also relates to a method for sequencing using a target-enriched sample, such as a target-enriched sample prepared using any one of the methods described herein. The method of the present disclosure can be used as part of a sequencing protocol, including a high-throughput single-molecule sequencing protocol. In some embodiments, the method of the present disclosure generates a library of enriched target nucleic acid molecules to be sequenced. The enriched target nucleic acid molecules in the library can optionally incorporate barcodes for molecular and sample identification, as described in U.S. Patent Application 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 disclosures of which are incorporated herein by reference in their entirety.

[0128] Referring to Figure 1, a method 100 for target enrichment by unidirectional dual-probe primer extension includes step 102 of preparing a nucleic acid library for analysis. In some embodiments, step 102 includes fragmentation (optional), adapter ligation (to each end of the nucleic acid molecule), and capture pre-amplification. Finally, the product of step 102 results in a library of nucleic acid molecules, each of which has a first end comprising a first adapter and a second end comprising a second adapter. In some embodiments, the first and second adapters can be the same or different.

[0129] In some embodiments, a nucleic acid library can be prepared from any source of nucleic acid molecules that contains one or more target nucleic acid molecules. Generally, the target nucleic acids contain regions or sequences of interest, and method 100 facilitates the preferential enrichment of one or more target nucleic acid molecules relative to non-target nucleic acid molecules in a nucleic acid library for downstream detection and analysis of these regions or sequences of interest.

[0130] After adapter ligation, the adapter-ligated (and optionally fragmented) nucleic acid molecules are amplified (pre-capture amplification). In some embodiments, pre-capture amplification is performed using any of the compositions described herein. For example, a suitable composition (e.g., a pre-capture amplification master mix 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 pre-capture master mix composition may include one or more primers, at least one of which has a high melting temperature (or high GC content), and / or 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 a single-stranded DNA-binding protein (e.g., in the amounts / concentrations described herein)).

[0131] In some embodiments, pre-capture amplification may be performed using a set of pre-capture primers (e.g., forward and reverse pre-capture primers), where at least one primer in the set of pre-capture primers has a high GC content and / or a high melting temperature (e.g., a melting temperature above 63°C, above about 66°C, above about 69°C, above about 72°C, above about 75°C, etc.). In some embodiments, the pre-capture primers introduced may have no restrictions on GC content and / or melting temperature. In some embodiments, up to about 25% of the primers utilized during pre-capture amplification are high-melting temperature and / or high-GC content primers. In some embodiments, up to about 20% of the primers utilized during pre-capture amplification are high-melting temperature and / or high-GC content primers. In some embodiments, up to about 15% of the primers utilized during pre-capture amplification are high-melting temperature and / or high-GC content primers. In some embodiments, up to about 12% of the primers utilized during pre-capture amplification are high-melting temperature and / or high-GC content primers. In some embodiments, up to about 10% of the primers utilized during capture pre-amplification are high melting temperature and / or high GC content primers. In some embodiments, up to about 9% of the primers utilized during capture pre-amplification are high melting temperature and / or high GC content primers. In some embodiments, up to about 8% of the primers utilized during capture pre-amplification are high melting temperature and / or high GC content primers. In some embodiments, up to about 7% of the primers utilized during capture pre-amplification are high melting temperature and / or high GC content primers. In some embodiments, up to about 6% of the primers utilized during capture pre-amplification are high melting temperature and / or high GC content primers. In some embodiments, up to about 5% of the primers utilized during capture pre-amplification are high melting temperature and / or high GC content primers. In some embodiments, up to about 4% of the primers utilized during capture pre-amplification are high melting temperature and / or high GC content primers.In some embodiments, up to about 3% of the primers utilized during capture pre-amplification are high melting temperature and / or high GC content primers. In some embodiments, up to about 2% of the primers utilized during capture pre-amplification are high melting temperature and / or high GC content primers. In some embodiments, up to about 1% of the primers utilized during capture pre-amplification are high melting temperature and / or high GC content primers.

[0132] In other embodiments, pre-capture amplification can be performed in the presence of a set of pre-capture primers (e.g., forward and reverse pre-capture primers), where at least one primer of the primer set (i) has a high GC content and / or a high melting temperature; and (ii) contains one or more modified dNTPs (7-deaza dGTP and / or 2-amino dATP). For example, the introduced primers can have a melting temperature above 63°C, above about 66°C, above about 69°C, above about 72°C, above about 75°C, etc.; the primers contain one or more modified dNTPs. In some embodiments, the introduced primers can have no restrictions on GC content and / or melting temperature; the primers also contain one or more modified dNTPs.

[0133] In yet other embodiments, capture pre-amplification is performed in the presence of one or more enhancers, such as betaine, DMSO, single-stranded DNA binding proteins, disaccharides, or any combination thereof. For example, capture pre-amplification can be performed using a composition comprising betaine (or a derivative or analog thereof), for example, a composition comprising betaine at a concentration ranging from about 0.1 mM to about 1 mM (e.g., 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.).

[0134] In further embodiments, the capture pre-amplification is performed in a composition comprising one or more pre-capture primers (e.g., forward and reverse pre-capture primers) having high GC content and / or a high melting temperature; 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 still 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 a further embodiment, the enhancer is betaine (or a derivative or analog thereof), and the concentration of betaine in the composition is about 0.5 mM.

[0135] In still further embodiments, capture pre-amplification can be performed in a composition comprising a mixture of unmodified dNTPs and modified dNTPs. In some embodiments, the ratio of unmodified dNTPs to modified dNTPs in any composition ranges from about 2:1 to about 1:2. In some embodiments, the ratio of unmodified dNTPs to modified dNTPs is about 1:1. In some embodiments, capture pre-amplification can be performed in a composition comprising one or more modified dNTPs (e.g., at a concentration ranging from about 0.2 mM to about 0.8 mM, from about 0.3 mM to about 0.7 mM, from about 0.3 mM to about 0.6 mM, etc.). In some embodiments, the concentration of each different modified dNTP in the composition is about 0.3 mM. In some embodiments, the one or more modified dNTPs include modified dGTP and / or modified dATP. In some embodiments, the 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.

[0136] In some embodiments, capture pre-amplification can be performed in the presence of a polymerase, betaine, and a mixture of unmodified and modified dNTPs. In these 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. In some embodiments, the modified dNTPs are selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate or 2-amino-2'-deoxyadenosine-5'-triphosphate.

[0137] In some embodiments, capture pre-amplification can be performed in the presence of a polymerase, betaine, an optional mixture of unmodified 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; 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 ranging from 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.

[0138] In some embodiments, capture pre-amplification can be performed in the presence of a polymerase, a mixture of unmodified 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 the 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. An example of a mixed capture pre-amplification master is shown in Table 1 below:

[0139] [Table 1]

[0140] Following the capture pre-amplification in step 102, step 104 of method 100 involves hybridizing a capture primer to a target nucleic acid present in a library of nucleic acid molecules, thereby forming an unextended capture primer-target complex. In some embodiments, the capture primer is a target-specific primer having a defined sequence complementary to the sequence of the target nucleic acid. An example of a target-specific primer is a gene-specific primer designed to hybridize to or near (e.g., upstream of, or 5' from) a gene of interest (e.g., cDNA, genomic DNA). The target nucleic acid can be RNA, DNA, or a combination thereof. The capture primer can be an oligonucleotide primer composed of ribonucleic acid, deoxyribonucleic acid, modified nucleic acid (e.g., biotinylated, locked nucleic acid, inosine, Seela base, etc.), or other nucleic acid analogs known in the art.

[0141] In some embodiments, the capture primer can include one or more modified bases, a capture moiety, or a combination thereof. If the capture primer includes a capture moiety, the capture primer can be attached to a solid support or free in solution (i.e., not bound to or otherwise attached to a solid support) prior to step 104, in which the capture primer is hybridized to the target nucleic acid. Other suitable capture moieties and their usefulness are described in U.S. Patent Application Publication No. 2020 / 0032244, the disclosure of which is incorporated herein by reference in its entirety. In embodiments in which the capture primer including a capture moiety is not attached to a solid support via a capture moiety, step 104 can be performed in solution. In embodiments in which the capture primer including a capture moiety is attached to a solid support via a capture moiety, step 104 can be performed in situ. Particularly in the case of an in situ reaction, the resulting unextended primer-target complex is attached to the solid support. Any non-target or target nucleic acid molecules not annealed to the capture primer remaining in solution can be removed by separating the solution from the solid support to which the primer-target complex is bound.

[0142] In step 106, a capture primer extension reaction is performed. In some embodiments, step 106 involves extending the hybridized capture primer with a first polymerase, thereby generating a capture primer extension product or complex comprising a 3' region of the extended capture primer that comprises the 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 may be separated by a wash step that removes unannealed, uncaptured target nucleic acid molecules from the reaction mixture.

[0143] In embodiments in which the capture primer hybridizes and is extended in the same step, referred to as "capture extension," the capture extension can occur using any of the compositions described herein, such as a composition comprising one or more capture primers; one or more polymerases; dNTPs (including unmodified dNTPs or a mixture of unmodified and modified dNTPs); and at least one enhancer (e.g., betaine). In other embodiments, the capture extension occurs in a composition comprising one or more capture primers, at least one of which has a high melting temperature and / or comprises one or more modified dNTPs; one or more polymerases; dNTPs (including unmodified dNTPs or a mixture of unmodified and modified dNTPs); and optionally at least one enhancer (e.g., betaine).

[0144] In some embodiments, capture extension may be performed in the presence of a set of capture primers, wherein at least one primer in the set of pre-capture primers has a high GC content and / or a high melting temperature (e.g., a melting temperature above 63°C, above about 66°C, above about 69°C, above about 72°C, above about 75°C, etc.). In some embodiments, the capture primers introduced may have no restrictions on GC content and / or melting temperature.

