Compositions and methods for assessing DNA damage in libraries and normalizing amplicon size bias - Patents.com

Abstract

Described herein are standards and methods for normalizing amplicon size bias. These standards can include unique molecular identifiers. In some embodiments, the standards and methods are for use with next generation sequencing (NGS) assays. Also described herein are methods for quantifying DNA damage in a sample containing DNA using fluorescence or for determining the presence of DNA damage in a library.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 167,171, filed March 29, 2021, and U.S. Provisional Patent Application No. 63 / 227,550, filed July 30, 2021, each of which is incorporated by reference in its entirety for any purpose.

[0002] explanation This application relates to standards and methods for assessing library damage and normalizing amplicon size bias in next generation sequencing (NGS) assays. This application also relates to the use of fluorescence to quantify DNA damage in samples containing DNA. [Background technology]

[0003] A common method for detecting and quantifying large insertion / deletion variants (indels) in genome editing or oncology applications involves targeted "long amplicon" PCR (LongAmp, >1 kb) followed by conversion to short-read libraries for long-read sequencing or (short-read) NGS. However, size-based biases in "long" PCR amplification complicate the process of accurately quantifying the relative frequency of large indel variants. Strategies that tag the ends of target DNA molecules with unique molecular identifiers before or during amplification require variants and UMIs to be identified in the same NGS read. Thus, tagging methods using long amplicon libraries require long-read sequencing or complex synthetic long-read library preparation. The post-amplification library conversion step for short-read NGS makes this UMI end tagging unsuitable, as short-read NGS can sever the variant sequence and the original amplicon UMI into separate reads.

[0004] These existing methods incorporate short-read NGS with UMI-containing synthetic DNA controls of various lengths to normalize amplicon size bias. The DNA controls are designed so that the identities of the standards and UMIs are included in the same NGS read. Running control assays with these standards or spiking in known amounts of these standards into each LongAmp assay allows for bioinformatics analysis of size-based PCR bias and facilitates better estimation of the frequency of large indels by taking into account the quantified PCR size bias.

[0005] Another issue with libraries for long-read sequencing (i.e., long-read libraries) is the presence of damaged library molecules. Assessment of the quality of long-read library preparation can be used to predict the success of subsequent workflow steps and sequencing. Long library molecules can easily be nicked or damaged during standard workflows, resulting in library molecules that are not associated with adapter sequences and therefore cannot be used in workflows that require adapters, such as sequencing. Library preparation steps can damage DNA, either by pipetting, storage, or other handling and / or procedural errors. If nicked DNA undergoes library preparation that requires both 5' and 3' adapters, the nicked DNA is unusable in downstream steps. Thus, unaccounted library damage can cause inaccurate estimation of library concentration, insufficient sequencing coverage, and overall poor sequencing assay criteria.

[0006] Library quality control (QC) methods for accurate quantification of undamaged library molecules during library preparation can help to solve this problem. The quantitative PCR (qPCR) QC methods described herein evaluate library preparation quality to avoid proceeding with subsequent workflow steps with libraries of inaccurate concentrations. Thus, these methods can avoid the loss of users' time, budget, and reagents and other consumables.

[0007] Furthermore, DNA damage from the environment, sample preparation and processing, or storage conditions can significantly affect the consistency of library preparation quality. For example, during the sequencing process, accumulation of DNA damage from exposure to low wavelength lasers and other chemicals between sequencing cycles can increase the sequencing error rate. Users may wish to assess this damage. Described herein is a method to quantify DNA damage using fluorescence. Other assays developed to quantify DNA damage using fluorescence (e.g., U.S. Patent Application Publication No. 2014 / 0030705, WO 2010028388, and U.S. Patent Application Publication No. 20090042205) have been hampered by low signal-to-noise ratios, likely due in part to non-specific binding of unincorporated fluorescent nucleotides. The method of the present invention for measuring DNA damage incorporates steps of dephosphorylation of dNTPs and binding / elution of repaired DNA from carboxylate or cellulose beads, improving the signal and allowing a larger dynamic range of the assay. Summary of the Invention

[0008] Described herein is a pool of nucleic acid standards of different lengths, the nucleic acid standards comprising a unique molecular identifier (UMI), as well as: a 5' universal oligonucleotide, the 5' universal oligonucleotide being the same for all standards; a 3' universal oligonucleotide, the 3' universal oligonucleotide being the same for all standards; and at least one region between the UMI and the 5' universal oligonucleotide and / or between the UMI and the 3' universal oligonucleotide; The length of at least one region determines the length of the standard.Also described herein are methods for quality control of the libraries.

[0009] Embodiment 1. A pool of nucleic acid standards of different lengths, the nucleic acid standards comprising a unique molecular identifier (UMI), and a. a 5' universal oligonucleotide, wherein the 5' universal oligonucleotide is the same for all standards; and b. a 3' universal oligonucleotide, the 3' universal oligonucleotide being the same for all standards; and c. at least one region between the UMI and the 5' universal oligonucleotide and / or between the UMI and the 3' universal oligonucleotide; The length of at least one region determines the length of the standard, the pool of standards.

[0010] Embodiment 2. The pool contains UMI, and a. a 5' universal oligonucleotide, wherein the 5' universal oligonucleotide is the same for all standards; and b. a 3' universal oligonucleotide, the 3' universal oligonucleotide being the same for all standards; The pool of standards described in embodiment 1, wherein the additional nucleic acid standards do not include at least one region between the UMI and the 5' universal oligonucleotide or between the UMI and the 3' universal oligonucleotide.

[0011] Embodiment 3. A pool of standards according to embodiment 1, wherein at least one region between the UMI and the 5' universal oligonucleotide and / or between the UMI and the 3' universal oligonucleotide comprises between 0.2 kb and 10 kb.

[0012] Embodiment 4. The pool of standards according to any one of embodiments 1 to 3, wherein the 5' universal oligonucleotide and / or the 3' universal oligonucleotide each comprises an amplicon amplified from a sequence of interest.

