EP4581153A1 - Amélioration de la performance de l'enrichissement des cibles de nouvelle génération - Google Patents

Amélioration de la performance de l'enrichissement des cibles de nouvelle génération

Info

Publication number
EP4581153A1
EP4581153A1 EP23764267.3A EP23764267A EP4581153A1 EP 4581153 A1 EP4581153 A1 EP 4581153A1 EP 23764267 A EP23764267 A EP 23764267A EP 4581153 A1 EP4581153 A1 EP 4581153A1
Authority
EP
European Patent Office
Prior art keywords
primers
composition
nucleic acid
capture
primer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23764267.3A
Other languages
German (de)
English (en)
Inventor
Florence Katharine CRARY-DOOLEY
Nitya Margaret FURTADO
Brian Christopher Godwin
Jingchuan Li
Junyan LIN
Ruben Gerhard VAN DER MERWE
Beijing WU
Liu XI
Donald E. SHARON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Roche Diagnostics GmbH
Original Assignee
F Hoffmann La Roche AG
Roche Diagnostics GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG, Roche Diagnostics GmbH filed Critical F Hoffmann La Roche AG
Publication of EP4581153A1 publication Critical patent/EP4581153A1/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • the present disclosure is directed to compositions and methods for improving uniformity of coverage and/or reducing GC bias during sequencing.
  • Next-generation sequencing is a massively parallel sequencing technology that offers high throughput, scalability, and speed.
  • Next-generation sequencing is instrumental for investigating the molecular basis of diseases (e.g., cancer) given its sensitivity and specificity.
  • Next generation sequencing application data can result in poor performance which can include low on-target rate, low genome equivalent recovery (dedup/unique depth), and low uniformity of coverage.
  • Low uniformity can be observed in target enrichment applications where either high GC or high AT content is being enriched. This is commonly described as GC bias.
  • Uniformity of coverage is a key metric and impacts the ability to evenly enrich and sequence regions of interest. Poor uniformity of coverage results in less sequencing coverage in poor performing regions which ultimately requires more sequencing to achieve target sequencing depth in these poorer performing regions. Poor on-target rate also leads to the need for more sequencing and ultimately a higher sequencing cost per sample.
  • nucleic acid molecules for next-generation sequencing involves multiple library preparation steps.
  • the nucleic acid molecule of interest is obtained, purified, fragmented, end-repaired, and A-tailed; adapters are then ligated; and then the libraries are purified and/or enriched, quantitated, normalized, and loaded onto the instrument.
  • GC bias may be introduced during PCR amplification of a nucleic library and/or during enrichment of the nucleic acid library. 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 workflows.
  • the present disclosure is directed to compositions and methods for improving uniformity of coverage and/or reducing GC bias during sequencing, especially in reducing GC bias during sequencing of target enriched samples.
  • Applicant has unexpectedly discovered that the use of primers having high melting temperatures and/or compositions which include one or more enhancers improves the uniformity of coverage and/or reduces GC bias during one or more downstream sequencing operations.
  • Applicant has also unexpectedly discovered that uniformity of coverage may be improved by optimizing temperature and time/temporal parameters during capture extension.
  • a first aspect of the present disclosure is a composition comprising a polymerase, one or more primers, unmodified dNTPs, and at least one enhancer.
  • the at least one enhancer is selected from betaine, dimethyl sulfoxide (DMSO), a disaccharide, and a single stranded DNA binding protein (SSB).
  • DMSO dimethyl sulfoxide
  • SSB single stranded DNA binding protein
  • the at least one enhancer is betaine.
  • a concentration of betaine in the composition ranges from between about 0.2 mM to about 0.8 mM. In some embodiments, a concentration of betaine in the composition ranges from between about 0.3 mM to about 0.6 mM. In some embodiments, a concentration of betaine in the composition is about 0.5mM.
  • the at least one enhancer is DMSO. In some embodiments, an amount of DMSO in the composition ranges from between about 1% (v/v) to about 10% (v/v). In some embodiments, an amount of DMSO in the composition ranges from between about 2% (v/v) to about 9% (v/v).
  • the one or more primers have a melting temperature (Tm) ranging from between about 57°C to about 95°C. In some embodiments, the one or more primers have a Tm ranging from between about 57°C to about 85°C. In some embodiments, the one or more primers have a Tm ranging from between about 57°C to about 75°C. In some embodiments, the one or more primers have a Tm ranging from between about 57°C to about 72°C. In some embodiments, the one or more primers have a Tm ranging from between about 57°C to about 69°C. In some embodiments, the one or more primers have a Tm ranging from between about 57°C to about 66°C.
  • Tm melting temperature
  • the one or more primers have a Tm ranging from between 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.).
  • high melting temperature and/or high GC content primers e.g., pre-amplification primers, capture primers, release primers, amplification primers, etc.
  • the one or more primers comprise one or more modified dNTPs.
  • the one or more modified dNTPs are selected from modified dGTPs and modified dATPs. In some embodiments, further comprising one or more modified dNTPs.
  • a concentration of the one or more modified dNTPs in the composition is about the same as a concentration of the unmodified dNTPs in the composition. In some embodiments, a concentration of the one or more modified dNTPs in the composition ranges from about 0.1 mM to about 0.5 mM. In some embodiments, a concentration of the one or modified dNTPs in the composition ranges from about 0.2 mM to about 0.4 mM.
  • the one or more modified dNTPs are modified dGTPs. In some embodiments, the modified dGTPs comprise 7-Deaza-2'-deoxyguanosine-5'- Triphosphates. In some embodiments, the one or more modified dNTPs are modified dATPs. In some embodiments, the modified dATPs comprise 2-Amino- 2'deoxyadenosine-5'-Triphosphates.
  • the composition further comprises a divalent cation.
  • the divalent cation is selected from the group consisting of Co 2+ , Mn 2+ , Mg 2+ , Cd 2+ , and Ca 2+ .
  • the composition further comprises one or more nucleic acid molecules.
  • the one or more nucleic acid molecules comprise a library of nucleic acid molecules, where each nucleic acid molecule in the library of nucleic acid molecules comprises first and second adapters.
  • the one or more primers comprise one or more capture primers, and wherein the one or more capture primers are capable of hybridizing to target nucleic acid sequences within the library of nucleic acid molecules.
  • a second aspect of the present disclosure is a composition
  • a composition comprising a polymerase, one or more primers, dNTPs, and optionally at least one enhancer, wherein the one or more primers include no limitation on the percent of guanine or cytosine bases, and wherein at least one primer of the one or more primers has a melting temperature greater than 63°C.
  • the at least one primer has a melting temperature greater than about 69°C.
  • the at least one primer has a melting temperature greater than about 75°C.
  • the at least one primer has a melting temperature greater than about 85°C.
  • the at least one primer has a melting temperature greater than about 95°C.
  • 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.).
  • 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 wherein a concentration of the betaine in the composition ranges from between about 0.2 mM to about 0.8 mM.
  • the dNTPs comprise a mixture of unmodified dNTPs and modified dNTPs.
  • the one or more modified dNTPs comprise 7-Deaza-2'-deoxyguanosine-5'-Triphosphate.
  • the one or more modified dNTPs comprise 2-Amino-2'deoxyadenosine-5'-Triphosphate.
  • a concentration of the one or more modified dNTPs in the composition ranges from between about 0.2 mM to about 0.8 mM.
  • the one or more primers comprise one or more modified dNTPs.
  • 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.
  • the composition further comprises a divalent cation.
  • the divalent cation is selected from the group consisting of Co2+, Mn2+, Mg2+, Cd2+, and Ca2+.
  • the composition further comprises one or more buffers.
  • the composition further comprises one or more polyols.
  • 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.
  • the one or more nucleic acid molecules comprise first and second adapters.
  • the one or more primers comprise one or more capture primers, and wherein the one or more capture primers are capable of hybridizing to target nucleic acid sequences of the nucleic acid molecules.
  • the one or more nucleic acid molecules comprise DNA.