[0145] In some embodiments, capture extension may be performed in the presence of a set of pre-capture primers (e.g., forward and reverse pre-capture primers), where at least one primer in the set of pre-capture primers 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 pre-capture primers introduced may have no restrictions on GC content and / or melting temperature.

[0146] In some embodiments, up to about 25% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers. In some embodiments, up to about 20% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers. In some embodiments, up to about 15% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers. In some embodiments, up to about 12% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers. In some embodiments, up to about 10% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers. In some embodiments, up to about 9% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers. In some embodiments, up to about 8% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers. In some embodiments, up to about 7% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers. In some embodiments, up to about 6% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers. In some embodiments, up to about 5% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers. In some embodiments, up to about 4% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers. In some embodiments, up to about 3% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers. In some embodiments, up to about 2% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers.In some embodiments, up to about 1% of the capture primers in any set of capture primers are high melting temperature and / or high GC content capture primers.

[0147] In other embodiments, capture extension can be performed in the presence of a set of capture primers, where at least one primer of the primer set (i) has a high GC content and / or a high melting temperature; and (ii) contains one or more modified dNTPs. For example, the introduced primer can have a melting temperature of 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.; the primer contains one or more modified dNTPs. In some embodiments, the introduced primer can have no restrictions on GC content and / or melting temperature; the primer also contains one or more modified dNTPs.

[0148] In yet another embodiment, the capture extension is carried out in the presence of one or more enhancers, such as betaine, DMSO, single-stranded DNA-binding protein, disaccharide, or any combination thereof. For example, the capture extension can be carried out using a composition containing betaine (or a derivative or analog thereof), for example, a composition containing betaine at a concentration ranging from about 0.1 mM to about 1 mM (e.g., 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.).

[0149] In further embodiments, capture extension may be performed in a composition comprising one or more capture primers with high GC content and / or high melting temperature; 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 still 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 a further embodiment, the enhancer is betaine (or a derivative or analog thereof), and the concentration of betaine in the composition is about 0.5 mM.

[0150] In still further embodiments, capture extension can be performed in a composition comprising a mixture of unmodified dNTPs and modified dNTPs. In some embodiments, the ratio of unmodified dNTPs to modified dNTPs in any composition ranges from about 2:1 to about 1:2. In some embodiments, the ratio of unmodified dNTPs to modified dNTPs is about 1:1. In some embodiments, capture extension can be performed in a composition comprising one or more modified dNTPs (e.g., at a concentration ranging from about 0.2 mM to about 0.8 mM, from about 0.3 mM to about 0.7 mM, from about 0.3 mM to about 0.6 mM, etc.). In some embodiments, the one or more modified dNTPs comprise modified dGTP and / or modified dATP. In some embodiments, the 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.

[0151] In some embodiments, capture extension can be performed in the presence of a polymerase, betaine, and a mixture of unmodified and modified dNTPs. In these 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. In some embodiments, the modified dNTPs are selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate or 2-amino-2'-deoxyadenosine-5'-triphosphate.

[0152] In some embodiments, capture extension can be performed in the presence of a polymerase, betaine, an optional mixture of unmodified 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; 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. In some embodiments, the divalent cation is present at a concentration of about 0.1 mM to about 0.4 mM.

[0153] In some embodiments, capture extension can be performed in the presence of a polymerase, a mixture of unmodified 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 the 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.

[0154] An example of a capture extension master mix that can be utilized when performing step 106 is shown in Table 2 below.

[0155] [Table 2]

[0156] In addition to providing compositions for performing step 106 of method 100, the present disclosure also provides methods for incubating samples. In particular, the present disclosure provides incubation programs (including denaturing, annealing, and extension portions) with various temperatures and times that can be utilized 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 utilized in conjunction with any of the compositions disclosed herein.

[0157] In some embodiments, the denaturation, annealing, and extension portions of the incubation can be carried out for a cumulative time ranging from about 20 minutes to about 40 minutes, e.g., from about 25 minutes to about 35 minutes. By way of example, the cumulative time for carrying out 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.

[0158] In some embodiments, the cumulative time for the annealing and extension portions of the incubation ranges from about 20 minutes to about 25 minutes. In some embodiments, the cumulative time for the annealing and extension portions of the incubation is about 23 minutes. In other embodiments, the cumulative time for the annealing and extension portions of the incubation is about 30 minutes to about 34 minutes. In other embodiments, the cumulative time for the annealing and extension portions of the incubation is about 32 minutes.

[0159] In some embodiments, annealing and extension are performed at temperatures ranging from about 60°C to about 80°C, as shown in Tables 3-7. In some embodiments, the incubation programs of the present disclosure include one or more pauses between annealing temperatures compared to the control incubation programs. For example, Program 1 adds pauses at regular intervals between 80°C and 60°C while maintaining the same overall program length as the control program (Program 5). Similarly, Program 2 adds pauses at higher temperature intervals between 80°C and 60°C while maintaining the same overall program length as the control program (Program 5). Program 3 adds pauses at higher temperature intervals between 80°C and 60°C, thereby increasing the total program time by 10 minutes, while matching the total incubation time of the control program (Program 5) at 60°C, 10 minutes. Program 4 matches the incubation time of the control program (Program 5) at 60°C for 10 minutes, but adds pauses at regular intervals between 80°C and 60°C, thereby increasing the total program time by 10 minutes.

[0160] [Table 3]

[0161] [Table 4]

[0162] [Table 5]

[0163] [Table 6]

[0164] [Table 7]

[0165] Method 100 further includes step 108 of capturing the capture primer extension complex. Capturing the capture primer extension complex can be accomplished in various ways, as disclosed herein, and can be accomplished before, simultaneously with, or after either step 104 or step 106 of method 100. As described above, the capture oligonucleotide can include a capture moiety that can be used to capture the capture primer onto a solid support before, during, or after step 104 or step 106 of method 100. In another example, extension of the capture primer after hybridization to the target nucleic acid includes incorporation of one or more modified nucleotides. The modified nucleotides can include a capture moiety, or can be configured such that downstream modifications of the modified nucleotides allow the capture moiety to be attached to or otherwise incorporated into the extended portion of the capture primer extension complex. Thus, the capture primer extension complex can be captured by a capture moiety associated with one or more modified nucleotides during or after step 106. The choice of whether the target nucleic acid, the annealed primer-target complex, or the target-extended primer complex is captured further determines whether steps 104 and 106 of the method are performed in solution or in situ.

[0166] Step 110 of method 110 involves enriching the capture primer extension complexes. In some embodiments, step 110 includes one or more purification and enrichment steps to recover the capture primer extension complexes from non-target nucleic acid molecules in the library and other molecules, 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 depleting (i.e., removing) other members of the library of nucleic acid molecules that are not the target nucleic acid molecule.

[0167] Hybridization of the release primer to 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 a region of interest within the target nucleic acid (as opposed to hybridizing to or being complementary to one or both of the first and second adapters introduced in step 102). In some embodiments, the target nucleic acid is part of a capture primer extension complex synthesized in step 112. For example, the release primer can hybridize to the target nucleic acid at a position 5′ (i.e., upstream) relative to the extended capture primer in the capture primer extension complex. The resulting unextended release primer-target complex in this case includes the extended capture primer, the target nucleic acid hybridized to the extended capture primer, and a second (unextended) oligonucleotide primer. When the capture primer extension product is attached to a solid support during step 112, the unextended release primer-target complex is also attached to the solid support. In other embodiments, the capture primer extension product is released from the solid support (e.g., after removal of non-target nucleic acid molecules from the reaction mixture) and is in solution to allow hybridization of the release primer in solution in step 112.

[0168] Step 114 of method 100 involves performing a release primer extension reaction. Following hybridization of the release primer to the target nucleic acid template in step 112, the release primer is extended by a second polymerase, thereby generating a release primer extension product or complex containing the target nucleic acid. The extended release primer includes a 3' region that includes the reverse complement of at least a portion of the target nucleic acid template. In some embodiments, extension of the release primer by the second polymerase releases the extended capture primer from its complex with the target nucleic acid. Releasing the extended capture oligonucleotide from the capture primer extension complex can include one or more of strand displacement (e.g., by a polymerase) or digestion (e.g., by a nuclease). For example, release of the extended capture oligonucleotide can be achieved using an enzyme with 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 the same. In other embodiments, the first polymerase and the second polymerase are different.

[0169] Steps 112 and 114 may be performed sequentially or simultaneously. Regardless of whether steps 112 and 114 are performed sequentially or simultaneously, any release primer utilized may have a high melting temperature and / or high GC content. In some embodiments, at least one release primer of the set of release primers has a melting temperature above 63°C, above about 66°C, above about 69°C, above about 72°C, above about 75°C, above about 80°C, etc. In some embodiments, multiple release primers of the set of release primers have melting temperatures above 63°C, above about 66°C, above about 69°C, above about 72°C, above about 75°C, above about 80°C, etc. In some embodiments, there are no limitations on the melting temperature and / or GC content of the release primer. In some embodiments, the release primer may include one or more modified dNTPs, such as two or more modified dNTPs, three or more modified dNTPs, four or more modified dNTPs, etc.