[0013] Embodiment 5. A pool of standards according to any one of embodiments 1 or 3-4, wherein at least one region between the UMI and the 5' universal oligonucleotide and / or between the UMI and the 3' universal oligonucleotide, respectively, contains an amplicon amplified from a sequence of interest.

[0014] Embodiment 6. A pool of standards according to any one of embodiments 1 or 3 to 5, wherein at least one region between the UMI and the 5' universal oligonucleotide and / or between the UMI and the 3' universal oligonucleotide, respectively, comprises any sequence.

[0015]

[0036] Embodiment 7. A pool of nucleic acid standards of different lengths, the nucleic acid standards comprising UMIs, as well as a. 5' partially overlapping oligonucleotides, which are identical over at least a portion of their sequence for all standards; and / or b. 3' partially overlapping oligonucleotides, which are identical over at least a portion of their sequence for all standards; The length of the 5' partially overlapping oligonucleotides and / or the 3' partially overlapping oligonucleotides determine the length of the standards, the pool of standards.

[0016] Embodiment 8. a. 5' partially overlapping oligonucleotides comprising at least a first portion of a sequence of interest; The pool of standards of embodiment 7, wherein the 3' partially overlapping oligonucleotides comprise at least a second portion of the sequence of interest.

[0017] Embodiment 9. The pool of standards according to any one of embodiments 7 to 8, wherein the 5' partially overlapping oligonucleotides and / or the 3' partially overlapping oligonucleotides each comprise a sequence that is 20 bp to 1 kb smaller than the sequence of interest.

[0018] Embodiment 10. The pool of standards according to any one of embodiments 7 to 9, wherein the 5' partially overlapping oligonucleotides and / or the 3' partially overlapping oligonucleotides each comprise an amplicon amplified from a sequence of interest.

[0019] Embodiment 11. A pool of standards according to any one of embodiments 1 to 10, wherein the standards comprise double-stranded nucleic acids.

[0020] Embodiment 12. A pool of standards according to any one of embodiments 1 to 11, wherein the standards comprise double-stranded DNA.

[0021] Embodiment 13. A pool of standards according to any one of embodiments 1 to 12, wherein each standard comprises a different UMI.

[0022] Embodiment 14. A pool of standards according to any one of embodiments 1 to 13, wherein the UMIs contained in the pool of standards are a random set of sequences comprising 16 to 20 base pairs.

[0023] Embodiment 15. The pool of standards according to embodiment 14, wherein the UMIs contained in the pool of standards are a random set of sequences comprising 18 base pairs.

[0024] 16. The pool of standards is 1×10 10 That's it, 10 x 10 10 or more, or 100 x 10 10 16. The pool of standards according to any one of the preceding embodiments, comprising the above standards, each standard comprising a different UMI.

[0025] Embodiment 17. A pool of standards according to any one of embodiments 1 to 16, wherein the number of standards in the pool is greater than the number of amplicons produced by the amplification reaction.

[0026] Embodiment 18. A pool of standards, wherein at least a first portion of the standards is from any one of embodiments 1 to 6 or 11 to 17, and at least a second portion of the standards is from any one of embodiments 7 to 17.

[0027] Embodiment 19. A method for generating a pool of nucleic acid standards, comprising: a. providing multiple copies of at least one sequence of interest comprising a nucleic acid; b. providing a series of oligonucleotides, each of which contains a UMI; c. providing a series of insertion oligonucleotides of various lengths; d. ligating (a) at least one sequence of interest, (b) at least one oligonucleotide comprising a UMI, and (c) at least one insertion amplicon to generate a plurality of nucleic acid standards of the pool of nucleic acid standards.

[0028] Embodiment 20. The method of embodiment 19, wherein at least one sequence of interest and / or insertion oligonucleotide is prepared by amplification.

[0029] Embodiment 21. The method of embodiment 19 or embodiment 20, wherein the sequence of interest, the oligonucleotide containing the UMI, and / or the insertion oligonucleotide contain a restriction enzyme cleavage site.

[0030] Embodiment 22. The method of embodiment 21, wherein the restriction enzyme cleavage site is proximal to the 5' and / or 3' ends of the sequence of interest, the oligonucleotide containing the UMI, and / or the insertion oligonucleotide.

[0031] Embodiment 23. The method according to embodiment 21 or embodiment 22, wherein the method further comprises cleaving the sequence of interest, the oligonucleotide containing the UMI, respectively, and / or the insertion oligonucleotide with a restriction enzyme prior to ligation.

[0032] Embodiment 24 The method of embodiment 23, wherein cleaving with a restriction enzyme generates sticky ends for ligation.

[0033] Embodiment 25. A method for generating a pool of nucleic acid standards, comprising: a. providing multiple copies of at least one sequence of interest comprising a nucleic acid; b. providing a series of oligonucleotides, each of which contains a UMI; c. ligating at least one sequence of interest (a) and at least one oligonucleotide containing a UMI (b).

[0034] Embodiment 26 The method of embodiment 25, wherein at least one sequence of interest is prepared by amplification.

[0035] Embodiment 27. The method of embodiment 25 or embodiment 26, wherein the oligonucleotide containing the sequence of interest and / or the UMI, respectively, contains a restriction enzyme cleavage site.

[0036] Embodiment 28. The method of embodiment 27, wherein the restriction enzyme cleavage site is proximal to the 5' and / or 3' ends of the oligonucleotide containing the sequence of interest and / or the UMI, respectively.

[0037] Embodiment 29. The method according to embodiments 27 to 28, wherein the method further comprises cleaving the oligonucleotides containing the sequence of interest and / or the UMI, respectively, with a restriction enzyme prior to ligation.

[0038] Embodiment 30 The method of embodiment 29, wherein cleaving with a restriction enzyme generates sticky ends for ligation.