  • a third aspect of the present disclosure is reaction tube (or container, vial, reaction chamber, etc.) comprising the composition of any one of the first and second aspects of the present disclosure described above or as disclosed herein.
  • a fourth aspect of the present disclosure is a use of a composition, such as any of the compositions noted in the first and second aspects of the disclosure described above or as further described herein, in the amplification of one or more nucleic acid molecules.
  • a fifth aspect of the present disclosure is a composition comprising one or more primers, input nucleic acid molecules, and at least one enhancer selected from the group consisting of betaine, DMSO, a disaccharide, and a single stranded DNA binding protein (SSB).
  • SSB single stranded DNA binding protein
  • the at least one enhancer is betaine. In some embodiments, a concentration of betaine in the composition ranges from between about 0.2 mM to about 0.8 mM. In some embodiments, a concentration of betaine in the composition ranges from between about 0.3 mM to about 0.6 mM. In some embodiments, the at least one enhancer is DMSO.
  • an amount of DMSO in the composition ranges from between about 1% (v/v) to about 10% (v/v). In some embodiments, an amount of DMSO in the composition ranges from between about 2% (v/v) to about 8% (v/v).
  • the one or more primers have a Tm ranging from between about 57°C to about 95°C. In some embodiments, the one or more primers have a Tm ranging from between about 57°C to about 85°C. In some embodiments, the one or more primers have a Tm ranging from between about 57°C to about 75°C. In some embodiments, the one or more primers have a Tm ranging from between about 57°C to about 72°C. In some embodiments, the one or more primers have a Tm ranging from between about 57°C to about 69°C. In some embodiments, the one or more primers have a Tm ranging from between 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.).
  • the one or more primers comprise one or more modified dNTPs.
  • the one or more modified dNTPs are selected from the group consisting of modified dGTPs and modified dATPs.
  • the composition further comprises a divalent cation.
  • the divalent cation is selected from the group consisting of Co 2+ , Mn 2+ , Mg 2+ , Cd 2+ , and Ca 2+ .
  • the composition further comprises one or more buffers.
  • 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.
  • a sixth aspect of the present disclosure is a composition comprising input nucleic acid molecules, one or more primers, and optionally at least one enhancer, wherein the one or more primers include no limitation on the percent of guanine or cytosine bases, and wherein at least one primer of the one or more primers has a melting temperature greater than 63 °C. In some embodiments, the at least one primer has a melting temperature greater than about 69°C. In some embodiments, the at least one primer has a melting temperature greater than about 75°C. In some embodiments, the at least one primer has a melting temperature greater than about 85°C. In some embodiments, the 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.).
  • the one or more primers include no limitation on the percent of guan
  • rein the one or more primers comprise one or more modified dNTPs.
  • the one or more modified dNTPs are selected from 7-Deaza-2'-deoxyguanosine-5'-Triphosphate and 2-Amino-2'deoxyadenosine-5'- Triphosphate.
  • the at least one optional enhancer is selected from betaine, DMSO, a disaccharide, and a single stranded DNA binding protein (SSB).
  • the at least one optional enhancer is betaine, and wherein a concentration of the betaine in the composition ranges from between about 0.2 mM to about 0.8 mM.
  • the composition further comprises a divalent cation.
  • the divalent cation is selected from the group consisting of Co2+, Mn2+, Mg2+, Cd2+, and Ca2+.
  • the composition further comprises one or more buffers.
  • the input nucleic acid molecules comprise a prepared nucleic acid library. In some embodiments, the input nucleic acid molecules comprise captured DNA. 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.
  • reaction tube or container, vial, reaction chamber, etc.
  • composition of any of the fifth or sixth aspects of the disclosure as noted above or as further described herein.
  • An eighth aspect of the present disclosure is a use of the composition of any one of the fifth or sixth aspects of the present disclosure noted above or as described herein for the preferential enrichment of one or more target nucleic acid molecules in a library of nucleic acid molecules.
  • 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.
  • the dNTPs comprise a mixture of unmodified and modified dNTPs.
  • the modified dNTPs include modified dGTPs and modified dATPs.
  • the sets of capture and release primers include one or more modified dNTPs.
  • the kit further comprises instructions to prepare a capture extension master mix, wherein the prepared capture extension master mix includes a concentration of betaine of about 0.5mM.
  • 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 sets of capture and release primers have no limits on melting temperatures and/or GC content. In some embodiments, both sets of capture and release primers have no limits on melting temperatures and/or GC content.
  • 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 of the set of capture primers has a melting temperature greater than about 65°C; and wherein at least one primer of the set of release primers has a melting temperature greater than about 65°C.
  • the at least one primer of the set of capture primers has a melting temperature greater than about 72°C; and wherein the at least one primer of the set of release primers has a melting temperature greater than about 72°C.
  • 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.).
  • the at least one primer of the set of capture primers comprises one or more modified dNTPs; and wherein the at least one primer of the set of release primers comprises one or more modified dNTPs.
  • 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.
  • An eleventh aspect of the present disclosure is 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 the target nucleic acid molecule in a library of nucleic acid molecules, where 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 producing the 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.
  • the capture primer hybridized to the target nucleic acid molecule is extended in a composition comprising at least one enhancer selected from the group consisting of betaine or a derivative or analog thereof, DMSO (or a solvent having similar chemical and/or physical properties, such as DMF), a single stranded DNA binding protein (such as a thermostable single-stranded DNA-binding protein, and a disaccharide (such as trehalose).
  • the at least one enhancer is betaine, and wherein the concentration of betaine ranges from between about 0.2 mM to about 0.8 mM.
  • the at least one enhancer is betaine, and wherein the concentration of betaine ranges from between about 0.3 mM to about 0.7 mM. In some embodiments, the at least one enhancer is betaine, and wherein the concentration of betaine ranges from between about 0.4 mM to about 0.6 mM.
  • the capture primer hybridized to the target nucleic acid molecule is extended in a composition comprising one or more modified dNTPs.
  • the one or more modified dNTPs are modified dGTPs.
  • the modified dGTPs comprise 7-Deaza-2'-deoxyguanosine-5'- Triphosphates.
  • the one or more modified dNTPs are modified dATPs.
  • the modified dATPs comprise 2-Amino- 2'deoxyadenosine-5'-Triphosphates.
  • the capture primer hybridized to the target nucleic acid molecule is extended in a composition comprising betaine and one or more modified dNTPs.
  • a concentration of betaine in the composition is about 0.5 mM.
  • the method further comprises capturing the capture primer extension complex.
  • the method further comprises hybridizing a release primer to the target nucleic acid.
  • the release primer is hybridized to the target nucleic acid in a composition comprising at least one enhancer.
  • the at least one enhancer is betaine.
  • the release primer is hybridized to the target nucleic acid in a composition comprising one or more dNTPs.
  • the release primer has a melting temperature greater than 63 °C. In some embodiments, the melting temperature greater than 65°C. In some embodiments, the melting temperature greater than 72°C.
  • the method further comprises extending the release primer hybridized to the target nucleic acid with a second polymerase.
  • a twelfth aspect of the present disclosure is a method of amplifying one or more nucleic acid molecules comprising: (i) obtaining a plurality of nucleic acid molecules; and (ii) performing a first amplification reaction in a first composition comprising the obtained plurality of nucleic acid molecules and a first set of primers, wherein at least one primer of the first set of primers has a melting temperature greater than 63°C.
  • each nucleic acid molecule of the obtained plurality of nucleic acid molecules comprises first and second adapters.