[0170] In some embodiments, up to about 25% of the release primers in any release primer set are release primers with high melting temperature and / or high GC content. In some embodiments, up to about 20% of the release primers in any release primer set are release primers with high melting temperature and / or high GC content. In some embodiments, up to about 15% of the release primers in any release primer set are release primers with high melting temperature and / or high GC content. In some embodiments, up to about 12% of the release primers in any release primer set are release primers with high melting temperature and / or high GC content. In some embodiments, up to about 10% of the release primers in any release primer set are release primers with high melting temperature and / or high GC content. In some embodiments, up to about 9% of the release primers in any release primer set are release primers with high melting temperature and / or high GC content. In some embodiments, up to about 8% of the release primers in any release primer set are release primers with high melting temperature and / or high GC content. In some embodiments, up to about 7% of the release primers in any release primer set are release primers with a high melting temperature and / or a high GC content. In some embodiments, up to about 6% of the release primers in any release primer set are release primers with a high melting temperature and / or a high GC content. In some embodiments, up to about 5% of the release primers in any release primer set are release primers with a high melting temperature and / or a high GC content. In some embodiments, up to about 4% of the release primers in any release primer set are release primers with a high melting temperature and / or a high GC content. In some embodiments, up to about 3% of the release primers in any release primer set are release primers with a high melting temperature and / or a high GC content. In some embodiments, up to about 2% of the release primers in any release primer set are release primers with a high melting temperature and / or a high GC content. In some embodiments, up to about 1% of the release primers in any release primer set are release primers with a high melting temperature and / or a high GC content.

[0171] In embodiments in which 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 concentration of betaine in any release primer hybridization master mix used during the performance of step 112 includes concentrations of betaine 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.

[0172] In embodiments in which steps 112 and 14 are performed simultaneously, the combined release primer hybridization and extension is performed in the presence of one or more enhancers, such as betaine, DMSO, single-stranded DNA-binding protein, disaccharide, or any combination thereof. For example, the combined release primer hybridization and extension can be performed using a composition comprising betaine (or a derivative or analog thereof), such as a composition comprising betaine at a concentration ranging from about 0.1 mM to about 1 mM (e.g., 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.).

[0173] In some embodiments, combined release primer hybridization and extension can be performed in a composition comprising a mixture of unmodified and modified dNTPs. In some embodiments, the ratio of unmodified dNTPs to modified dNTPs in any composition ranges from about 2:1 to 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 comprise 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.

[0174] In further embodiments, the combined release primer hybridization and extension is performed in a composition comprising one or more capture extensions having a high GC content and / or a high melting temperature; 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 still 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 a further embodiment, the enhancer is betaine (or a derivative or analog thereof), and the concentration of betaine in the composition is about 0.5 mM.

[0175] In yet other embodiments, combined release primer hybridization and extension can be performed in the presence of a polymerase, betaine, and a mixture of unmodified and modified dNTPs. In these 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. In some embodiments, the modified dNTPs are selected from 7-deaza-2'-deoxyguanosine-5'-triphosphate or 2-amino-2'-deoxyadenosine-5'-triphosphate.

[0176] In some embodiments, combined release primer hybridization and extension can be performed in the presence of a polymerase, betaine, an optional mixture of unmodified 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; 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. In some embodiments, the divalent cation is present at a concentration of about 0.1 mM to about 0.4 mM.

[0177] In some embodiments, combined release primer hybridization and extension may be performed in the presence of a polymerase, a mixture of unmodified 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 the 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.

[0178] In some embodiments, step 114 can further include terminating the primer extension reaction to control the length of the extended-release primer. In particular, the length of the extended-release primer product can be actively controlled by techniques such as inactivating the polymerase added in step 114, or passively controlled by allowing the reaction to go to completion, such as by consuming a limiting reactant, or by controlling / selecting the size of fragments of nucleic acid molecules in a library of nucleic acid molecules.

[0179] If the extended capture primer included one or more capture moieties attached to a solid support, then release of the extended capture oligonucleotide in step 114 results in the released primer extension complex being free in solution as opposed to being attached to a solid support. Thus, as described in step 110 of method 100, one or more purification techniques can be performed after step 114 to recover unbound second extension products or complexes comprising the target nucleic acid from the extended capture primer attached to the support, the second polymerase, other reaction components, etc., and combinations thereof.

[0180] Method 100 further includes a post-capture amplification step 116, which may involve linear or exponential amplification (e.g., PCR). Generally, step 116 involves amplifying the target nucleic acid using a third polymerase, a first amplification primer, and a second amplification primer. In some embodiments, the first and second amplification primers are designed to be complementary to the sequences of the adapters incorporated into the target nucleic acid molecules in the library of nucleic acid molecules in step 102. For example, the first amplification primer may have a 3' end complementary to the first adapter, and the second amplification primer may have a 3' end complementary to the second adapter. However, the primers for amplification may include any sequence present in the target nucleic acid to be amplified (e.g., gene / target-specific primers, universal primers, etc.) and may support synthesis of one or both strands (i.e., both the top and bottom strands of a double-stranded nucleic acid molecule corresponding to the template for the amplification reaction). In some embodiments, post-capture amplification may be performed with compositions similar to those utilized in pre-capture amplification, including any of the compositions listed herein.

[0181] In some embodiments, step 116 allows for selective amplification of target nucleic acid molecules from a library of nucleic acid molecules, as opposed to amplification of either the extended capture or release primer derived from the target nucleic acid. In one example, a 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 resulting from the reaction contains at least one uracil nucleotide, but the target nucleic acid template may be a DNA template lacking uracil nucleotides. A uracil-incompatible polymerase is then included in step 116 to amplify the target nucleic acid. The uracil-incompatible polymerase can amplify target nucleic acids lacking uracil nucleotides. However, the uracil-incompatible polymerase cannot replicate the uracil-containing extended oligonucleotide primer. Alternatively or additionally, the uracil-containing product can be selectively digested or otherwise degraded, thereby leaving only the original molecule from the library of nucleic acid molecules.

[0182] After the step of amplification 116, method 100 can include a step 118 of analyzing the amplified target nucleic acid molecules. Step 116 can include any method for determining the nucleic acid sequence of one or more products of method 100. Step 116 can further include sequence alignment, identification of sequence variations, counting unique primer extension products, etc., or a combination thereof.

[0183] In some embodiments, the amplicons generated after step 116 are sequenced, such as by next-generation sequencing. As used herein, the term "next-generation sequencing" refers to sequencing technologies that offer high-throughput sequencing compared to traditional Sanger electrophoresis and capillary electrophoresis-based approaches, where the sequencing process is performed in parallel, producing, for example, thousands or millions of relatively small sequence reads at a time. Some examples of next-generation sequencing technologies include, but are not limited to, sequencing-by-synthesis, sequencing-by-ligation, and sequencing-by-hybridization. These technologies produce shorter reads (anywhere from about 25 to about 500 bp) but can produce hundreds of thousands or millions of reads in a relatively short time. Examples of such sequencing devices available from Illumina (San Diego, California) include, but are not limited to, iSEQ, MiniSEQ, MiSEQ, NextSEQ, and NoveSEQ. Illumina's next-generation sequencing technology is believed to enable rapid sequencing using clonal amplification and sequencing-by-synthesis (SBS) chemistry. This process simultaneously identifies DNA bases as they are incorporated into nucleic acid strands. Each base emits a unique fluorescent signal as it is added to the growing strand, which is used to determine the order of the DNA sequence. A non-limiting example of a sequencing device available from ThermoFisher Scientific (Waltham, Massachusetts) includes the Ion Personal Genome Machine™ (PGM™) system. Ion Torrent sequencing is believed to measure the direct release of H+ (protons) from the incorporation of individual bases by DNA polymerase. A non-limiting example of a sequencing device available from Pacific Biosciences (Menlo Park, California) includes the PacBio Sequel System. A non-limiting example of a sequencing device available from Roche (Pleasanton, California) is the Roche 454.Next-generation sequencing methods can also include nanopore sequencing. Three nanopore sequencing approaches have generally been pursued: strand sequencing, in which DNA bases are identified as they pass sequentially through a nanopore; exonuclease-based nanopore sequencing, in which nucleotides are enzymatically cleaved one by one from a DNA molecule and monitored as they are captured and passed through a nanopore; and nanopore sequencing by synthesis (SBS), in which distinguishable polymer tags are attached to nucleotides and registered in the nanopore during enzyme-catalyzed DNA synthesis. Common to all these methods is the need for precise control of reaction kinetics so that each base is determined sequentially. Strand sequencing requires a method to slow the passage of DNA through the nanopore and decode multiple bases within the channel; for this purpose, a ratchet approach utilizing molecular motors has been developed. Exonuclease-based sequencing requires the release of each nucleotide close enough to the pore to ensure its capture and passage through the pore at a rate slow enough to obtain a valid ionic current signal. Furthermore, these methods all rely on the differences between four natural bases: two relatively similar purines and two similar pyrimidines. The nanopore SBS approach utilizes synthetic polymer tags attached to nucleotides that are specifically designed to generate unique and easily distinguishable ionic current blocking signatures for sequencing. In some embodiments, sequencing nucleic acid molecules by nanopore sequencing includes preparing a nanopore sequencing complex and determining a polynucleotide sequence. Methods for preparing nanopores and nanopore sequencing are described in U.S. Patent Application Publication No. 2017 / 0268052, and International Publication Nos. 2014 / 074727, 2006 / 028508, 2012 / 083249, and 2014 / 074727, the disclosures of which are incorporated herein by reference in their entirety.In some embodiments, tagged nucleotides can be used in determining polynucleotide sequences (see, e.g., PCT Publication Nos. WO / 2020 / 131759, WO / 2013 / 191793, and WO / 2015 / 148402, the disclosures of which are incorporated herein by reference in their entireties). Analysis of data generated by sequencing is generally performed using software and / or statistical algorithms that perform various data transformations, such as converting signal emissions to base calls, converting base calls to consensus sequences of nucleic acid templates, etc. Such software, statistical algorithms, and their uses are described in detail in U.S. Patent Application Publication Nos. 2009 / 0024331 and 2017 / 0044606 and PCT Publication No. WO / 2018 / 034745, the disclosures of which are incorporated herein by reference in their entireties. [Example]

[0184] Example 1 - Effect of Varying Primer Maximum Melting Temperature (TM) Limits on Enrichment of High % GC Targets material NA12878 cell line DNA Hotspot Panel HyperPlus Library Prep Kit KAPA UDI Primer Mix KAPA Universal UMI Adapter

[0185] method Panels were designed for three high %GC targets with no constraint on %GC, a minimum Tm of 57°C, and various maximum Tm constraints (eg, 66°C, 69°C, 72°C, no constraint).