[0039] Embodiment 31. A method for normalizing amplicon size bias, comprising: a. combining a sample containing a target nucleic acid with a pool of nucleic acid standards of different lengths, each standard containing a UMI; b. amplifying amplicons of sequences of interest contained in the standard and target nucleic acids; c. sequencing the amplicons of the standards and the sequence of interest to generate sequencing data; d. determining a bias profile based on amplicon size using sequencing data from the standard; e. Normalizing the amplicon size bias using the bias profile.

[0040] Embodiment 32. The method of embodiment 31, wherein the standards in the pool of nucleic acid standards range from 0.2 kb to 20 kb base pairs.

[0041] Embodiment 33 The method of embodiment 31 or embodiment 32, wherein each standard contained in the pool of nucleic acid standards comprises a different UMI.

[0042] Embodiment 34. The method of any one of embodiments 31 to 33, wherein the UMIs included in the pool of standards are a random set of sequences comprising 16 to 20 base pairs.

[0043] Embodiment 35. The method of any one of embodiments 31 to 34, wherein the UMIs included in the pool of standards are a random set of sequences comprising 18 base pairs.

[0044] 36. The pool of standards is 1×10 10 That's it, 10 x 10 10 or more, or 100 x 10 10 The method of any one of embodiments 31 to 35, comprising the above standard substances, each standard substance comprising a different UMI.

[0045] Embodiment 37. The method of any one of embodiments 31 to 36, wherein the number of standards in the pool of standards is greater than the number of amplicons generated by the amplification.

[0046] Embodiment 38. The method according to any one of embodiments 31 to 37, wherein the pool of nucleic acid standards comprises a pool of nucleic acid standards according to any one of embodiments 1 to 18.

[0047] Embodiment 39. The method according to any one of embodiments 31 to 37, wherein the pool of nucleic acid standards comprises a first portion comprising a pool of nucleic acid standards according to any one of embodiments 1 to 6 or 11 to 17, and a second portion comprising a pool of nucleic acid standards according to any one of embodiments 7 to 17.

[0048] Embodiment 40. The method of any one of embodiments 31 to 39, wherein the sequence of interest comprises a restriction enzyme cleavage site that is not at the 5' and / or 3' end of the sequence of interest or that is not adjacent to the 5' and / or 3' end of the sequence of interest.

[0049] Embodiment 41. The method of any one of embodiments 31 to 40, wherein the sequence of interest may contain an insertion or deletion mutation.

[0050] Embodiment 42. The method of any one of embodiments 31 to 41, wherein the sequence of interest has been subjected to gene editing, and optionally, the sequence of interest comprises a cleavage site introduced by gene editing.

[0051] Embodiment 43. The method according to any one of embodiments 31 to 42, wherein amplifying an amplicon of the sequence of interest comprises amplifying an amplicon from the target nucleic acid using a pair of PCR primers that bind to primer binding sequences at the ends of the sequence of interest.

[0052] Embodiment 44. The method of any one of embodiments 31 to 43, wherein the standard comprises the same primer binding sequence as that at the end of the sequence of interest.

[0053] Embodiment 45. The method of any one of embodiments 31 to 44, further comprising generating a library of fragments after amplification and prior to sequencing.

[0054] Embodiment 46 The method of any one of embodiments 31 to 45, wherein generating the library of fragments is by tagmentation.

[0055] Embodiment 47. The method of any one of embodiments 31 to 46, wherein the sequencing data from the standard used to determine the bias profile is the unique molecular count of the UMI contained in the standard.

[0056] Embodiment 48. A method for determining the presence of DNA damage in a library comprising one or more library molecules, each library molecule comprising a double-stranded DNA insert having a hairpin adaptor at each end of the insert, the method comprising: a. denaturing a first strand and a second strand of a double-stranded DNA insert contained in a library molecule; b. annealing a forward primer and a reverse primer to the library molecules; c. amplifying to generate library amplicons; d. assessing the presence of DNA damage based on the number of library amplicons generated.

[0057] Embodiment 49. The method of embodiment 48, wherein the forward primer and / or the reverse primer binds to one or more sequences contained in one or both hairpin adaptors.

[0058] Embodiment 50. The method of embodiment 48 or embodiment 49, wherein the forward primer binds to a sequence contained in a hairpin adaptor attached to a first end of the double-stranded DNA insert, and the reverse primer binds to a sequence contained in a hairpin adaptor attached to a second end of the double-stranded DNA insert.

[0059] Embodiment 51. The method of any one of embodiments 48 to 50, wherein the number of generated library amplicons is estimated by measuring the cycle of quantification (Cq) value.

[0060] Embodiment 52. The method of any one of embodiments 48 to 51, wherein the higher the number of library amplicons, the lower the Cq value.

[0061] Embodiment 53. The method of any one of embodiments 48 to 52, wherein a library with a lower Cq value has less DNA damage.

[0062] Embodiment 54. The method of any one of embodiments 51 to 53, further comprising determining conditions for analysis of the library based on the Cq value.

[0063] Embodiment 55. The method of embodiment 54, wherein the analysis is sequencing.

[0064] Embodiment 56. The method of any one of embodiments 48 to 55, wherein the amplifying is optimized for amplifying library molecules that are 5 kb or more, 10 kb or more, 15 kb or more, 20 kb or more, 25 kb or more, or 30 kb or more.

[0065] Embodiment 57. The method of any one of embodiments 48 to 56, wherein amplifying is performed using a polymerase that is optimized for amplifying long amplicons.

[0066] Embodiment 58. The method of embodiment 57, wherein the polymerase is optimized for amplification of amplicons of 20 kb or more or 30 kb or more.

[0067] Embodiment 59. The method of embodiment 57 or embodiment 58, wherein the polymerase has a higher processivity or extension rate compared to wild-type Taq polymerase.

[0068] Embodiment 60. The method of embodiment 59, wherein the polymerase comprises one or more mutations or fusions that increase processivity or extension rate.