  • the first composition further comprises betaine. In some embodiments, wherein a concentration of betaine in the first composition is about 0.5 mM.
  • a thirteenth aspect of the present disclosure is a method of amplifying one or more nucleic acid molecules comprising: (i) obtaining a plurality of nucleic acid molecules; (ii) 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.
  • a concentration of betaine in the first composition is about 0.5 mM.
  • at least one primer of the first set of primers has a high melting temperature and/or a high GC content.
  • each nucleic acid molecule of the obtained plurality of nucleic acid molecules comprises first and second adapters, and wherein the method further comprises enriching the obtained plurality of nucleic acid molecules for one or more target nucleic acid molecules.
  • the method further comprises performing a second amplification reaction in a second composition comprising the one or more target nucleic acid molecules, betaine, and a mixture of unmodified dNTPs and modified dNTPs.
  • the obtained plurality of nucleic acid molecules is a target enriched library comprising a plurality of target nucleic acid molecules.
  • a fourteenth aspect of the present disclosure is a kit for the enrichment of at least one target nucleic acid in a library of nucleic acid molecules, the kit comprising: a first oligonucleotide complementary to a target nucleic acid in library of nucleic acid molecules, each of the nucleic acid molecules in the library of nucleic acid molecules having a first end comprising a first adapter and a second end comprising a second adapter; a second oligonucleotide complementary to the target nucleic acid; a first amplification primer; and a second amplification primer; and where at least one of the first oligonucleotide or the second oligonucleotide has a high melting temperature and/or a high GC content.
  • both the first and second oligonucleotides have a high melting temperature and/or a high GC content.
  • a seventeenth aspect of the present disclosure is a composition consisting essentially of a polymerase, one or more primers having no limits on melting temperature and/or GC content, a mixture of unmodified dNTPs and modified dNTPs, and at least one enhancer.
  • the at least one enhancer is betaine.
  • the composition further comprises one or more nucleic acid molecules.
  • An eighteenth aspect of the present disclosure is a composition consisting of a polymerase, one or more primers having no limits on melting temperature and/or GC content, a mixture of unmodified dNTPs and modified dNTPs, and at least one enhancer.
  • the at least one enhancer is betaine.
  • the composition further comprises one or more nucleic acid molecules.
  • a nineteenth aspect of the present disclosure is a reaction tube comprising the composition of any one of the sixteenth, seventeenth, or eighteenth aspects of the present disclosure as noted above or as described herein.
  • a twentieth aspect of the present disclosure is a use of the composition of any one of the sixteenth, seventeenth, or eighteenth aspects of the present disclosure as noted above or as described herein in the amplification of one or more nucleic acid molecules, such as a library of nucleic acids or captured nucleic acid molecules.
  • a twenty-first aspect of the present disclosure is a method of amplifying one or more nucleic acid molecules, wherein the amplification is performed in the presence of any one of the compositions of the sixteenth, seventeenth, or eighteenth aspects of the present disclosure as noted above or as described herein.
  • a step of thermocycling is performed for a time period ranging from between about 25 minutes to about 35 minutes, such as for about 34 minutes.
  • FIG. 1 provides a flow chart illustrating an embodiment of a method for enrichment of at least one target nucleic acid molecule in a library of nucleic acid molecules according to the present disclosure. The steps illustrated may be performed in the presence of any of the compositions described herein.
  • FIG. 2A depicts that primer availability in target areas with high %GC is limited when primer %GC is restricted to between 20% and 80%, and primer Tm is restricted to between 57°C and 63°C. Primer coverage over primary targets is incomplete with these restrictions to primer %GC and primer Tm.
  • FIG. 2B illustrates that by removing limitations on primer %GC content and primer Tm increases primer availability in target areas having high %GC content and improves primer coverage in these areas.
  • FIG. 2C shows that increasing the maximum primer Tm permitted during primer design decreases the number of target bases not covered by primers.
  • FIG. 3A illustrates samples enriched with primer test panels designed with primer Tm ranging from between about 57°C to about 63°C (Current Primer Database), from between about 57°C to about 66°C (Max Tm 66), from between about 57°C to about 69°C (Max Tm 69), from between about 57°C to about 62°C (Max Tm 72°C), and from about 57°C to "no Tm limit" (No Max Tm).
  • FIG. 3B illustrates samples enriched with primer test panels designed with primer Tm from between about 57°C to about 63°C (Current Primer Database), from between about 57°C to about 66°C (Max Tm 66), from between about 57°C to about 69°C (Max Tm 69), from between about 57°C to about 62°C (Max Tm 72°C), and from about 57°C to no limit (No Max Tm).
  • Normalized sequencing coverage in high %GC targets (65%, 75%, and 77%) is most improved in samples enriched with the No Max Tm test panel compared to samples enriched with the Current Primer Database
  • FIG. 4 depicts samples prepared into libraries with and without Betaine (PCR1 + B, PCR1 - B) and 7-deaza dGTP (PCR1 + 7, PCR1 - 7). Libraries were then used as input into HyperPETE with and without Betaine in capture extension (CapExt + B, CapExt - B). Yields for PCR1 + B with PCR1 -7 pre-capture input libraries were higher than PCR1 - B with PCR1 -7 pre-capture input libraries. Yields for PCR1 + 7 pre-capture input libraries were comparatively lower
  • FIG. 6B shows samples prepared into libraries with and without 7-deaza dGTP (PCR1 + 7-deaza dGTP, PCR1 - 7-deaza dGTP). Libraries were then used as input into HyperPETE with and without Betaine in capture extension (Capture Extension + Betaine, Capture Extension - Betaine). For PCR1 - 7-deaza dGTP samples, normalized coverage across three high %GC targets is more uniform for Capture Extension + Betaine samples than for Capture Extension - Betaine samples.
  • FIG. 7 illustrates samples prepared into libraries with and without Betaine (PCR1 + Betaine, PCR1 - Betaine) and 7-deaza dGTP (PCR1 + 7-deaza dGTP, PCR1 - 7- deaza dGTP).
  • Libraries were then used as input into HyperPETE with and without Betaine in capture extension (Capture Extension + Betaine, Capture Extension - Betaine).
  • PCR1 + 7-deaza dGTP samples had the highest normalized coverage for high %GC targets (75%, 80%) followed by PCR1 - 7-deaza dGTP + Capture Extension + Betaine samples.
  • PCR1 + Betaine samples had higher coverage in low %GC (30%) targets compared to all other sample groups.
  • FIG. 12 illustrates normalized position deduped coverage across high GC percentage regions of interest.
  • FIG. 13 illustrates normalized position deduped coverage by regions of interest.
  • FIG. 14 sets forth sequencing quality control metrics.
  • FIG. 15 illustrates normalized position deduped coverage across high GC percentage regions of interest.
  • FIG. 16 illustrates normalized position deduped coverage by regions of interest.
  • FIG. 17 provides sequencing quality control metrics for DMSO titration with a 1.7Mb panel.
  • FIG. 18 illustrates DMSO titration with a 1.7Mb panel, showing normalized position deduped coverage and high GC percentage.
  • oligonucleotide refers to an oligomer of nucleotide or nucleoside monomer units wherein the oligomer optionally includes non-nucleotide monomer units, and/or other chemical groups attached at internal and/or external positions of the oligomer.
  • the oligomer can be natural or synthetic and can include naturally-occurring oligonucleotides, or oligomers that include nucleosides with non-naturally-occurring (or modified) bases, sugar moieties, phosphodiester-analog linkages, and/or alternative monomer unit chiralities and isomeric structures (e.g., 5'- to 2'-linkage, L-nucleosides, a-anomer nucleosides, P-anomer nucleosides, locked nucleic acids (LNA), peptide nucleic acids (PNA)).
  • an oligonucleotide may be 10 to 20, 11 to 30, 31 to 40, 41 to 50, 51-60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200 nucleotides in length.
  • DNA polymerase refers to an enzyme that performs template- directed synthesis of polynucleotides.
  • a DNA polymerase can add free nucleotides only to the 3' end of the newly forming strand. This results in elongation of the newly forming strand in a 5'-3' direction. No known DNA polymerase is able to begin a new chain (de novo).
  • DNA polymerase can add a nucleotide only on to a pre-existing 3 '-OH group, and, therefore, needs a primer at which it can add the first nucleotide.