[0186] Twenty-four libraries were generated according to the KAPA HyperPETE somatic tissue DNA workflow v1.0, Chapter 4. Library preparation from DNA using 10 ng of non-formalin-damaged NA24143 cell line DNA (exceptions listed below): During step 5 (0.8X purification after ligation using KAPA HyperPure Beads), pool the adapter-ligated DNA samples and aliquot this pool to 20 μL per sample for step 6.

[0187] Primer extension target enrichment was performed on 10 libraries according to Chapter 5 of the KAPA HyperPETE somatic tissue DNA workflow v1.0 (the disclosure of which is incorporated herein by reference in its entirety). Primer extension target enrichment (PETE) (as described herein) was performed (except as listed below): During step 2 (capture extension reaction), 5 μL of five small custom capture test panels (Table 8) targeting three high %GC ROIs using various maximum Tm limits were added to four samples per condition. 5 μL of hot spot capture panel was added, but water was not included in the capture extension reaction master mix. During step 6 (release primer hybridization), 20 μL of 1x Wash & Resuspension Buffer and 10 μL of hot spot release panel were added to the release primer hybridization master mix instead of 30 μL. 10 μL of the five test capture panels were added to four corresponding samples per condition.

[0188] Primer extension target enriched 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 samples.

[0189] [Table 8]

[0190] result Results for the current primer database are shown in Figure 2A. Similarly, results for the "No Max Tm Panel" are shown in Figure 2B. Figure 2C illustrates primer availability. Increasing the primer max Tm increased primer availability in high GC regions (a) and reduced the number of target bases not covered by the capture and release primer spans (b). As shown in Figure 3A, samples enriched with the no max Tm test panel had the highest percentage of bases within a 2-fold range and a panel exonic region percentage of over 1000X. A higher primer max Tm was found to improve normalized coverage for targets above 65% GC compared to the current primer database with a max Tm of 57°C (see Figure 3D). A small custom panel designed without a max primer Tm had the highest normalized coverage in high GC targets.

[0191] conclusion Applicant has found that target enrichment in the presence of primers synthesized without constraints on melting temperature and / or GC content improves the enrichment of GC-rich target nucleic acid molecules, ultimately resulting in improved coverage uniformity when the GC-rich target enriched nucleic acid molecules are sequenced.

[0192] Example 2 - Effect of 7-deaza dGTP and betaine on PCR and capture extension the purpose The effect of adding 7-deaza-dGTP to the library prep amplification and betaine to both the library prep amplification and capture extension reactions on enrichment of high % GC targets is tested.

[0193] material NA24143 cell line DNA Hotspot Panel Small custom panel (3 high %GC ROIs) HyperPlus Library Prep Kit KAPA UDI Primer Mix KAPA Universal UMI Adapter

[0194] method Sixteen libraries were generated according to Chapter 4 of the KAPA HyperPETE somatic tissue DNA workflow v1.0 (the disclosure of which is incorporated herein by reference in its entirety). Library preparation from DNA using 10 ng of non-formalin-damaged NA12878 cell line DNA (exceptions are listed below): During step 5 (0.8X purification after ligation using KAPA HyperPure Beads), adapter-ligated DNA samples were eluted using 12 μL of 10 mM Tris-HCl pH 8.0 instead of the 20 μL recommended by the manufacturer. The adapter-ligated DNA samples were pooled, and the pool was aliquoted to 12 μL per sample for step 6. During step 6 (amplification with KAPA UDI primer mix (also referred to as PCR1)), 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. Then, within each set of eight samples, 5 μL of water was added to four samples, and 5 μL of 5 M betaine was added to the other four samples. During step 7 (post-amplification 1× purification using KAPA HyperPure Beads), the pre-capture library was eluted with 15 μL of 10 mM Tris-HCl pH 8.0 instead of the 25 μL specified in the instructions.

[0195] Primer extension target enrichment was performed on the 16 libraries according to Chapter 5, Primer Extension Target Enrichment (PETE), of the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0, with the following exceptions: During step 2 (capture extension reaction), 5 µL of each pre-capture input library was used instead of the indicated 10–15 µL, which resulted in less than 500 ng of pre-capture input library per sample for some samples. In addition to 5 µL of the hotspot capture panel, 5 µL of a small custom capture panel targeting three high %GC ROIs was added to the capture extension reaction master mix. The capture extension reaction master mix did not contain water; rather, 5 µL of either water or 5 M betaine was added to the 5 µL pre-capture input library so that each combination of conditions had two replicates (Table 9). During step 6 (release primer hybridization), 20 µL of 1X Wash & Resuspension Buffer was added to the release primer hybridization master mix instead of 30 µL, and 10 µL of small custom release panel was added along with 10 µL of hotspot release panel.

[0196] Primer extension target enriched 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 samples.

[0197] [Table 9]

[0198] result The pre-capture input library concentration was lower in samples with 7-deaza-dGTP added (PCR1+7) compared to samples without (PCR1-7) (see Figure 4). Due to the lower pre-capture input library yield, the input to PETE was also lower in samples with 7-deaza-dGTP. Compared to samples without betaine in the capture extension reaction under the shared PCR1 conditions, the PETE library yield was lower for samples with betaine in the capture extension reaction (CapExtx+B) (see Figure 4).

[0199] The percentage of on-target reads was similar across all test conditions. Samples containing betaine in PCR1 had higher deduplication depths and larger average fragment lengths. The percentage of bases within a 2-fold range, the percentage of panel exon regions greater than 1000X, and the percentage error rate were higher in samples with 7-deaza-dGTP. For samples without 7-deaza-dGTP, samples containing betaine in the capture extension reaction had a higher percentage of bases within a 2-fold range and the percentage of exon regions greater than 1000X (see Figure 5).

[0200] Figures 6A and 6B show normalized coverage across high GC targets. In particular, Figure 6A shows that coverage across three high GC ROIs targeted by a small custom panel of samples with 7-deaza dGTP is more uniform than that across samples without 7-deaza dGTP. Figure 6B shows that for samples without 7-deaza dGTP, coverage across three high GC ROIs targeted by a small custom panel of samples with betaine in the capture extension reaction is more uniform than that across samples without betaine in the capture extension reaction.

[0201] Figure 7 shows the normalized coverage of targets by %GC. The normalized coverage of targets with GC greater than 70% is higher in samples with 7-deaza dGTP than in samples without 7-deaza dGTP. For samples without 7-deaza dGTP, the normalized coverage of targets with GC greater than 70% is higher in samples with betaine in the capture extension reaction than in samples without betaine in the capture extension reaction.

[0202] conclusion Applicants have found that the addition of either 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 variant calling effect of the higher error rate percentage seen in samples with 7-deaza dGTP.

[0203] Example 3 - Optimization of the capture and extension program the purpose The effect of different capture extension incubation times and temperatures on enrichment of high % GC targets is tested.

[0204] material NA24143 cell line DNA Hotspot Panel Small custom panel (3 high %GC ROIs) HyperPlus Library Prep Kit KAPA UDI Primer Mix KAPA Universal UMI Adapter

[0205] method Ten libraries were generated according to Chapter 4 of the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 (Library Preparation from DNA using 10 ng of non-formalin-damaged NA24143 cell line DNA) (with exceptions listed below): During step 5 (0.8X purification after ligation using KAPA HyperPure Beads), adapter-ligated DNA samples were pooled and this pool was aliquoted at 20 μL per sample for step 6.

[0206] Primer extension target enrichment was performed on 10 libraries according to Chapter 5 of the KAPA HyperPETE somatic tissue DNA workflow v1.0 (the disclosure of which is incorporated herein by reference in its entirety). Primer extension target enrichment (PETE) (except as listed below): During step 2 (capture extension reaction), 5 μL of the hotspot capture panel and 5 μL of a small custom capture panel targeting three high %GC ROIs were added to the capture extension reaction master mix. No water was included in the capture extension reaction master mix. Two samples were incubated in each of the four test capture extension programs detailed in Table 10 or the control capture extension program using the thermal cycler program in step 2.2. During step 6 (release primer hybridization), 20 μL of 1X Wash & Resuspension Buffer was added to the release primer hybridization master mix instead of 30 μL, and 10 μL of the small custom release panel was added along with the 10 μL hotspot release panel. Primer extension target enriched 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 samples.

[0207] [Table 10]

[0208] Figure 8 shows sequencing quality control metrics. Compared to samples from programs indicated in the instructions for use (IFU), samples from test programs 1-4 had higher percent on-target reads, deduplication depth, percent bases within 2-fold range, and percent panel exon regions greater than 1000X. Average fragment length was higher for the IFU program. Program 3 samples had the highest percent bases within 2-fold range and percent panel exon regions greater than 1000X.

[0209] Figure 9 shows the normalized coverage per target. Normalized coverage of targets with GC greater than 70% is higher for samples from test programs 1-4 compared to IFU samples, with program 3 samples having the highest normalized coverage for targets with GC greater than 75%. Test programs 1-4 samples have lower normalized coverage for targets with GC less than 40% compared to IFU samples, with program 1 samples having the lowest coverage for targets with GC less than 35%.

[0210] conclusion Test program 3 (Table 10), which had a longer incubation time at a higher temperature, provided the greatest improvements in normalized coverage of high % GC targets, percent bases within a 2-fold range, and percent panel exon regions greater than 1000X compared to the other test programs and IFU programs. Applicants have found that longer incubation times and higher temperatures can provide improved normalized coverage of high % GC targets.