[0069] Embodiment 61 The method of embodiment 59 or embodiment 60, wherein the polymerase has an extension rate of greater than 3 kb / min.

[0070] Embodiment 62. The method of any one of embodiments 48 to 61, wherein the amplifying is exponential.

[0071] Embodiment 63. The method of any one of embodiments 48 to 62, wherein the amplification is performed for 30 or more cycles or for 40 or more cycles.

[0072] Embodiment 64. The method of any one of embodiments 48 to 63, wherein the DNA damage comprises one or more nicks in the library molecule.

[0073] Embodiment 65. The method of embodiment 64, wherein the one or more nicks are within the insert.

[0074] Embodiment 66 The method of embodiment 64 or embodiment 65, wherein the Cq value is higher when a higher percentage of library molecules in the library contain one or more nicks.

[0075] Embodiment 67. The method of any one of embodiments 64 to 66, wherein the DNA damage comprises two or more nicks in the library molecule, the nicks being in the same strand of the double-stranded DNA insert.

[0076] Embodiment 68. The method of any one of embodiments 64 to 66, wherein the DNA damage comprises two or more nicks in the library molecule, the nicks being in both strands of the double-stranded DNA insert.

[0077] Embodiment 69. The method of any one of embodiments 48 to 68, wherein the forward primer and / or the reverse primer are unable to generate an amplicon corresponding to the complete sequence of the library molecule if the library molecule contains one or more nicks.

[0078] Embodiment 70. The method of embodiment 69, wherein amplicons generated from library molecules containing nicks lack sequences for binding to the forward primer and / or the reverse primer.

[0079] Embodiment 71. The method of any one of embodiments 64 to 70, wherein library molecules containing nicks generate fewer amplicons during amplification compared to library molecules that do not contain nicks.

[0080] Embodiment 72. The method of any one of embodiments 64 to 71, further comprising generating a double-stranded break from the nick before annealing the forward primer and the reverse primer. Embodiment 73 The method of embodiment 72, wherein generating the double-stranded break is carried out using an enzymatic reaction.

[0081] Embodiment 74. The method of embodiment 73, wherein the enzymatic reaction is carried out by an endonuclease.

[0082] Embodiment 75. The method of embodiment 74, wherein the endonuclease is T7 endonuclease.

[0083] Embodiment 76. The method of any one of embodiments 72 to 75, wherein a library molecule containing a double-stranded break does not generate an amplicon corresponding to the complete sequence of the library molecule during amplification.

[0084] Embodiment 77. The method of any one of embodiments 72 to 76, wherein the amplicons generated from the library molecules containing the double-stranded break lack a sequence for binding to the forward primer and / or the reverse primer.

[0085] Embodiment 78. A method for quantifying DNA damage in a sample containing DNA using fluorescence, comprising: a. i. an aliquot of the sample containing DNA; ii. one or more DNA repair enzymes; iii. combining dNTPs, where one or more dNTPs are fluorescently labeled; b. preparing repaired DNA; c. Dephosphorylate the phosphate from dNTPs; d. binding the repaired DNA to carboxylate or cellulose beads; e. eluting the bound repaired DNA from the carboxylate or cellulose beads with a resuspension buffer; f. Measuring the fluorescence of the repaired DNA to determine the amount of DNA damage.

[0086] Embodiment 79. The method of embodiment 78, wherein higher fluorescence of repaired DNA indicates more DNA damage.

[0087] Embodiment 80 The method of embodiment 78 or embodiment 79, wherein the fluorescence of repaired DNA is linear over a range of different amounts of DNA damage.

[0088] Embodiment 81. The method according to any one of embodiments 78 to 80, wherein the assay is capable of assessing DNA damage induced by manipulation by evaluating an aliquot of the same sample before and after the manipulation of the sample. Embodiment 82. The method of embodiment 81, wherein the operation is sequencing of the sample.

[0089] Embodiment 83. The method of embodiment 81 or embodiment 82, wherein measuring the fluorescence of the repaired DNA comprises creating a standard curve of dilutions of the repaired DNA and measuring the fluorescence of the dilutions of the repaired DNA.

[0090] Embodiment 84. The method of any one of embodiments 78 to 83, wherein measuring the fluorescence of the repaired DNA comprises comparing the fluorescence of the repaired DNA against a separate standard curve of dilutions of one or more fluorescently labeled dNTPs alone to determine the number of fluorescent dye molecules contained in the repaired DNA.

[0091] Embodiment 85. The method of embodiment 84, further comprising calculating a normalized number of fluorophores contained in the repaired DNA by dividing the determined number of fluorophores by the mass of the repaired DNA.

[0092] Embodiment 86 The method of any one of embodiments 78 to 85, wherein the DNA is a library comprising genomic DNA, cDNA, or fragmented double-stranded DNA.

[0093] Embodiment 87. The method of embodiment 86, wherein the DNA is genomic DNA and cDNA, and the method further comprises preparing a library after determining the amount of DNA damage.

[0094] Embodiment 88. The method of embodiment 87, wherein a library is prepared where the amount of DNA damage is less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of total nucleotides.

[0095] Embodiment 89. The method of any one of embodiments 78 to 88, wherein a library is not prepared if the amount of DNA damage is 5% or more, 4% or more, 3% or more, 2% or more, or 1% or more of the total nucleotides.

[0096] Embodiment 90. A method according to any one of embodiments 78 to 89, in which the repaired DNA is bound to carboxylate beads or cellulose beads and eluted two or more times before measuring fluorescence.

[0097] Embodiment 91. The method according to embodiment 90, in which the repaired DNA is bound to carboxylate or cellulose beads and eluted twice before measuring fluorescence.

[0098] Embodiment 92. The method of any one of embodiments 78 to 91, wherein the carboxylate beads or cellulose beads are magnetic.

[0099] Embodiment 93. The method of any one of embodiments 78 to 92, wherein adjusting the repaired DNA is performed at 37°C.