  • unmodified dNTP or "unmodified nucleoside triphosphate” refer to the four deoxyribonucleotide triphosphates dATP (deoxyadenosine triphosphate), dCTP (deoxycytidine triphosphate), dGTP (deoxyguanosine triphosphate) and dTTP (deoxythymidine triphosphate) that are normally used as building blocks in the synthesis of DNA. s
  • sequence when used in reference to a nucleic acid molecule, refers to the order of nucleotides (or bases) in the nucleic acid molecules. In cases, where different species of nucleotides are present in the nucleic acid molecule, the sequence includes an identification of the species of nucleotide (or base) at respective positions in the nucleic acid molecule. A sequence is a property of all or part of a nucleic acid molecule. The term can be used similarly to describe the order and positional identity of monomeric units in other polymers such as amino acid monomeric units of protein polymers.
  • sequencing refers to the determination of the order and position of bases in a nucleic acid molecule. More particularly, the term “sequencing” refers to biochemical methods for determining the order of the nucleotide bases, adenine, guanine, cytosine, and thymine, in a DNA oligonucleotide. Sequencing, as the term is used herein, can include without limitation parallel sequencing or any other sequencing method known of those skilled in the art, for example, chain-termination methods, rapid DNA sequencing methods, wandering-spot analysis, Maxam-Gilbert sequencing, dye- terminator sequencing, or using any other modern automated DNA sequencing instruments.
  • target or target sequence refer to nucleic acid molecule sequences of interest, e.g., those which hybridize to oligonucleotide probes.
  • the term “universal primer” refers to a primer that can hybridize to and support amplification of target polynucleotides having a shared complementary universal primer binding site. Similar, the term “universal primer pair” refers to a forward and reverse primer pair that can hybridize to and support PCR amplification of target polynucleotides having shared complementary forward and reverse universal primer binding sites. Such universal primer(s) and universal primer binding site(s) can allow single or double primer mediated universal amplification (e.g., universal PCR) of target polynucleotide regions of interest.
  • the headings provided herein are for convenience only and do not interpret the scope or meaning of the disclosed embodiments.
  • compositions that include one or more components which facilitate improving uniformity of coverage and/or reducing GC bias during one or more downstream sequencing operations.
  • the compositions described herein are useful in the preparation of target enriched samples and/or the amplification of such target enriched samples.
  • Methods of preparing target enriched samples and/or the amplification of target enriched samples are disclosed in U.S. Patent Publication Nos. 2018/0016630 and 2020/0032244 and in International Application Nos. PCT/US2017/041748 and PCT/EP2018/08527, the disclosures of which are hereby incorporated herein in their entireties.
  • compositions of the present disclosure may 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 subsequently during post-capture amplification of the prepared target enriched sample.
  • the present disclosure is also directed to methods of preparing target enriched samples and methods of amplifying one or more targets in the prepared target enriched samples.
  • the steps of pre-capture, capture extension, release primer hybridization and extension, and/or post-capture amplification are conducted in the presence of one or more components which improve uniformity of coverage and/or reduce GC bias during one or more downstream sequencing operations.
  • the one or more components which increase uniformity of coverage and/or reduce GC bias are dNTP mixtures which include unmodified dNTPs and one or more modified dNTPs; or primers which include one or more modified dNTPS.
  • dNTP mixtures which include unmodified dNTPs and one or more modified dNTPs; or primers which include one or more modified dNTPS.
  • primers which include one or more modified dNTPS.
  • any combination of any of these components may be used during any hybridization and/or extension reaction, as described in further detail herein.
  • the present disclosure also provides for methods of improving uniformity of coverage and/or reducing GC bias during sequencing by optimizing certain parameters of certain incubation steps utilized during the preparation of target enriched samples. For instance, the present disclosure provides methods of incubating samples at higher temperatures and/or for longer durations as compared with control incubation programs. These and other embodiments are described further herein.
  • compositions for use in hybridization and/or extension reactions where the compositions comprise one or more components which facilitate improving a uniformity of coverage and/or reducing GC bias during sequencing.
  • the one or more components which facilitate improving the uniformity of coverage during sequencing include (i) primers which have high GC content and/or high melting temperatures; (ii) primers which include one or modified dNTPs; (iii) enhancers; and/or (iv) dNTP mixtures which include unmodified and modified dNTPs.
  • enhancers include, but are not limited to, betaine, DMSO, single stranded DNA binding proteins, disaccharides (e.g., trehalose), and combinations thereof. Each of these components are described herein.
  • the compositions of the present disclosure include both (a) primers which have high GC content and/or high melting temperatures; and (b) one or more enhancers. In other embodiments, the compositions of the present disclosure include both (a) primers which have high GC content and/or high melting temperatures; and (b) primers which include one or more modified dNTPs. In yet other embodiments, the compositions of the present disclosure include both (a) primers which include one or modified dNTPs; and (b) one or more enhancers. In further embodiments, the compositions of the present disclosure include both (a) one or more enhancers; and (b) mixtures of modified and unmodified dNTPs.
  • compositions of the present disclosure include (a) primers which have high GC content and/or high melting temperatures; (b) primers which include one or modified dNTPs; and (c) one or more enhancers.
  • compositions of the present disclosure include (a) primers which have high GC content and/or high melting temperatures; (b) primers which include one or modified dNTPs; and (c) mixtures of unmodified dNTPs and modified dNTPs.
  • the compositions include one or more primers; one or more polymerases; deoxynucleoside triphosphates (dNTPs) (e.g., unmodified dNTPs or mixtures of unmodified and modified dNTPs); and at least one enhancer.
  • the compositions include one or more primers, where at least one primer of the one or more primers has a high melting temperature (or a high GC content) and/or includes one or more modified dNTPs; one or more polymerases; dNTPs (e.g., unmodified dNTPs or mixtures of unmodified and modified dNTPs); and optionally at least one enhancer.
  • the compositions further include 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.
  • Primers including any of the pre-capture primers (e.g., pre-capture forward and precapture reverse primers), capture primers, release primers, and/or post-capture amplification primers described herein, may be synthesized to include at least one modified dNTP, including any of the modified dNTPs described herein.
  • primers including 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, may be synthesized to include a high GC content and/or to have a high melting temperature.
  • primers including any of the precapture primers (e.g., pre-capture forward and reverse primers), capture primers, release primers, and/or post-capture amplification primers described herein, may be synthesized to include (i) a high GC content and/or to have a high melting temperature; and (ii) one or more modified dNTPs.
  • compositions of the present disclosure include one or more primers, wherein at least one primer of the one or more primers has a high GC content and/or a high melting temperature.
  • high GC content it is meant that the at least one primer of the one or more primers has a GC content of greater than about 70%.
  • primers included within any set of primers may have melting temperatures ranging from between about 57°C to about 63 °C, from between about 57°C to about 64°C, from between about 57°C to about 65°C, from between about
  • 57°C to about 70°C from between about 57°C to about 71 °C, from between about 57°C to about 72°C, from between about 57°C to about 73°C, from between about
  • At least one primer in the set of primers having any of the aforementioned temperature ranges has a melting temperature greater than 63°C.
  • any of the primers within the set of primers may include one or more modified dNTPs, e.g. one or more modified dGTPs or dATPs.