[0211] Example 4 - Effect of 7-deaza dGTP, 2-amino dATP, DMSO, trehalose, betaine and single-stranded binding protein on capture extension The objective of this study was to test the effect of adding 7-deaza dGTP and 2-amino dATP to library prep amplification and DMSO, trehalose, betaine, and single-stranded DNA binding protein to capture extension reactions on enrichment of high % GC targets.

[0212] material NA12878 cell line DNA Panel without maximum Tm KAPA HyperPETE Hotspot Panel 2-aminodATP 7-deaza-dGTP Single-stranded DNA binding protein Betaine Trehalose DMSO KAPA HyperPETE Reagent Kit KAPA HyperCapture Bead Kit KAPA HyperPlus Library Prep Kit KAPA UDI Primer Mix KAPA Universal UMI Adapter

[0213] method Sixteen libraries were generated according to Chapter 4 of the KAPA HyperPETE somatic tissue DNA workflow v1.0. Library preparation from DNA using 10 ng of non-formalin-damaged NA24143 cell line DNA (exceptions listed below): During step 5 (0.8X post-ligation purification using KAPA HyperPure Beads), adapter-ligated DNA samples were pooled and this pool was aliquoted at 20 μL per sample for step 6. In four replicates, 2-amino-dATP was added to 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 PCR1 amplification at a concentration of 0.3 mM. In another four replicates, 7-deaza-dGTP was added to PCR1 amplification at a concentration of 0.3 mM (Table 11). In the final four replicates, no additives were included in PCR1 amplification.

[0214] Primer extension target enrichment was performed on 10 μL of each of the 16 libraries according to Chapter 5 of the KAPA HyperPETE somatic tissue DNA workflow v1.0. Primer Extension Target Enrichment (PETE) (except as listed below): During Step 2 (Capture Extension Reaction), equal concentrations of the high %GC ROI capture panel without max Tm and the hotspot capture panel were added to the capture extension reaction master mix. During Step 6 (Release Primer Hybridization), the volume of the 1x Wash & Resuspension Buffer was reduced to accommodate equal concentrations of the hotspot release panel and the no-max Tm release panel in the release primer hybridization master mix.

[0215] Twenty-four libraries were generated according to Chapter 4 of the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0 (Library Preparation from DNA using 10 ng of non-formalin-damaged NA24143 cell line DNA) (with exceptions listed below): During step 5 (0.8X purification after ligation using KAPA HyperPure Beads), adapter-ligated DNA samples were pooled and this pool was aliquoted at 20 μL per sample for step 6.

[0216] Primer extension target enrichment was performed on 10 μL of each of the 16 libraries according to Chapter 5, Primer Extension Target Enrichment (PETE), of the KAPA HyperPETE Somatic Tissue DNA Workflow v1.0, except as listed below: During step 2 (capture extension reaction), equal concentrations of the high %GC ROI capture panel and hotspot capture panel without max Tm were added to the capture extension reaction master mix. Amounts of 100 ng, 200 ng, or 400 ng of single-stranded DNA binding protein were added to three replicates each. Betaine was added to three replicates to achieve a final concentration of 0.5 M. Trehalose was added to three replicates to achieve a final concentration of 0.1 M. DMSO was added to three replicates at a final concentration of 5% or 10% (Table 111). Three replicates did not contain any capture extension additives. During step 6 (Release Primer Hybridization), the volume of the 1x Wash & Resuspension Buffer was reduced to accommodate equal concentrations of Hot Spot Release Panel and Max Tm No Release Panel in the Release Primer Hybridization Master Mix.

[0217] Primer-extended target-enriched libraries were sequenced on a NextSeq500 and analyzed using a Roche internal pipeline with 8M total subsampled reads. Normalized coverage at each position for each sample was calculated by dividing the coverage at a position by the average coverage across samples.

[0218] [Table 11]

[0219] result The on-target read percentage was similar between the 7-deaza dGTP (7DdGTP)-only sample and the 7DdGTP and 2-amino dATP (2AdATP) sample, and slightly higher than the control sample (see Figure 11). The control sample and the 2AdATP-only sample had similar on-target read percentages. The sample with 7DdGTP alone had a much higher deduplication depth than the control sample, while the sample with 2AdATP, with or without 7DdGTP, had a lower deduplication depth than the control sample. The sample with 7DdGTP with or without 2AdATP had a slightly higher percentage of bases within a 2-fold range than the control sample and the 2AdATP-only sample. The sample with 7DdATP had a much higher panel exon region percentage of 1000X or more than the control sample, followed by the 2AdATP and 7DdATP sample. The sample with 2AdATP alone had a slightly lower panel exon region percentage of 1000X or more than the control sample. Samples spiked with 7DdATP alone had the same percent error rate as the control sample, while samples spiked with 2AdATP with or without 7DdATP had a higher percent error rate compared to the control sample.

[0220] The normalized positional de-duplication coverage across the three high percent GC targets in the maximum Tm-free panel was more uniform and closer to the targeted normalized coverage of 1, as shown by the dotted lines, for samples spiked with 7DdTP with or without 2AdATP compared to samples spiked with only 2AdATP and the control sample (Figure 12).

[0221] Normalized positional deduplication coverage was higher for high percent GC targets in samples captured using the no-max Tm panel than in samples captured using a panel designed with the current primer database from previous experiments. However, the median normalized coverage level was still below the ideal of 1 (see Figure 13). Adding 7DdGTP to PCR1 increased the normalized coverage for the three targets to above 1.

[0222] The percent on-target reads was significantly reduced by the addition of 10% DMSO (DMSO10) compared to all other samples with similar on-target read percentages, and slightly increased by the addition of betaine (see Figure 14). The addition of DMSO10 significantly reduced deduplication depth compared to all other samples with similar deduplication depths. The percent bases within a 2x range and the percent panel exon regions greater than 1000x were significantly reduced by the addition of DMSO10 compared to all other samples with similar performance. The percent error rate increased by the addition of DMSO10 compared to all other samples with similar error rate percentages.

[0223] Compared to the control sample, for samples with DMSO5 or betaine added, the normalized position-deduplication coverage across the three high percent GC targets in the max Tm-less panel was more uniform and closer to the target-normalized coverage of 1, as shown by the dotted lines (see Figure 15). Samples with DMSO10 added had the opposite profile for CEBPB exon 1 and EGFR exon 1 compared to the control sample, with high coverage areas in the control sample being low coverage in the DMSO10 sample, and vice versa.

[0224] Normalized positional deduplication coverage was higher for high percent GC targets in samples captured using the no-max Tm panel than in samples captured using a panel designed with the current primer database from previous experiments. However, the median normalized coverage level was still below the ideal of 1 (see Figure 16). With the addition of betaine in the capture extension, the normalized coverage for the three targets was closer to the ideal of 1 compared to the sample without betaine (no-max Tm panel). With the addition of DMSO5 in the capture extension, the normalized coverage for the three targets was much higher than the ideal of 1.

[0225] conclusion The addition of 7-deaza-dGTP to PCR1, betaine in the capture extension, and 5% DMSO in the capture extension all improved coverage in high-percent GC 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 some slight decreases in sequencing QC metric performance.

[0226] Example 6 - Titration of DMSO in Capture Extension the purpose The objective of this study was to perform a titration of 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.

[0227] material NA12891 cell line DNA 1.7Mb panel DMSO KAPA HyperPETE Reagent Kit KAPA HyperCapture Bead Kit KAPA HyperPlus Library Prep Kit KAPA UDI Primer Mix KAPA Universal UMI Adapter High-sensitivity D1000 reagent NextSeq500 / 550 HO Reagent

[0228] method Eighteen libraries were prepared from 10 ng of non-formalin-damaged NA12891 cell line DNA according to Chapter 4 of the KAPA HyperPETE somatic tissue DNA workflow v1.0, except that to limit the sample to differences in the library preparation process, libraries were pooled after ligation cleanup and realiquoted in 20 μL volumes. For enrichment, we followed Chapter 5 of the KAPA HyperPETE somatic tissue DNA workflow v1.0, with several notable modifications to facilitate the use of a custom-made 1.7 Mb HyperPETE panel, which is outside the size range of commercially available HyperPETE panels. The 1.7 Mb panel was designed using an extended primer database with 0-100% GC and a Tm of 57-100°C.

[0229] Library input to primer extension target enrichment (PETE) was normalized to 1000 ng of library per sample. PETE workflow modifications included diluting capture primers to 0.37x per primer concentration versus the standard 1x per primer concentration, thus keeping the total capture primer concentration at 2x that of the largest commercially available panel, the HyperPETE Pan Cancer Panel. Other modifications included using 3x HyperPure Bead Clean-up to enrich Cot DNA and adding twice the amount of human Cot DNA in the capture extension step by using 300 µL of capture beads versus 100 µL in the standard protocol. In the capture extension step, a master mix was created containing 0–5% DMSO (Table 12). For the PCR amplification step, 13 cycles were used based on the panel size. PETE libraries were sequenced on a NextSeq500 and analyzed using a Roche internal pipeline subsampling at 60M total reads / sample.

[0230] [Table 12]

[0231] result The on-target rate increased with increasing amounts of DMSO in the capture extension (see Figure 17). Deduplication depth remained similar up to 2% DMSO and then began to decrease. Due to uniformity, the % of bases within a 2-fold range decreased as the amount of DMSO increased. The % panel exon region above 300x metric was best at 1% and 2% DMSO and began to decrease as the amount of DMSO increased.

[0232] Normalized coverage for higher % GC targets improved with increasing DMSO in the capture extension (see Figure 18). 2% DMSO also had minimal adverse effects on coverage for low GC / high AT targets. Data are not shown for 1% and 3%, but the trends were similar.

[0233] conclusion Addition of 2% DMSO in capture extension using a 1.7 Mb panel improved coverage of high GC targets while minimizing adverse effects on low GC targets, and also improved or maintained similar enrichment performance based on sequencing quality metrics.