[0100] Embodiment 94. The method of any one of embodiments 78 to 93, wherein preparing the repaired DNA is carried out for 10 minutes or more, 20 minutes or more, 30 minutes or more, 45 minutes or more, or 60 minutes or more.

[0101] Embodiment 95. The method according to any one of embodiments 78 to 94, wherein dephosphorylating the phosphate from the dNTP is carried out using an enzyme.

[0102] Embodiment 96. The method of embodiments 78 to 95, wherein the enzyme for dephosphorylating phosphates from dNTPs is shrimp alkaline phosphatase (SAP) or calf intestinal alkaline phosphatase (CIP).

[0103] Embodiment 97. The method of any one of embodiments 78 to 96, wherein the one or more DNA repair enzymes comprise a DNA polymerase.

[0104] Embodiment 98. The method of embodiment 97, wherein the DNA polymerase has 5'-3' polymerase activity but lacks 5'-3' exonuclease activity.

[0105] Embodiment 99. The method of embodiment 97, wherein the DNA polymerase is Bst DNA polymerase, large fragment.

[0106] Embodiment 100. The method of any one of embodiments 78 to 99, wherein the one or more DNA repair enzymes comprises a ligase.

[0107] Embodiment 101. The method of embodiment 100, wherein the ligase is Taq ligase.

[0108] Embodiment 102. The method of any one of embodiments 78 to 101, wherein the DNA damage comprises a nick in double-stranded DNA.

[0109] Embodiment 103. The method of any one of embodiments 78 to 102, wherein the one or more DNA repair enzymes comprise T4 pyrimidine dimer glycosylase (PDG).

[0110] Embodiment 104. The method of any one of embodiments 78 to 103, wherein the DNA damage comprises thymine dimers.

[0111] Embodiment 105. The method of embodiment 104, wherein the thymine dimers are induced by ultraviolet irradiation.

[0112] Embodiment 106. The method of any of embodiments 78 to 105, wherein the one or more DNA repair enzymes comprise uracil DNA glycosylase (UDG) and an apurinic or apyrimidinic site lyase.

[0113] Embodiment 107. The method of any one of embodiments 78 to 106, wherein the DNA damage comprises uracil.

[0114] Embodiment 108. The method of any of embodiments 78 to 107, wherein the one or more DNA repair enzymes comprise a formamidopyrimidine DNA glycosylase (FPG) and an apurinic or apyrimidinic site lyase.

[0115] Embodiment 109. The method of embodiment 78 to 108, wherein the DNA damage comprises oxidized bases.

[0116] Embodiment 110. The method of any one of embodiments 78 to 109, wherein the dNTPs include dATP, dGTP, dCTP, and dTTP or dUTP.

[0117] Embodiment 111. The method of any one of embodiments 78 to 110, wherein all dNTPs are fluorescently labeled.

[0118] Embodiment 112. The method according to embodiments 78 to 111, wherein dUTP and dCTP are fluorescently labeled.

[0119] Embodiment 113. The method of embodiment 112, wherein the fluorescent label is Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 633, fluorescein isothiocyanate (FITC), or tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC).

[0120] Additional objects and advantages will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice. The objects and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0121] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the scope of the appended claims.

[0122] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments and, together with the description, serve to explain the principles described herein. [Brief description of the drawings]