  • up to about 1% of the primers within any set of primers have a high GC content and/or a high melting temperature. In some embodiments, up to about 2% of the primers within any set of primers have a high GC content and/or a high melting temperature. In some embodiments, up to about 3% of the primers within any set of primers have a high GC content and/or a high melting temperature. In some embodiments, up to about 4% of the primers within any set of primers have a high GC content and/or a high melting temperature. In some embodiments, up to about 5% of the primers within any set of primers have a high GC content and/or a high melting temperature.
  • up to about 6% of the primers within any set of primers have a high GC content and/or a high melting temperature. In some embodiments, up to about 7% of the primers within any set of primers have a high GC content and/or a high melting temperature. In some embodiments, up to about 8% of the primers within any set of primers have a high GC content and/or a high melting temperature. In some embodiments, up to about 9% of the primers within any set of primers have a high GC content and/or a high melting temperature. In some embodiments, up to about 10% of the primers within any set of primers have a high GC content and/or a high melting temperature.
  • two or more modified dNTPs may be used in any of the primers within any of the compositions described herein.
  • the primers may include two or more modified dNTPs that are the same.
  • the primers may include two or more modified dNTPs that are different.
  • forward and reverse primers may include the same or different dNTP analogs.
  • up to about 5% of the nucleoside triphosphates included within any primer are modified dNTPs.
  • up to about 10% of the nucleoside triphosphates included within any primer are modified dNTPs.
  • the primers are target-specific primers that have a sequence that is complementary to a sequence of a target nucleic acid.
  • a target-specific primer is a gene-specific primer designed to hybridize to or nearby (e.g., upstream of, or 5' to) a gene (e.g., cDNA, genomic DNA) of interest.
  • the target nucleic acid can be RNA, DNA, or a combination thereof.
  • compositions of the present disclosure include one or more dNTPs, including unmodified dNTPs and/or modified dNTPs.
  • dNTPs are selected from the group consisting of unmodified dNTPs (dCTP, dATP, dGTP, dTTP, and dUTP).
  • unmodified dNTPs dCTP, dATP, dGTP, dTTP, and dUTP.
  • one or more modified dNTPs are used instead of or in addition to unmodified dNTPs.
  • a composition may comprise between about 0.1 mM and about 0.7 mM of unmodified dNTPs; and may further comprise one or more modified dNTPs. In other embodiments, a composition may comprise between about 0.1 mM and about 0.6mM of unmodified dNTPs; and may further comprise one or more modified dNTPs. In yet other embodiments, a composition may comprise between about 0.2 mM and about 0.5 mM of unmodified dNTPs; and may further comprise one or more modified dNTPs.
  • a composition may comprise between about 0.1 mM and about 0.7 mM of unmodified dNTPs; and may further comprise between about 0.1 mM to about 0.7 mM of one or more modified dNTPs. In other embodiments, a composition may comprise between about 0.1 mM and about 0.6 mM of unmodified dNTPs; and may further comprise between about 0.1 mM to about 0.6 mM of one or more modified dNTPs. In yet other embodiments, a composition may comprise between about 0.2 mM and about 0.5 mM of unmodified dNTPs; and may further comprise between about 0.2 mM to about 0.5 mM of one or more modified dNTPs.
  • a composition may comprise between about 0.2 mM and about 0.4 mM of unmodified dNTPs; and may further comprise between about 0.2 mM to about 0.4 mM of one or more modified dNTPs. In even further embodiments, a composition may comprise about 0.3 mM of unmodified dNTPs; and may further comprise between about 0.2 mM to about 0.5 mM of one or more modified dNTPs. In even further embodiments, a composition may comprise about 0.3 mM of unmodified dNTPs; and may further comprise about 0.3 mM of one or more modified dNTPs.
  • the enhancer is betaine and a concentration of betaine in any composition ranges from between about 0.4 mM to about 0.6 mM. In some embodiments, the enhancer is betaine and a concentration of betaine in any composition is at least about 0.1 mM, at least about 0.2mM, 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.
  • the enhancer is DMSO and the DMSO may be present in any composition in an amount 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 up to about 5% (v/v). In some embodiments, the enhancer is DMSO and the DMSO may be present in any composition in an amount up to about 4% (v/v). In some embodiments, the enhancer is DMSO and the DMSO may be present in any composition in an amount up to about 3% (v/v). In some embodiments, the enhancer is DMSO and the DMSO may be present in any composition in an amount up to about 2% (v/v). In some embodiments, the enhancer is DMSO and the DMSO may be present in any composition in an amount up to about 1 (v/v).
  • the enhancer is DMSO and an amount of DSMO in any composition ranges from between about 0.1% (v/v) to about 10% (v/v) of the composition. In other embodiments, the enhancer is DMSO and an amount of DSMO in any composition ranges from between about 0.5% (v/v) to about 10% (v/v) of the composition. In other embodiments, the enhancer is DMSO and an amount of DSMO in any composition ranges from between about 1% (v/v) to about 10% (v/v) of the composition. In other embodiments, the enhancer is DMSO and an amount of DSMO in any composition ranges from between about 2% (v/v) to about 8% (v/v) of the composition. In other embodiments, the enhancer is DMSO and an amount of DSMO in any composition ranges from between about 3% (v/v) to about 7% (v/v) of the composition.
  • the single-stranded DNA-binding protein is a thermostable binding protein, such as some derived from a hyperthermophilic microorganism (e.g., ET SSB, available from New England BioLabs, Inc.).
  • the single-stranded DNA-binding protein is derived from Escherichia coli, Drosophila or Xenopus, a T4 bacteriophage-derived 32, 41, 44, 45 or 61 gene protein or an RPA protein in a eukaryote.
  • a non-limiting example of a single-stranded DNA binding protein is the gene 2.5 protein of bacteriophage T7 and equivalent such T7- type phage proteins or other proteins.
  • the compositions of the present disclosure includes a disaccharide, such as trehalose.
  • the disaccharide is present at a concentration ranging from between about 0M to about IM. In some embodiments, the disaccharide is present at a concentration ranging from between about 0.1M to about IM. In some embodiments, the disaccharide is present at a concentration ranging from between about 0.1M to about 0.8M. In some embodiments, the disaccharide is present at a concentration ranging from between about 0.2M to about 0.8M.
  • a cumulative time period for conducting the denaturing, annealing and extension portions of step 106 of method 100 may 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.
  • a cumulative time period for conducting the annealing and extension portions of incubation ranges from between about 20 minutes to about 25 minutes.
  • a cumulative time period for conducting the annealing and extension portions of incubation is about 23 minutes.
  • a cumulative time period for conducting the annealing and extension portions of incubation from between about 30 minutes to about 34 minutes.
  • a cumulative time period for conducting the annealing and extension portions of incubation is about 32 minutes.
  • up to about 4% of the release primers of any set of release primers are high melting temperature and/or high GC content release primers. In some embodiments, up to about 3% of the release primers of any set of release primers are high melting temperature and/or high GC content release primers. In some embodiments, up to about 2% of the release primers of any set of release primers are high melting temperature and/or high GC content release primers. In some embodiments, up to about 1% of the release primers of any set of release primers are high melting temperature and/or high GC content release primers.
  • step 112 may be conducted 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).
  • enhancer selected from betaine (or a derivative or analog thereof), DMSO, a disaccharide, and a single stranded DNA binding protein (SSB).
  • the at least one enhancer is betaine
  • a concentration of betaine in any release primer hybridization master mix used during the performance of step 112 includes a concentration 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.
  • the combined release primer hybridization and extension may take place in the presence of one or more enhancers, e.g., betaine, DMSO, single stranded DN binding proteins, di saccharides, or any combination thereof.
  • enhancers e.g., betaine, DMSO, single stranded DN binding proteins, di saccharides, or any combination thereof.
  • combined release primer hybridization and extension may be performed utilizing a composition which includes betaine (or a derivative or analog thereof), such as a composition including betaine at a concentration ranging from between about 0.1 mM to about 1 mM (e.g., a concentration of about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, etc.).