[0234] Additional Embodiments Additional Embodiment 1. A composition comprising: (a) a polymerase; (b) one or more primers; (c) unmodified dNTPs; and (d) at least one enhancer.

[0235] Additional Embodiment 2. The composition of additional embodiment 1, wherein the at least one enhancer is selected from the group consisting of betaine, dimethyl sulfoxide (DMSO), a disaccharide, and a single-stranded DNA binding protein (SSB).

[0236] Additional Embodiment 3. The composition of any one of the preceding additional embodiments, wherein at least one enhancer is betaine.

[0237] Additional Embodiment 4. The composition of Additional Embodiment 3, wherein the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM.

[0238] Additional Embodiment 5. The composition of Additional Embodiment 3, wherein the concentration of betaine in the composition ranges from about 0.3 mM to about 0.6 mM.

[0239] Additional Embodiment 6. The composition of additional embodiment 3, wherein the concentration of betaine in the composition is about 0.5 mM.

[0240] Additional embodiment 7. The composition of additional embodiment 2, wherein at least one enhancer is DMSO.

[0241] Additional Embodiment 8. The composition of additional embodiment 7, wherein the amount of DMSO in the composition ranges from about 1% (v / v) to about 10% (v / v).

[0242] Additional Embodiment 9. The composition of additional embodiment 7, wherein the amount of DMSO in the composition ranges from about 2% (v / v) to about 9% (v / v).

[0243] Additional Embodiment 10. The composition of any one of the preceding additional embodiments, wherein one or more primers have a Tm in the range of about 57°C to about 95°C.

[0244] Additional Embodiment 11. The composition of any one of the preceding additional embodiments, wherein one or more primers have a Tm in the range of about 57°C to about 85°C.

[0245] Additional Embodiment 12. The composition of any one of the preceding additional embodiments, wherein one or more primers have a Tm in the range of about 57°C to about 75°C.

[0246] Additional Embodiment 13. The composition of any one of the preceding additional embodiments, wherein one or more primers have a Tm in the range of about 57°C to about 72°C.

[0247] Additional Embodiment 14. The composition of any one of the preceding additional embodiments, wherein one or more primers have a Tm in the range of about 57°C to about 69°C.

[0248] Additional Embodiment 15. The composition of any one of the preceding additional embodiments, wherein one or more primers have a Tm in the range of about 57°C to about 66°C.

[0249] Additional Embodiment 16. The composition of any one of the preceding additional embodiments, wherein one or more primers have a Tm in the range of about 57°C to about 63°C.

[0250] Additional Embodiment 17 The composition of any one of the preceding additional embodiments, wherein one or more primers comprise one or more modified dNTPs.

[0251] Additional Embodiment 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.

[0252] Additional Embodiment 19 The composition of any one of the preceding additional embodiments, further comprising one or more modified dNTPs.

[0253] Additional Embodiment 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.

[0254] Additional Embodiment 21. The composition of Additional Embodiment 19, wherein the concentration of the one or more modified dNTPs in the composition ranges from about 0.1 mM to about 0.5 mM.

[0255] Additional Embodiment 22. The composition of additional embodiment 19, wherein the concentration of the one or modified dNTPs in the composition ranges from about 0.2 mM to about 0.4 mM.

[0256] Additional embodiment 23 The composition of additional embodiment 19, wherein the one or more modified dNTPs is modified dGTP.

[0257] Additional Embodiment 24. The composition of additional embodiment 23, wherein the modified dGTP comprises 7-deaza-2'-deoxyguanosine-5'-triphosphate.

[0258] Additional embodiment 25 The composition of additional embodiment 19, wherein the one or more modified dNTPs is modified dATP.

[0259] Additional Embodiment 26. The composition of additional embodiment 25, wherein the modified dATP comprises 2-amino-2'deoxyadenosine-5'-triphosphate.

[0260] Additional Embodiment 27. The composition of any one of additional embodiments 1-26, further comprising a divalent cation.

[0261] Further embodiment 28. The divalent cation is Co 2+ , Mn 2+ , Mg 2+ , Cd 2+ , and Ca 2+ 28. The composition of further embodiment 27, selected from the group consisting of:

[0262] Additional Embodiment 29. The composition of any one of the preceding additional embodiments, wherein the composition further comprises one or more buffers.

[0263] Additional Embodiment 30. The composition of any one of the preceding additional embodiments, wherein the composition further comprises one or more polyols.

[0264] Additional Embodiment 31 The composition of any one of the preceding additional embodiments, wherein the one or more primers comprise pre-capture forward and reverse primers.

[0265] Additional Embodiment 32 The composition of any one of the preceding additional embodiments, wherein the one or more primers comprises a capture primer.

[0266] Additional Embodiment 33 The composition of any one of the preceding additional embodiments, wherein the capture primer comprises a capture moiety.

[0267] Additional Embodiment 34. The composition of any one of the preceding additional embodiments, wherein the composition further comprises an input nucleic acid molecule.

[0268] Additional embodiment 35. The composition of additional embodiment 34, wherein the input nucleic acid molecules comprise a library of nucleic acid molecules, and each nucleic acid molecule in the library of nucleic acid molecules comprises a first and a second adaptor.

[0269] Additional embodiment 36. The composition of additional embodiment 34, wherein the one or more primers comprise one or more capture primers, and the one or more capture primers are capable of hybridizing to a target nucleic acid sequence within the library of nucleic acid molecules.

[0270] Additional embodiment 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 do not include a limitation on the percentage of guanine bases or cytosine bases, and at least one primer of the one or more primers has a melting temperature greater than 63°C.

[0271] Additional Embodiment 38. The composition of additional embodiment 37, wherein at least one primer has a melting temperature greater than about 69°C.

[0272] Additional Embodiment 39. The composition of additional embodiment 37, wherein at least one primer has a melting temperature greater than about 75°C.

[0273] Additional Embodiment 40. The composition of additional embodiment 37, wherein at least one primer has a melting temperature greater than about 85°C.

[0274] Additional embodiment 41. The composition of additional embodiment 37, wherein at least one primer has a melting temperature greater than about 95°C.

[0275] Additional Embodiment 42. The composition of any one of additional embodiments 37-41, wherein at least one optional enhancer is selected from the group consisting of betaine, DMSO, disaccharides, and single-stranded DNA binding proteins (SSBs).

[0276] Additional Embodiment 43. The composition of additional embodiment 42, wherein the at least one optional enhancer is betaine, and the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM.

[0277] Additional Embodiment 44. The composition of any one of additional embodiments 37-43, wherein the dNTPs comprise a mixture of unmodified and modified dNTPs.

[0278] Additional Embodiment 45. The composition of additional embodiment 44, wherein the one or more modified dNTPs comprise 7-deaza-2'-deoxyguanosine-5'-triphosphate.

[0279] Additional Embodiment 46. The composition of additional embodiment 44, wherein the one or more modified dNTPs comprise 2-amino-2'deoxyadenosine-5'-triphosphate.

[0280] Additional Embodiment 47. The composition of any one of additional embodiments 44-46, wherein the concentration of the one or more modified dNTPs in the composition ranges from about 0.2 mM to about 0.8 mM.

[0281] Additional Embodiment 48. The composition of any one of additional embodiments 37-41, wherein one or more primers comprise one or more modified dNTPs.

[0282] Additional Embodiment 49. 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.

[0283] Additional Embodiment 50. The composition of any one of additional embodiments 37-49, further comprising a divalent cation.

[0284] Additional Embodiment 51. The composition of additional embodiment 50, wherein the divalent cation is selected from the group consisting of Co2+, Mn2+, Mg2+, Cd2+, and Ca2+.

[0285] Additional Embodiment 52. The composition of any one of additional embodiments 37-51, wherein the composition further comprises one or more buffers.

[0286] Additional Embodiment 53. The composition of any one of additional embodiments 37-52, wherein the composition further comprises one or more polyols.

[0287] Additional Embodiment 54. The composition of any one of additional embodiments 37-53, wherein the one or more primers comprise pre-capture forward and reverse primers.

[0288] Additional Embodiment 55. The composition of any one of additional embodiments 37-53, wherein one or more primers is a capture primer.

[0289] Additional Embodiment 56. The composition of any one of additional embodiments 37-55, wherein the composition further comprises an input nucleic acid molecule.

[0290] Additional embodiment 57. The composition of additional embodiment 56, wherein the input nucleic acid molecule comprises a first and a second adaptor.

[0291] Additional Embodiment 58. The composition of additional embodiment 56, wherein the one or more primers comprise one or more capture primers, and the one or more capture primers are capable of hybridizing to a target nucleic acid sequence of the nucleic acid molecule.

[0292] Additional embodiment 59. The composition of additional embodiment 56, wherein the input nucleic acid molecule comprises DNA.

[0293] Additional Embodiment 60. A reaction vessel comprising the composition of any one of Additional Embodiments 1-59.

[0294] Additional embodiment 61. Use of the composition of any one of additional embodiments 1-33 and 37-55 in amplifying an input nucleic acid molecule.

[0295] Additional embodiment 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).

[0296] Additional embodiment 63. The composition of additional embodiment 62, wherein at least one enhancer is betaine.

[0297] Additional Embodiment 64. The composition of additional embodiment 63, wherein the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM.

[0298] Additional Embodiment 65. The composition of additional embodiment 63, wherein the concentration of betaine in the composition ranges from about 0.3 mM to about 0.6 mM.

[0299] Additional embodiment 66. The composition of additional embodiment 62, wherein at least one enhancer is DMSO.

[0300] Additional Embodiment 67. 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).

[0301] Additional Embodiment 68. 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).

[0302] Additional Embodiment 69. The composition of additional embodiment 62, wherein one or more primers have a Tm in the range of about 57°C to about 95°C.