[0123] [Figure 1] A representative standard method for large indel detection is shown. Such a method involves low cycle PCR (low cycle, ~1 kb wild type amplicon) around the cut site with PCR conditions optimized for long amplicons (~10 kb). After amplification, Nextera library preparation (LP) is performed on the PCR amplicons. Amplicon analysis involves "de novo" amplicon assembly and quantification of unique gene editing events (i.e., events that generate unique amplicons). [Figure 2A]Summary of long amplification (LongAmp) insert controls that can be prepared using universal UMI double-stranded (ds) DNA oligonucleotides. UMI dsDNA oligonucleotides can be commercially supplied (such as gBlock gene fragments from Integrated DNA Technologies) (A). The oligonucleotides can be used to prepare LongAmp insert controls (B). RS (such as RS1) refers to the restriction site. N18 refers to the UMI sequence containing 18 random nucleotides. LA-fwd and LA-rev refer to the forward and reverse primers for the LongAmp reaction, respectively. Controls 1, 2, 3, and n contain 0.2 kb, 1 kb, 2 kb, and 10 kb inserts, respectively. The light area of ​​the 10 kb standard indicates that this standard is not drawn to scale. [Figure 2B] Summary of long amplification (LongAmp) insert controls that can be prepared using universal UMI double-stranded (ds) DNA oligonucleotides. UMI dsDNA oligonucleotides can be commercially supplied (such as gBlock gene fragments from Integrated DNA Technologies) (A). The oligonucleotides can be used to prepare LongAmp insert controls (B). RS (such as RS1) refers to the restriction site. N18 refers to the UMI sequence containing 18 random nucleotides. LA-fwd and LA-rev refer to the forward and reverse primers for the LongAmp reaction, respectively. Controls 1, 2, 3, and n contain 0.2 kb, 1 kb, 2 kb, and 10 kb inserts, respectively. The light area of ​​the 10 kb standard indicates that this standard is not drawn to scale. [Diagram 3]A method for generating an upstream universal PCR adapter amplicon and a downstream universal PCR adapter amplicon is shown. These amplicons can be used as 5' universal oligonucleotides and 3' universal oligonucleotides, respectively. Using primers that contain RS1 and RS2 and bind to complementary strands in the 5' or 3' regions of the target sequence of interest, LA-amp forward and reverse primers can be used to generate an upstream universal PCR adapter amplicon (5' region) and a downstream universal PCR adapter amplicon (3' region), respectively (e.g., using LA-fwd / RS1 primer for the upstream amplicon and LA-rev / RS2 for the downstream amplicon). The "cleavage sites" shown refer to cleavage sites introduced into a representative sequence of interest via gene editing (e.g., using the CRISPR Cas system), and insertions and deletions can often occur around such cleavage sites used in gene editing. Other sequences of interest (e.g., sequences contained in samples from cancer patients being evaluated for insertion / deletion mutations) do not have cleavage sites introduced. [Figure 4] FIG. 1 shows a method for preparing insert amplicons of different sizes using tailed PCR primers. The method uses a set of two primers (i.e., two primers, e.g., one containing the RS1 / RS3 sequence, or two primers, e.g., one containing RS2 / RS4, as shown), that contain a sequence of a restriction enzyme cleavage site (RS) and bind to a primer binding sequence within the sequence of interest. The size of the insert amplicon and the insert amplicon can be controlled by the selection of primers based on the primer binding site with the sequence of interest. In this figure, upstream refers to the sequence in the 5' portion of the sequence of interest, and downstream refers to the sequence in the 3' portion of the sequence of interest. An insert amplicon pair can refer to an upstream insert amplicon and a downstream insert amplicon. The light area of ​​the 10 kb standard indicates that this standard is not drawn to scale. [Diagram 5]A method for making a deletion standard is shown. Primers that bind to RS3 and RS4 on the complementary strand of the sequence of interest can be used to generate a deletion amplicon using LA-amp forward and LA-amp reverse primers (e.g., LA-fwd / RS3 primer or LA-rev / RS4). A deletion amplicon pair can refer to an upstream deletion amplicon and a downstream deletion amplicon. Restriction sites corresponding to RS3 and RS4 can then be used to generate suitable ends for ligating the truncated amplicon to a universal UMI dsDNA oligonucleotide (shown in FIG. 6A) to generate the LongAmp deletion standard shown in FIG. 6B. [Figure 6A] Summary of long amplification (LongAmp) deletion controls that can be prepared using universal UMI double-stranded (ds) DNA oligonucleotides. UMI dsDNA oligonucleotides can be commercially supplied (such as gBlock gene fragments from Integrated DNA Technologies) (A). Using this oligonucleotide, LongAmp deletion standards can be prepared (B). Controls 1, 2, 3, and n contain deletions of -20 base pairs (bp), -50 bp, or approximately -1 kb, respectively. [Figure 6B] Summary of long amplification (LongAmp) deletion controls that can be prepared using universal UMI double-stranded (ds) DNA oligonucleotides. UMI dsDNA oligonucleotides can be commercially supplied (such as gBlock gene fragments from Integrated DNA Technologies) (A). Using this oligonucleotide, LongAmp deletion standards can be prepared (B). Controls 1, 2, 3, and n contain deletions of -20 base pairs (bp), -50 bp, or approximately -1 kb, respectively. [Figure 7] Shown is the mass of a control input that may be present in a LongAmp reaction to avoid duplication of UMI sequences. [Figure 8A]Representative individual standards that may be included in a pool of nucleic acid standards of different lengths are shown. All of these standards may include a UMI, as well as LA-rev and LA-fwd primer binding sequences. Table 1 below provides a description of the labeled regions and oligonucleotides included in the standards. Full-length standards may include a 5' universal oligonucleotide and a 3' universal oligonucleotide (100 and 101) (A). Insertion standards may include a 5' universal oligonucleotide, a 3' universal oligonucleotide, and the region between the UMI and the 5' universal oligonucleotide and the region between the UMI and the 3' universal oligonucleotide (100, 101, and 102 and 103) (B). Insertion standards may also include either the region between the UMI and the 5' universal oligonucleotide or the region between the UMI and the 3' universal oligonucleotide, but not both regions (shown in the lower standard in 8B, which includes 100, 101, and 103, but not 102). Deletion standards may include a 5' partially overlapping oligonucleotide and a 3' partially overlapping oligonucleotide (104 and 105) (C). The deletion standards can include either 5' partially overlapping oligonucleotides or 3' partially overlapping oligonucleotides, but not both (as shown in the lower standard of 8C including 104 but not including 105). As described herein, the pool of nucleic acid standards can include any or all of the different types of standards shown herein. [Figure 8B]Representative individual standards that may be included in a pool of nucleic acid standards of different lengths are shown. All of these standards may include a UMI, as well as LA-rev and LA-fwd primer binding sequences. Table 1 below provides a description of the labeled regions and oligonucleotides included in the standards. Full-length standards may include a 5' universal oligonucleotide and a 3' universal oligonucleotide (100 and 101) (A). Insertion standards may include a 5' universal oligonucleotide, a 3' universal oligonucleotide, and the region between the UMI and the 5' universal oligonucleotide and the region between the UMI and the 3' universal oligonucleotide (100, 101, and 102 and 103) (B). Insertion standards may also include either the region between the UMI and the 5' universal oligonucleotide or the region between the UMI and the 3' universal oligonucleotide, but not both regions (shown in the lower standard in 8B, which includes 100, 101, and 103, but not 102). Deletion standards may include a 5' partially overlapping oligonucleotide and a 3' partially overlapping oligonucleotide (104 and 105) (C). The deletion standards can include either 5' partially overlapping oligonucleotides or 3' partially overlapping oligonucleotides, but not both (as shown in the lower standard of 8C including 104 but not including 105). As described herein, the pool of nucleic acid standards can include any or all of the different types of standards shown herein. [Figure 8C]Representative individual standards that may be included in a pool of nucleic acid standards of different lengths are shown. All of these standards may include a UMI, as well as LA-rev and LA-fwd primer binding sequences. Table 1 below provides a description of the labeled regions and oligonucleotides included in the standards. Full-length standards may include a 5' universal oligonucleotide and a 3' universal oligonucleotide (100 and 101) (A). Insertion standards may include a 5' universal oligonucleotide, a 3' universal oligonucleotide, and the region between the UMI and the 5' universal oligonucleotide and the region between the UMI and the 3' universal oligonucleotide (100, 101, and 102 and 103) (B). Insertion standards may also include either the region between the UMI and the 5' universal oligonucleotide or the region between the UMI and the 3' universal oligonucleotide, but not both regions (shown in the lower standard in 8B, which includes 100, 101, and 103, but not 102). Deletion standards may include a 5' partially overlapping oligonucleotide and a 3' partially overlapping oligonucleotide (104 and 105) (C). The deletion standards can include either 5' partially overlapping oligonucleotides or 3' partially overlapping oligonucleotides, but not both (as shown in the lower standard of 8C including 104 but not including 105). As described herein, the pool of nucleic acid standards can include any or all of the different types of standards shown herein.