  • betaine or a derivative or analog thereof
  • combined release primer hybridization and extension may be performed in a composition which includes a mixture of unmodified dNTPs and modified dNTPs.
  • a ratio of unmodified dNTPs to modified dNTPs in any composition ranges from between about 2: 1 to about 1 :2. In some embodiments, the ratio of unmodified dNTPs to modified dNTPs is about 1 : 1.
  • the one or more modified dNTPs include modified dGTPs and/or modified dATPs.
  • the one or more modified dNTPs are 7-Deaza-2'-deoxyguanosine-5'-Triphosphates and/or 2-Amino- 2'deoxyadenosine-5'-Triphosphates. In some embodiments, a concentration of 7- Deaza-2'-deoxyguanosine-5'-Triphosphates and/or 2-Amino-2'deoxyadenosine-5'- Triphosphates is about the same as the concentration of unmodified dNTPs in the composition.
  • combined release primer hybridization and extension takes place in a composition which includes one or more capture extension that have a high GC content and/or a high melting temperature; and where the composition further includes at least one enhancer.
  • the enhancer is betaine (or a derivative or analog thereof) and a concentration of betaine in the composition ranges from between about 0.2 mM to about 0.8 mM. In other embodiments, the enhancer is betaine (or a derivative or analog thereof) and a concentration of betaine in the composition ranges from between about 0.3 mM to about 0.7 mM.
  • the enhancer is betaine (or a derivative or analog thereof) and a concentration of betaine in the composition ranges from between about 0.4 mM to about 0.6 mM. In further embodiments, the enhancer is betaine (or a derivative or analog thereof) and a concentration of betaine in the composition is about 0.5 mM.
  • combined release primer hybridization and extension may take place in the presence of a polymerase, betaine, and a mixture of unmodified and modified dNTPs.
  • the betaine is present at a concentration ranging from about 0.4 mM to about 0.6mM; and where a ratio of the unmodified dNTPs to modified dNTPs is about 1 : 1.
  • the modified dNTPs are selected from 7-Deaza-2'-deoxyguanosine-5'-Triphosphates or 2-Amino- 2'deoxyadenosine-5'-Triphosphates.
  • combined release primer hybridization and extension may take place 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.
  • the betaine is present at a concentration ranging from about 0.4 mM to about 0.6mM; and where a ratio of the unmodified dNTPs to modified dNTPs is about 1 : 1.
  • step 114 can further include termination of the primer extension reaction in order to control the length of the extended-release primer.
  • the length of the extended-release primer product can be controlled actively through techniques such as inactivating the polymerase added in the step 114, or passively by enabling the reaction to go to completion such as through the consumption of limiting reactants or by controlling/selecting the size of the fragments of the nucleic acid molecules in the library of nucleic acid molecules.
  • the method 100 further includes a step 116 of post-capture amplification, involving linear or exponential amplification (e.g., PCR).
  • the step 116 includes amplifying the target nucleic acid with a third polymerase, a first amplification primer, and a second amplification primer.
  • 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 the step 102.
  • the first amplification primer can have a 3' end complementary to the first adapter and the second amplification primer can have a 3' end complementary to the second adapter.
  • the primers for amplification can include any sequences that are present within the target nucleic acid being amplified (e.g., gene/target specific primers, universal primers, or the like) and can support synthesis of one or both strands (i.e., both the top and bottom strands of a double-stranded nucleic acid molecules corresponding to the template of the amplification reaction).
  • post-capture amplification may be conducted in a composition similar to those utilize in precapture amplification, including any of those compositions recited herein.
  • the uracil incompatible polymerase can amplify the target nucleic acid having no uracil nucleotides; however, the uracil incompatible polymerase will be incapable of replicating the uracil-containing extended oligonucleotide primers.
  • uracil-containing products can be selectively digested or otherwise degraded, thereby leaving behind only the original molecules from the library of nucleic acid molecules.
  • the method 100 can include a step 118 of analyzing the amplified target nucleic acid molecules.
  • the step 116 can include any method for determining the nucleic acid sequence of one or more products of the method 100.
  • the step 116 can further include sequences alignment, identification of sequence variations, counting of unique primer extension products, the like, or combinations thereof.
  • Ion Torrent sequencing measures 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, CA) includes the PacBio Sequel Systems.
  • a non-limiting example of a sequencing device available from Roche (Pleasanton, CA) is the Roche 454.
  • Next-generation sequencing methods may also include nanopore sequencing methods.
  • strand sequencing in which the bases of DNA 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 by and pass through the nanopore
  • SBS nanopore sequencing by synthesis
  • Strand sequencing requires a method for slowing down the passage of the DNA through the nanopore and decoding a plurality of bases within the channel; ratcheting approaches, taking advantage of molecular motors, have been developed for this purpose.
  • Exonuclease-based sequencing requires the release of each nucleotide close enough to the pore to guarantee its capture and its transit through the pore at a rate slow enough to obtain a valid ionic current signal.
  • both of these methods rely on distinctions among the four natural bases, two relatively similar purines and two similar pyrimidines.
  • the nanopore SBS approach utilizes synthetic polymer tags attached to the nucleotides that are designed specifically to produce unique and readily distinguishable ionic current blockade signatures for sequence determination.
  • sequencing of nucleic acid molecules comprises via nanopore sequencing comprises preparing nanopore sequencing complexes and determining polynucleotide sequences.
  • Methods of preparing nanopores and nanopore sequencing are described in U.S. Patent Application Publication No. 2017/0268052, and PCT Publication Nos. WO20 14/074727, W02006/028508, WO2012/083249, and WO/2014/074727, the disclosures of which are hereby incorporated by reference herein in their entireties.
  • tagged nucleotides may be used in the determination of the polynucleotide sequences (see, e.g., PCT Publication No.
  • EXAMPLE 1 EFFECT OF VARIOUS PRIMER MAXIMUM MELTING TEMPERATURE (TM) LIMITS ON THE ENRICHMENT OF HIGH %GC TARGETS
  • Panels were designed to three high %GC targets using no limits on %GC, a minimum Tm of 57°C and various max Tm limits (e.g., 66°C, 69°C, 72°C, no limit).
  • Primer extension target enrichment was performed on the ten libraries according to the KAPA HyperPETE Somatic Tissue DNA Workflow vl.O Chapter 5 (the disclosure of which is hereby incorporated by reference herein in its entirety).
  • Primer Extension Target Enrichment (PETE) (described herein) was performed with exceptions listed as follows: During Step 2, Capture Extension Reaction, 5pL of five small custom capture test panels targeting three high %GC ROIs using various max Tm limits, Table 8, was added to four samples per condition. 5pL of Hot Spot Capture Panel was added, but no water was included in the Capture Extension Reaction Master Mix.
  • Step 6 Release Primer Hybridization, 20pL instead of 30pL of IX Wash & Resuspension Buffer and lOpL of Hot Spot Release Panel was added to the Release Primer Hybridization Master Mix. lOpL of five test capture panels were added to corresponding four samples per condition.
  • Primer Extension Target Enrichment libraries were sequenced on aNextSeq 550 and analyzed. Normalized coverage at each position for each sample was calculated by dividing the coverage at a position by the average coverage for the whole sample.
  • FIG. 2A The results for the current primer database are illustrated in FIG. 2A.
  • FIG. 2B the results for the "no Max Tm Panel" are illustrated in FIG. 2B.
  • FIG. 2C depicts primer availability. Increasing the primer max Tm increased the availability of primers in high %GC regions (a) and reduced the number of targeted bases not covered by capture and release primer spans (b).
  • the small custom panel designed with no max primer Tm had the highest normalized coverage in high %GC targets.
  • Primer extension target enrichment was performed on the ten libraries according to the KAPA HyperPETE Somatic Tissue DNA Workflow vl.O Chapter 5 (the disclosure of which is hereby incorporated by reference herein in its entirety).