[0303] Additional Embodiment 70. The composition of additional embodiment 62, wherein one or more primers have a Tm in the range of about 57°C to about 85°C.

[0304] Additional Embodiment 71. The composition of additional embodiment 62, wherein one or more primers have a Tm in the range of about 57°C to about 75°C.

[0305] Additional Embodiment 72. The composition of additional embodiment 62, wherein one or more primers have a Tm in the range of about 57°C to about 72°C.

[0306] Additional Embodiment 73. The composition of additional embodiment 62, wherein one or more primers have a Tm in the range of about 57°C to about 69°C.

[0307] Additional Embodiment 74. The composition of additional embodiment 62, wherein one or more primers have a Tm in the range of about 57°C to about 66°C.

[0308] Additional embodiment 75. The composition of additional embodiment 62, wherein one or more primers comprise one or more modified dNTPs.

[0309] Additional Embodiment 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.

[0310] Additional Embodiment 77. The composition of any one of additional embodiments 62-76, further comprising a divalent cation.

[0311] Further embodiment 78. The divalent cation is Co 2+ , Mn 2+ , Mg 2+ , Cd 2+ , and Ca 2+ 78. The composition of further embodiment 77, selected from the group consisting of:

[0312] Additional Embodiment 79. The composition of any one of additional embodiments 62-78, wherein the composition further comprises one or more buffers.

[0313] Additional Embodiment 80. The composition of any one of additional embodiments 62-79, wherein the input nucleic acid molecule comprises a prepared nucleic acid library.

[0314] Additional Embodiment 81. The composition of any one of additional embodiments 62-79, wherein the input nucleic acid molecule comprises a captured nucleic acid molecule.

[0315] Additional Embodiment 82. The composition of additional embodiment 81, wherein the captured nucleic acid molecule comprises a complex of a nucleic acid molecule comprising the target nucleic acid sequence and an extended capture primer hybridized to at least a portion of the target nucleic acid sequence.

[0316] Additional embodiment 83. A composition comprising: (a) an input nucleic acid molecule; (b) one or more primers; and, optionally, (c) at least one enhancer, wherein the one or more primers do not include a limitation on the percentage of guanine bases or cytosine bases, and at least one primer of the one or more primers has a melting temperature greater than 63°C.

[0317] Additional Embodiment 84. The composition of additional embodiment 83, wherein at least one primer has a melting temperature greater than about 69°C.

[0318] Additional Embodiment 85. The composition of additional embodiment 83, wherein at least one primer has a melting temperature greater than about 75°C.

[0319] Additional Embodiment 86. The composition of additional embodiment 83, wherein at least one primer has a melting temperature greater than about 85°C.

[0320] Additional Embodiment 87. The composition of additional embodiment 83, wherein at least one primer has a melting temperature greater than about 95°C.

[0321] Additional Embodiment 88. The composition of any one of additional embodiments 83-87, wherein one or more primers comprise one or more modified dNTPs.

[0322] Additional Embodiment 89. 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.

[0323] Additional Embodiment 90. The composition of any one of additional embodiments 83-89, wherein at least one optional enhancer is selected from the group consisting of betaine, DMSO, disaccharides, and single-stranded DNA binding proteins (SSBs).

[0324] Additional Embodiment 91. The composition of additional embodiment 90, wherein the at least one optional enhancer is betaine, and the concentration of betaine in the composition ranges from about 0.2 mM to about 0.8 mM.

[0325] Additional Embodiment 92. The composition of any one of additional embodiments 83-91, further comprising a divalent cation.

[0326] Additional Embodiment 93. The composition of additional embodiment 92, wherein the divalent cation is selected from the group consisting of Co2+, Mn2+, Mg2+, Cd2+, and Ca2+.

[0327] Additional Embodiment 94. The composition of any one of additional embodiments 83-93, wherein the composition further comprises one or more buffers.

[0328] Additional Embodiment 95. The composition of any one of additional embodiments 83-94, wherein the input nucleic acid molecule comprises a prepared nucleic acid library.

[0329] Additional Embodiment 96. The composition of any one of additional embodiments 83-94, wherein the input nucleic acid molecule comprises captured DNA.

[0330] Additional Embodiment 97. The composition of additional embodiment 96, wherein the captured nucleic acid molecule comprises a complex of a nucleic acid molecule comprising the target nucleic acid sequence and an extended capture primer hybridized to at least a portion of the target nucleic acid sequence.

[0331] Additional Embodiment 98. A reaction vessel comprising the composition of any one of additional embodiments 62-97.

[0332] Additional embodiment 99. Use of the composition of any one of additional embodiments 62-79 and 83-94 for preferential enrichment of one or more target nucleic acid molecules in a library of nucleic acid molecules.

[0333] Additional embodiment 100. A kit comprising: (a) a set of capture primers; (b) a set of release primers; (c) a polymerase; (d) dNTPs; and (e) betaine or a derivative or analog thereof.

[0334] Additional Embodiment 101. The kit of additional embodiment 100, wherein the dNTPs comprise a mixture of unmodified and modified dNTPs.

[0335] Additional Embodiment 102. The kit of additional embodiment 101, wherein the modified dNTPs comprise modified dGTP and modified dATP.

[0336] Additional Embodiment 103. The kit of any one of additional embodiments 100-102, wherein the set of capture and release primers comprises one or more modified dNTPs.

[0337] Additional Embodiment 104. The kit of 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.

[0338] Additional Embodiment 105. The kit of any one of additional embodiments 100-104, further comprising one or more buffers.

[0339] Additional Embodiment 106. The kit of any one of additional embodiments 100-105, further comprising one or more divalent cations.

[0340] Additional Embodiment 107. The kit of any one of additional embodiments 100-106, further comprising one or more polyols.

[0341] Additional Embodiment 108. The kit of any one of additional embodiments 100-107, wherein at least one of the sets of capture primers and release primers has no limitations on melting temperature and / or GC content.

[0342] Additional Embodiment 109. The kit of any one of additional embodiments 100-108, wherein both the capture primer and the set of release primers have no limitations on melting temperature and / or GC content.

[0343] Additional embodiment 110. A kit comprising: (a) a set of capture primers; (b) a set of release primers; (c) a polymerase; and (d) dNTPs, wherein at least one primer in the set of capture primers has a melting temperature greater than about 65°C; and at least one primer in the set of release primers has a melting temperature greater than about 65°C.

[0344] Additional Embodiment 111. The kit of additional embodiment 110, wherein at least one primer in the set of capture primers has a melting temperature greater than about 72°C; and at least one primer in the set of release primers has a melting temperature greater than about 72°C.

[0345] Additional Embodiment 112. The kit of any one of additional embodiments 110-111, wherein at least one primer in the set of capture primers comprises one or more modified dNTPs; and at least one primer in the set of release primers comprises one or more modified dNTPs.

[0346] Additional Embodiment 113. The kit of any one of additional embodiments 110-112, further comprising 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.

[0347] Additional embodiment 114. A method of 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 library of nucleic acid molecules, wherein each nucleic acid molecule in the library of nucleic acid molecules has a first end comprising a first adapter and a second end comprising a second adapter; and (b) extending the hybridized capture primer with a first polymerase to produce a capture primer extension complex, wherein the extending capture primer has a melting temperature greater than 63°C.

[0348] Additional Embodiment 115. The method of additional embodiment 114, wherein the melting temperature of the capture primer is greater than 66°C.

[0349] Additional Embodiment 116 The method of additional embodiment 114, wherein the melting temperature of the capture primer is greater than 72°C.

[0350] Additional Embodiment 117 The method of additional embodiment 114, wherein the melting temperature of the capture primer is greater than 75°C.

[0351] Additional Embodiment 118. The method of any one of additional embodiments 114-117, wherein 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, a single-stranded DNA binding protein, and a disaccharide.

[0352] Additional Embodiment 119. The method of additional embodiment 118, wherein the at least one enhancer is betaine and the concentration of the betaine ranges from about 0.2 mM to about 0.8 mM.

[0353] Additional Embodiment 120. The method of additional embodiment 118, wherein the at least one enhancer is betaine and the concentration of the betaine ranges from about 0.3 mM to about 0.7 mM.

[0354] Additional Embodiment 121. The method of additional embodiment 118, wherein the at least one enhancer is betaine and the concentration of the betaine ranges from about 0.4 mM to about 0.6 mM.

[0355] Additional Embodiment 122 The method of any one of additional embodiments 114-117, wherein the capture primer hybridized to the target nucleic acid molecule is extended in a composition comprising one or more modified dNTPs.

[0356] Additional Embodiment 123 The method of additional embodiment 122, wherein the one or more modified dNTPs is modified dGTP.

[0357] Additional Embodiment 124 The method of additional embodiment 123, wherein the modified dGTP comprises 7-deaza-2'-deoxyguanosine-5'-triphosphate.

[0358] Additional Embodiment 125 The method of additional embodiment 122, wherein the one or more modified dNTPs is modified dATP.

[0359] Additional Embodiment 126. The method of additional embodiment 125, wherein the modified dATP comprises 2-amino-2'deoxyadenosine-5'-triphosphate.

[0360] Additional Embodiment 127. The method of any one of additional embodiments 114-117, wherein the capture primer hybridized to the target nucleic acid molecule is extended in a composition comprising betaine and one or more modified dNTPs.

[0361] Additional Embodiment 128 The method of additional embodiment 127, wherein the concentration of betaine in the composition is about 0.5 mM.

[0362] Additional Embodiment 129. The method of any one of additional embodiments 114-128, further comprising capturing the capture primer extension complex.

[0363] Additional Embodiment 130. The method of any one of additional embodiments 114-129, further comprising hybridizing a release primer to the target nucleic acid.