[0124] [Table 1] [Figure 9]Summary of a quantitative PCR (qPCR) assay for assessing DNA damage in long libraries. The assay uses forward and reverse primers that bind to sequences within hairpin adaptors contained in library molecules. Libraries that do not contain DNA damage (e.g., nicks) generate more signal (i.e., produce more full-length amplicons). As shown in the figure, an exemplary assay can include exponential amplification using a LongAmp PCR-optimized polymerase (e.g., PrimeStar GXL DNA polymerase, Takara). [Figure 10A] Average quantification cycle (Cq) and % damage results from the QC assay for libraries treated with different concentrations of nickase are shown. Cq (A) and % damage (B) results for the 10 ng library, and Cq (C) and % damage (D) results for the 20 ng library are shown. [Figure 10B] Average quantification cycle (Cq) and % damage results from the QC assay for libraries treated with different concentrations of nickase are shown. Cq (A) and % damage (B) results for the 10 ng library, and Cq (C) and % damage (D) results for the 20 ng library are shown. [Figure 10C] Average quantification cycle (Cq) and % damage results from the QC assay for libraries treated with different concentrations of nickase are shown. Cq (A) and % damage (B) results for the 10 ng library, and Cq (C) and % damage (D) results for the 20 ng library are shown. [Figure 10D] Average quantification cycle (Cq) and % damage results from the QC assay for libraries treated with different concentrations of nickase are shown. Cq (A) and % damage (B) results for the 10 ng library, and Cq (C) and % damage (D) results for the 20 ng library are shown. [Figure 11]The results of methods to convert nicks in library molecules to double-stranded breaks, for example using a combination of Vibrio vulnificus nuclease (VVN) and a mutant T7 endonuclease, are shown. Endo = endonuclease. [Figure 12A] The difference in Cq values ​​between the libraries treated with and without the endonuclease mutants is summarized. (A) Summary of Cq values. (B) Summary of automated electrophoresis results using TapeStation®, Agilent. [Figure 12B] The difference in Cq values ​​between the libraries treated with and without the endonuclease mutants is summarized. (A) Summary of Cq values. (B) Summary of automated electrophoresis results using TapeStation®, Agilent. [Figure 13A] Results are shown when SMRTbell templates were evaluated by quantitative PCR (qPCR) and then sequenced on a PacBio Sequel 2 system to determine whether qPCR Cq correlated with sequencing criteria. Samples are ordered from lowest Cq to highest Cq. (A) Mean Cq. (B) Total power. (C) Variation (P1%). Correlation was observed for qPCR Cq and total power (gigabase, GB), with lower Cq indicating higher power (except for one outlier, library 8, which had the lowest Cq). In general, the average Cq value for the libraries was between 2 and 3. The qPCR results predicted library 13 to be of low quality, which is confirmed by the relatively poor sequencing results. [Figure 13B]Results are shown when SMRTbell templates were evaluated by quantitative PCR (qPCR) and then sequenced on a PacBio Sequel 2 system to determine whether qPCR Cq correlated with sequencing criteria. Samples are ordered from lowest Cq to highest Cq. (A) Mean Cq. (B) Total power. (C) Variation (P1%). Correlation was observed for qPCR Cq and total power (gigabase, GB), with lower Cq indicating higher power (except for one outlier, library 8, which had the lowest Cq). In general, the average Cq value for the libraries was between 2 and 3. The qPCR results predicted library 13 to be of low quality, which is confirmed by the relatively poor sequencing results. [Figure 13C] Results are shown when SMRTbell templates were evaluated by quantitative PCR (qPCR) and then sequenced on a PacBio Sequel 2 system to determine whether qPCR Cq correlated with sequencing criteria. Samples are ordered from lowest Cq to highest Cq. (A) Mean Cq. (B) Total power. (C) Variation (P1%). Correlation was observed for qPCR Cq and total power (gigabase, GB), with lower Cq indicating higher power (except for one outlier, library 8, which had the lowest Cq). In general, the average Cq value for the libraries was between 2 and 3. The qPCR results predicted library 13 to be of low quality, which is confirmed by the relatively poor sequencing results. [Figure 14A] Data are shown using another set of SMRTbell templates assessed by qPCR and then sequenced on a PacBio Sequel 2 system. (A) Mean Cq values, samples ordered from lowest Cq to highest Cq. (B) Total power (GB). (C) P1 percent. A correlation is observed for qPCR Cq and total power, with lower Cq indicating higher power (except for one outlier, library 14, which had the lowest Cq). The mean Cq value for most libraries was 3-4. qPCR predicts library 10 to be of low quality, which is confirmed by sequencing. [Figure 14B]Data are shown using another set of SMRTbell templates assessed by qPCR and then sequenced on a PacBio Sequel 2 system. (A) Mean Cq values, samples ordered from lowest Cq to highest Cq. (B) Total power (GB). (C) P1 percent. A correlation is observed for qPCR Cq and total power, with lower Cq indicating higher power (except for one outlier, library 14, which had the lowest Cq). The mean Cq value for most libraries was 3-4. qPCR predicts library 10 to be of low quality, which is confirmed by sequencing. [Figure 14C] Data are shown using another set of SMRTbell templates assessed by qPCR and then sequenced on a PacBio Sequel 2 system. (A) Mean Cq values, samples ordered from lowest Cq to highest Cq. (B) Total power (GB). (C) P1 percent. A correlation is observed for qPCR Cq and total power, with lower Cq indicating higher power (except for one outlier, library 14, which had the lowest Cq). The mean Cq value for most libraries was 3-4. qPCR predicts library 10 to be of low quality, which is confirmed by sequencing. [Figure 15A] Figure 1 shows qPCR QC assay results for several PacBio SMRTbell libraries prior to sequencing, and the data correlated with total Gb output. Total output increases at lower Cq values, suggesting that this QC assay may serve as a useful tool for predicting sequencing performance. Cq values ​​and Gb measurements for library fractions (F#) from library 20 (A), library 21 (B), and library 22 (C). [Figure 15B] Figure 1 shows qPCR QC assay results for several PacBio SMRTbell libraries prior to sequencing, and the data correlated with total Gb output. Total output increases at lower Cq values, suggesting that this QC assay may serve as a useful tool for predicting sequencing performance. Cq values ​​and Gb measurements for library fractions (F#) from library 20 (A), library 21 (B), and library 22 (C). [Figure 15C] Figure 1 shows qPCR QC assay results for several PacBio SMRTbell libraries prior to sequencing, and the data correlated with total Gb output. Total output increases at lower Cq values, suggesting that this QC assay may serve as a useful tool for predicting sequencing performance. Cq values ​​and Gb measurements for library fractions (F#) from library 20 (A), library 21 (B), and library 22 (C). [Figure 16] DNA damage detection workflow. The signal-to-noise ratio of the assay was increased by using both shrimp alkaline phosphatase (SAP) digestion and a stringent dual SPRI bead-based purification step (i.e., two purifications with carboxylate beads) to greatly reduce nonspecific binding of unincorporated fluorescent nucleotides. [Figure 17] Results of SAP digestion and a single SPRI bead-based purification step are shown. Single SPRI-purified sheared genomic DNA showed reduced non-specific binding of fluorescent nucleotides when treated with SAP prior to purification (+SAP) in contrast to no SAP treatment (-SAP). [Figure 18] This shows that two bead-based purification steps substantially reduced non-specific binding of fluorescent nucleotides. [Figure 19A] A comparison of the effectiveness of a commercially available repair mix (PreCR Repair mix (NEB), shown in panel (A)) and the method of the present invention (shown in panel (B)) using a DNA repair enzyme mix containing Taq ligase (40 U), Bst polymerase large fragment (8 U), and T4 PDG (1 U). [Figure 19B] A comparison of the effectiveness of a commercially available repair mix (PreCR Repair mix (NEB), shown in panel (A)) and the method of the present invention (shown in panel (B)) using a DNA repair enzyme mix containing Taq ligase (40 U), Bst polymerase large fragment (8 U), and T4 PDG (1 U). [Figure 20]Measurement of ultraviolet (UV) damage to genomic DNA samples. As the light energy is increased and the exposure time is increased, the amount of fluorescence also increases in samples repaired with a custom DNA repair enzyme mix containing Taq ligase, Bst polymerase, and the UV damage-specific repair enzyme T4 pyrimidine dimer glycosylase (T4 PDG). [Figure 21] 1 shows the measurement of nicking damage to genomic DNA samples, where increasing amounts of nicking enzyme (Nt.BspQI) generally increase the fluorescent signal in samples repaired with Taq ligase and Bst polymerase using the assay of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0125] Long amplification PCR can be used for targeted long indel detection in sequences of interest from target nucleic acid. However, PCR is biased towards smaller amplicons, such as those with small insertion and deletion mutations, and towards longer amplicons, such as long insertions. This bias is inherent to PCR methods, since long amplicons take longer to synthesize new strands of nucleic acid, which makes it less likely that long amplicons will be generated in a PCR cycle, compared to short amplicons. Furthermore, long amplicons have a lower success rate of producing complete amplicons before events can stop replication. In other words, the amplification of long amplicons can fail at a higher rate than shorter amplicons. For example, the longer the time that polymerase must act to produce an amplicon, the more likely that the polymerase will not reach the end of the amplicon due to random dropouts, encounters with DNA damage, or lack of time when considering processing speed.