  • Primer Extension Target Enrichment (PETE) with exceptions listed as follows: During Step 2, Capture Extension Reaction, 5pL of Hot Spot Capture Panel, 5pL of a small custom capture panel targeting three high %GC ROIs was 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 four test capture extension programs detailed in Table 10 or a control capture extension program according to the thermocycler program in Step 2.2.
  • Primer Extension Target Enrichment libraries were sequenced on aNextSeq 550 and analyzed. Normalized coverage at each position for each sample was calculated by dividing the coverage at a position by the average coverage for the whole sample
  • EXAMPLE 4 EFFECT OF 7-DEAZA DGTP, 2-AMINO DATP, DMSO, TREHALOSE, BETAINE, AND SINGLE STRANDED BINDING PROTEIN ON CAPTURE EXTENSION
  • the purpose of this study was to test the effects of adding 7-deaza dGTP and 2-amino dATP to library preparation amplification as well as DMSO, Trehalose, Betaine, and single stranded DNA binding protein to the capture extension reaction on the enrichment of high %GC targets.
  • PETE Primer Extension Target Enrichment
  • 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).
  • DMSO dimethyl sulfoxide
  • SSB single stranded DNA binding protein
  • composition of additional embodiment 3 wherein a concentration of betaine in the composition ranges from between about 0.2 mM to about 0.8 mM.
  • composition of additional embodiment 3 wherein a concentration of betaine in the composition ranges from between about 0.3 mM to about 0.6 mM.
  • composition of additional embodiment 2 wherein the at least one enhancer is DMSO. Additional Embodiments.
  • composition of additional embodiment 7, wherein an amount of DMSO in the composition ranges from between about 1% (v/v) to about 10% (v/v).
  • composition of additional embodiment 7, wherein an amount of DMSO in the composition ranges from between about 2% (v/v) to about 9% (v/v).
  • composition of any one of the preceding additional embodiments, wherein the one or more primers have a Tm ranging from between about 57°C to about 63°C. Additional Embodiment!?.
  • the composition of any one of the preceding additional embodiments, wherein the one or more primers comprise one or more modified dNTPs.
  • composition of additional embodiment 17, wherein the one or more modified dNTPs are selected from the group consisting of modified dGTPs and modified dATPs.
  • composition of additional embodiment 19 wherein a concentration of the one or more modified dNTPs in the composition is about the same as a concentration of the unmodified dNTPs in the composition.
  • composition of additional embodiment 19 wherein a concentration of the one or more modified dNTPs in the composition ranges from about 0.1 mM to about 0.5 mM.
  • composition of additional embodiment 19 wherein a concentration of the one or modified dNTPs in the composition ranges from about 0.2 mM to about 0.4 mM.
  • composition of additional embodiment 23, wherein the modified dGTPs comprise 7-Deaza-2'-deoxyguanosine-5'- Triphosphates.
  • composition of additional embodiment 25 wherein the modified dATPs comprise 2-Amino-2'deoxyadenosine-5'- Triphosphates. Additional Embodiment!?.
  • composition of additional embodiment 27, wherein the divalent cation is selected from the group consisting of Co 2+ , Mn 2+ , Mg 2+ , Cd 2+ , and Ca 2+ .
  • composition of any one of the preceding additional embodiments, wherein the one or more primers comprise pre-capture forward and reverse primers.
  • composition of additional embodiment 34 wherein the input nucleic acid molecules comprise a library of nucleic acid molecules, where each nucleic acid molecule in the library of nucleic acid molecules comprises first and second adapters.
  • 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 include no limitation on the percent of guanine or cytosine bases, and wherein at least one primer of the one or more primers has a melting temperature greater than 63°C.
  • composition of additional embodiment 37, wherein the at least one primer has a melting temperature greater than about 69°C.
  • composition of additional embodiment 37, wherein the at least one primer has a melting temperature greater than about 75°C.
  • composition of additional embodiment 37, wherein the at least one primer has a melting temperature greater than about 85°C.
  • composition of additional embodiment 37, wherein the at least one primer has a melting temperature greater than about 95°C.
  • the at least one optional enhancer is selected from the group consisting of betaine, DMSO, a disaccharide, and a single stranded DNA binding protein (SSB).
  • 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.
  • Additional Embodiment58 The composition of additional embodiment 56, wherein the one or more primers comprise one or more capture primers, and wherein the one or more capture primers are capable of hybridizing to target nucleic acid sequences of the nucleic acid molecules.
  • composition of additional embodiment 56, wherein the input nucleic acid molecules comprise DNA.
  • Additional Embodiment61 Use of the composition of any one of additional embodiments 1 - 33 and 37 - 55 in the amplification of input nucleic acid molecules.
  • a composition comprising: (a) one or more primers, (b) input nucleic acid molecules, and (c) at least one enhancer selected from the group consisting of betaine, DMSO, a disaccharide, and a single stranded DNA binding protein (SSB).
  • SSB single stranded DNA binding protein
  • Additional Embodiment63 The composition of additional embodiment 62, wherein the at least one enhancer is betaine. Additional Embodiment64. The composition of additional embodiment 63, wherein a concentration of betaine in the composition ranges from between about 0.2 mM to about 0.8 mM.
  • Additional Embodiment68 The composition of additional embodiment 66, wherein an amount of DMSO in the composition ranges from between about 2% (v/v) to about 8% (v/v).
  • composition of additional embodiment 62, wherein the one or more primers have a Tm ranging from between about 57°C to about 95°C.
  • composition of additional embodiment 62, wherein the one or more primers have a Tm ranging from between about 57°C to about 85°C.
  • composition of additional embodiment 62, wherein the one or more primers have a Tm ranging from between about 57°C to about 75°C.
  • Additional Embodiment72 The composition of additional embodiment 62, wherein the one or more primers have a Tm ranging from between about 57°C to about 72°C.
  • Additional Embodiment73 The composition of additional embodiment 62, wherein the one or more primers have a Tm ranging from between about 57°C to about 69°C.
  • composition of additional embodiment 62, wherein the one or more primers have a Tm ranging from between about 57°C to about 66°C.
  • composition of additional embodiment 62, wherein the one or more primers comprise one or more modified dNTPs.
  • composition of additional embodiment 75, wherein the one or more modified dNTPs are selected from the group consisting of modified dGTPs and modified dATPs.
  • composition of additional embodiment 77, wherein the divalent cation is selected from the group consisting of Co 2+ , Mn 2+ , Mg 2+ , Cd 2+ , and Ca 2+ .
  • composition of additional embodiment 81 wherein 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.
  • a composition comprising: (a) input nucleic acid molecules, and (b) one or more primers, and optionally (c) at least one enhancer, wherein the one or more primers include no limitation on the percent of guanine or cytosine bases, and wherein at least one primer of the one or more primers has a melting temperature greater than 63°C.
  • Additional Embodiment84 The composition of additional embodiment 83, wherein the at least one primer has a melting temperature greater than about 69°C.
  • composition of additional embodiment 83, wherein the at least one primer has a melting temperature greater than about 75°C.
  • composition of additional embodiment 83, wherein the at least one primer has a melting temperature greater than about 85°C.
  • composition of additional embodiment 83, wherein the at least one primer has a melting temperature greater than about 95°C.
  • 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.
  • composition of additional embodiment 92, wherein the divalent cation is selected from the group consisting of Co2+, Mn2+, Mg2+, Cd2+, and Ca2+.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne des compositions et des kits pour l'amplification par PCR. La présente invention concerne également des procédés d'amplification de molécules d'acide nucléique visant à améliorer l'uniformité de la couverture et/ou à réduire le biais GC pendant les opérations de séquençage en aval.
EP23764267.3A 2022-09-02 2023-08-28 Amélioration de la performance de l'enrichissement des cibles de nouvelle génération Pending EP4581153A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202263374400P 2022-09-02 2022-09-02
PCT/EP2023/073555 WO2024046992A1 (fr) 2022-09-02 2023-08-28 Amélioration de la performance de l'enrichissement des cibles de nouvelle génération