[0364] Additional Embodiment 131. The method of additional embodiment 130, wherein the release primer hybridizes to the target nucleic acid in a composition comprising at least one enhancer.

[0365] Additional embodiment 132. The method of additional embodiment 131, wherein at least one enhancer is betaine.

[0366] Additional Embodiment 133 The method of additional embodiment 130, wherein the release primer hybridizes to the target nucleic acid in a composition comprising one or more dNTPs.

[0367] Additional Embodiment 134 The method of additional embodiment 130, wherein the release primer has a melting temperature greater than 63°C.

[0368] Additional Embodiment 135. The method of additional embodiment 134, wherein the melting temperature is greater than 65°C.

[0369] Additional Embodiment 136. The method of additional embodiment 134, wherein the melting temperature is greater than 72°C.

[0370] Additional Embodiment 137 The method of additional embodiment 130, further comprising extending the release primer hybridized to the target nucleic acid with a second polymerase.

[0371] Additional embodiment 138. A method for amplifying one or more nucleic acid molecules, 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 primer set, wherein at least one primer of the first primer set has a melting temperature greater than 63°C.

[0372] Additional Embodiment 139. The method of additional embodiment 138, wherein each nucleic acid molecule of the resulting plurality of nucleic acid molecules comprises a first and a second adaptor.

[0373] Additional Embodiment 140. The method of any one of additional embodiments 138-139, wherein the first composition further comprises betaine.

[0374] Additional Embodiment 141. The method of additional embodiment 140, wherein the concentration of betaine in the first composition is about 0.5 mM.

[0375] Additional Embodiment 142 The method of additional embodiment 139, further comprising enriching the obtained plurality of nucleic acid molecules for one or more target nucleic acid molecules.

[0376] Additional Embodiment 143. The method of 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 primer set, wherein at least one primer of the second primer set has a melting temperature greater than 63°C.

[0377] Additional Embodiment 144. The method of additional embodiment 143, wherein the second composition further comprises betaine.

[0378] Additional Embodiment 145. The method of additional embodiment 144, wherein the concentration of betaine in the second composition is about 0.5 mM.

[0379] Additional Embodiment 146 The method of additional embodiment 138, wherein the obtained plurality of nucleic acid molecules is a target enriched library comprising a plurality of target nucleic acid molecules.

[0380] Additional Embodiment 147. The method of additional embodiment 146, further comprising sequencing the amplified target enriched library.

[0381] Additional embodiment 148. A method for amplifying one or more nucleic acid molecules, 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.

[0382] Additional Embodiment 149. The method of additional embodiment 148, wherein the concentration of betaine in the first composition is about 0.5 mM.

[0383] Additional Embodiment 150. The method of any one of additional embodiments 148-149, wherein at least one primer of the first primer set has a high melting temperature and / or a high GC content.

[0384] Additional embodiment 151. The method of additional embodiment 148, wherein each nucleic acid molecule of the obtained plurality of nucleic acid molecules comprises a first and a second adaptor, and the method further comprises enriching the obtained plurality of nucleic acid molecules for one or more target nucleic acid molecules.

[0385] Additional embodiment 152. The method of additional embodiment 151, further comprising carrying out 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.

[0386] Additional Embodiment 153 The method of additional embodiment 148, wherein the obtained plurality of nucleic acid molecules is a target enriched library comprising a plurality of target nucleic acid molecules.

[0387] Additional Embodiment 154. The method of additional embodiment 153, further comprising sequencing the amplified target enriched library.

[0388] Additional Embodiment 155. A kit for enriching at least one target nucleic acid in a library of nucleic acid molecules, comprising: (a) a first oligonucleotide complementary to the target nucleic acid in the library of nucleic acid molecules, wherein each of the nucleic acid molecules in the library of nucleic acid molecules has 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, wherein at least one of the first oligonucleotide or the second oligonucleotide has a high melting temperature and / or a high GC content.

[0389] Additional Embodiment 156. The kit of additional embodiment 155, wherein both the first and second oligonucleotides have a high melting temperature and / or a high GC content.

[0390] Additional embodiment 157. A composition comprising: (a) a library of nucleic acid molecules comprising at least one target nucleic acid, wherein each of the nucleic acid molecules in the library of nucleic acid molecules has a first end comprising a first adaptor, a second end comprising a second adaptor, and a region of interest intermediate the first adaptor and the second adaptor; (b) an extended first oligonucleotide hybridized to the region of interest of the target nucleic acid, the extended first oligonucleotide comprising at least one capture moiety; a solid support attached to the at least one capture moiety; (c) a second oligonucleotide hybridized to the target nucleic acid at a position 5' relative to 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.

[0391] Additional embodiment 158. A composition comprising: (a) a polymerase; (b) one or more primers having no limitations on melting temperature and / or GC content; (c) a mixture of unmodified and modified dNTPs; and (d) at least one enhancer.

[0392] Additional Embodiment 159. The composition of additional embodiment 158, wherein at least one enhancer is betaine.

[0393] Additional Embodiment 160. The composition of any one of additional embodiments 158 and 159, further comprising one or more nucleic acid molecules.

[0394] Additional embodiment 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 and modified dNTPs, and at least one enhancer.

[0395] Additional Embodiment 162. The composition of additional embodiment 161, wherein at least one enhancer is betaine.

[0396] Additional Embodiment 163. The composition of any one of additional embodiments 161 and 162, further comprising one or more nucleic acid molecules.

[0397] Additional embodiment 164. A composition consisting of a polymerase, one or more primers with no limitations on melting temperature and / or GC content, a mixture of unmodified and modified dNTPs, and at least one enhancer.

[0398] Additional Embodiment 165. The composition of additional embodiment 164, wherein at least one enhancer is betaine.

[0399] Additional Embodiment 166. The composition of any one of additional embodiments 164 and 165, further comprising one or more nucleic acid molecules.

[0400] Additional Embodiment 167. A reaction vessel comprising the composition of any one of Additional Embodiments 158-166.

[0401] Additional embodiment 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.

[0402] Additional Embodiment 169. A method of amplifying one or more nucleic acid molecules, wherein the amplification is performed in the presence of any one of the compositions of additional embodiments 158, 159, 161, 162, 164, and 165.

[0403] Additional embodiment 170. The method of 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) an unmodified dNTP, (d) at least one enhancer selected from the group consisting of betaine, dimethyl sulfoxide (DMSO), trehalose, and single-stranded DNA-binding proteins, and (e) optionally one or more modified dNTPs.

2. The composition according to claim 1, wherein the at least one enhancer is DMSO, and the amount of DMSO in the composition is in the range of about 1% (v / v) to about 3% (v / v).

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

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

5. The composition according to claim 1, wherein the at least one enhancer is the single-chain binding protein, and the concentration of the single-chain binding protein in the composition is in the range of about 1 ng / mL to about 9 ng / mL.

6. The composition according to claim 1, wherein one or more primers are capture primers, and the capture primers include a capture portion.

7. The composition 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) an unmodified dNTP and (ii) 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-aminodATP, and (d) optionally at least one enhancer selected from the group consisting of betaine or its derivatives or analogs, DMSO, single-stranded DNA-binding proteins and disaccharides, wherein the one or more primers are not limited to a percentage of guanine bases or cytosine bases, and at least one of the one or more primers has a melting temperature greater than 63°C.

9. The composition according to claim 8, wherein the modified dNTP comprises both 7-deaza-dGTP and 2-amino-dATP, and the 7-deaza-dGTP and 2-amino-dATP are present at concentrations ranging from about 0.2 mM to about 0.4 mM, respectively.

10. A method for producing a capture-primer extension complex comprising a target nucleic acid molecule and a capture primer, comprising: (a) hybridizing the capture primer to a portion of the target nucleic acid molecules in a library of nucleic acid molecules such that each of the nucleic acid molecules in the library of nucleic acid molecules has a first end comprising a first adapter and a second end comprising a second adapter; 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 selected from the group consisting of betaine or its derivatives or analogs, DMSO, single-stranded DNA-binding proteins, and disaccharides.

11. The method according to claim 10, wherein the at least one enhancer is DMSO, and the amount of DMSO in the composition is in the range of about 1% (v / v) to about 3% (v / v).

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

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

14. The method according to claim 10, wherein the at least one enhancer is the single-chain binding protein, and the concentration of the single-chain binding protein in the composition is in the range of about 9 ng / mL to about 21 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, (a) Obtaining multiple nucleic acid molecules; (b) Performing a first amplification reaction to amplify the obtained plurality of nucleic acid molecules, wherein the first amplification is as follows: (i) polymerase; (ii) One or more primers; (iii) dNTP mixture containing the following: (a) Unmodified dNTP, and (b) Modified dNTPs selected from the group consisting of the following: (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 its derivatives or analogues, DMSO, single-stranded DNA-binding proteins, and disaccharides. This includes carrying out a first amplification reaction using a first composition containing, A method wherein the one or more primers are not limited to a percentage of guanine bases or cytosine bases, and at least one of the one or more primers has a melting temperature above 63°C.

17. The composition according to claim 16, wherein the modified dNTP comprises both 7-deaza-dGTP and 2-amino-dATP, and the 7-deaza-dGTP and 2-amino-dATP are present at concentrations ranging from about 0.2 mM to about 0.4 mM, respectively.

18. The method according to claim 16, further comprising enriching the plurality of nucleic acid molecules obtained for one or more target nucleic acid molecules before carrying out the first amplification reaction.

19. The method further includes carrying out a second amplification reaction, wherein the second amplification is as follows: (i) polymerase; (ii) One or more primers; (iii) dNTP mixture containing the following: (a) Unmodified dNTP, and (b) Modified dNTPs selected from the group consisting of the following: (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 its derivatives or analogues, DMSO, single-stranded DNA-binding proteins, and disaccharides. The method according to any one of claims 16 to 18, carried out using a second composition comprising the above.

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