Publications (1)

Publication Number Publication Date
EP4581153A1 true EP4581153A1 (fr) 2025-07-09

Family

ID=87889453

Family Applications (1)

Application Number Title Priority Date Filing Date
EP23764267.3A Pending EP4581153A1 (fr) 2022-09-02 2023-08-28 Amélioration de la performance de l'enrichissement des cibles de nouvelle génération

Country Status (4)

Country Link
EP (1) EP4581153A1 (fr)
JP (1) JP2025528488A (fr)
CN (1) CN119895049A (fr)
WO (1) WO2024046992A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025043073A1 (fr) * 2023-08-22 2025-02-27 Molecular Assemblies, Inc. Procédés et matières pour la synthèse de polynucléotides
WO2025264831A1 (fr) * 2024-06-18 2025-12-26 Illumina, Inc. Procédés pour augmenter la qualité de séquençage de régions riches en gc
WO2025264836A1 (fr) * 2024-06-18 2025-12-26 Illumina, Inc. Procédés pour augmenter la qualité de séquençage de régions riches en gc

Family Cites Families (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4000159A (en) 1974-06-28 1976-12-28 Phillips Petroleum Company Preparation of n,n-disubstituted thioamides
US5176995A (en) 1985-03-28 1993-01-05 Hoffmann-La Roche Inc. Detection of viruses by amplification and hybridization
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
ATE452992T1 (de) * 2004-02-04 2010-01-15 Qiagen North American Holdings Zusammensetzungen auf dutp-basis zurverringerung der primeraggregatbildung während der nukleinsäureamplifikation
US7238485B2 (en) 2004-03-23 2007-07-03 President And Fellows Of Harvard College Methods and apparatus for characterizing polynucleotides
US7393665B2 (en) 2005-02-10 2008-07-01 Population Genetics Technologies Ltd Methods and compositions for tagging and identifying polynucleotides
CA2689626C (fr) 2007-06-06 2016-10-25 Pacific Biosciences Of California, Inc. Methodes et procedes pour identifier des bases dans des procedes d'incorporation en fonction de la sequence
US8592150B2 (en) * 2007-12-05 2013-11-26 Complete Genomics, Inc. Methods and compositions for long fragment read sequencing
WO2011106368A2 (fr) * 2010-02-23 2011-09-01 Illumina, Inc. Procédés d'amplification destinés à minimiser le biais spécifique de séquence
ES2523140T3 (es) 2010-09-21 2014-11-21 Population Genetics Technologies Ltd. Aumento de la confianza en las identificaciones de alelos con el recuento molecular
WO2012083249A2 (fr) 2010-12-17 2012-06-21 The Trustees Of Columbia University In The City Of New York Séquençage d'adn par une synthèse utilisant des nucléotides modifiés et une détection par nanopores
EP3674412A1 (fr) 2012-06-20 2020-07-01 The Trustees of Columbia University in the City of New York Séquençage d'acide nucléique par détection de nanopores de molécules d'étiquettes
US9605309B2 (en) 2012-11-09 2017-03-28 Genia Technologies, Inc. Nucleic acid sequencing using tags
CN105980828B (zh) 2013-12-13 2021-12-17 文塔纳医疗系统公司 用于生物标本组织处理的染色试剂和其它液体及关联技术
CN106029962A (zh) * 2014-02-11 2016-10-12 豪夫迈·罗氏有限公司 靶向测序和uid过滤
MA39774A (fr) 2014-03-24 2021-05-12 Roche Sequencing Solutions Inc Procédés chimiques pour produire des nucléotides étiquetés
EP3332024B1 (fr) 2015-08-06 2020-10-28 F.Hoffmann-La Roche Ag Enrichissement d'une cible par extension d'une unique amorce de sonde
CA2995422A1 (fr) 2015-08-12 2017-02-16 The Chinese University Of Hong Kong Sequencage monomoleculaire d'adn plasmatique
ES2924487T3 (es) 2016-01-29 2022-10-07 Hoffmann La Roche Un adaptador de conformación en Y novedoso para secuenciación de ácidos nucleicos y procedimiento de uso
US20170268052A1 (en) 2016-02-29 2017-09-21 Genia Technologies, Inc. Polymerase-template complexes
US10907204B2 (en) 2016-07-12 2021-02-02 Roche Sequencing Solutions, Inc. Primer extension target enrichment
WO2018034745A1 (fr) 2016-08-18 2018-02-22 The Regents Of The University Of California Appel de bases de séquençage par nanopores
WO2019121842A1 (fr) 2017-12-21 2019-06-27 F. Hoffmann-La Roche Ag Enrichissement d'une cible par extension d'amorce à double sonde unidirectionnelle
CN113316585B (zh) 2018-12-19 2024-06-04 豪夫迈·罗氏有限公司 3’保护的核苷酸
EP4251770A4 (fr) * 2021-02-08 2024-05-29 Singular Genomics Systems, Inc. Procédés et compositions pour le séquençage de polynucléotides complémentaires

Also Published As

Publication number Publication date
CN119895049A (zh) 2025-04-25
WO2024046992A1 (fr) 2024-03-07
JP2025528488A (ja) 2025-08-28

Similar Documents

Publication Publication Date Title
US20230099446A1 (en) Fast pcr for str genotyping
EP4581153A1 (fr) Amélioration de la performance de l'enrichissement des cibles de nouvelle génération
JP6225123B2 (ja) 非特異的核酸増幅を低減するための方法及びキット
JP7587013B2 (ja) 次世代シーケンシングライブラリーの標的捕捉中の、ポイズンプライマーを使用した非標的ライブラリー分子の標的化された枯渇
CA2778449C (fr) Amorces d'amplification avec des bases non-standard ayant une specificite de reaction augmentee
JP2020508692A (ja) Ｄｎａおよびｒｎａの同時濃縮を含むプライマー伸長標的濃縮およびそれに対する向上
WO2014140309A1 (fr) Procédés d'amplification et de séquençage à l'aide de tthprimpol thermiquement stable
US10443094B2 (en) Solid phase isothermal amplification
JP7013069B2 (ja) 低塩条件下での等温増幅
US10358673B2 (en) Method of amplifying nucleic acid sequences
US20240279732A1 (en) Targeted next-generation sequencing via anchored primer extension
US20250368983A1 (en) Controlling for tagmentation sequencing library insert size using archaeal histone-like proteins
WO2026082489A1 (fr) Pcr universelle destinée à renforcer la récupération de molécules dans la préparation d'une bibliothèque d'adn acellulaire et flux de travail d'enrichissement en cibles
Dambrowitz Applications of DNA indexing

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20250402

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC ME MK MT NL NO PL PT RO RS SE SI SK SM TR

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)