[0126] Due to a known bias towards long amplicons, long amplification (LongAmp) PCR cannot be used to accurately determine the relative frequency of different events. Thus, the results of LongAmp amplification cannot quantify the relative number of a particular mutation in the original target nucleic acid sample, because the sizes of the amplicons associated with different mutations are amplified differently.

[0127] The standards and methods described herein can help normalize this amplicon size bias.

[0128] Furthermore, the present disclosure also describes quality control (QC) methods for assessing library quality. In some embodiments, libraries, such as those for long-read sequencing, are assessed prior to sequencing. In some embodiments, the library comprises library molecules that include double-stranded DNA inserts with hairpin adapters at both ends of the insert. In some embodiments, the library is generated by fragmenting target DNA and incorporating hairpin adapters at both ends of the fragments, such as by tagmentation or ligation.

[0129] I. Standards for normalizing amplicon size bias In some embodiments, a pool of nucleic acid standards of different lengths can be used in methods for normalizing amplicon size bias. In some embodiments, these diffusion standards include unique molecular identifiers (UMIs).

[0130] In some embodiments, the pool of nucleic acids can include a variety of different sequences contained within a sequence of interest.

[0131] In some embodiments, the number of standards in the pool is greater than the number of amplicons produced by the amplification reaction. In some embodiments, the amplification reaction is an amplification of a sequence of interest.

[0132] In some embodiments, at least a first portion of the standards is from one pool of standards and at least a second portion of the standards is from another pool of standards.

[0133] In some embodiments, the standards are double-stranded. In some embodiments, the standards comprise double-stranded DNA. In some embodiments, each standard comprises a different UMI.

[0134] In some embodiments, an amplification primer binding sequence is included at or adjacent to one or both ends of each standard. Throughout this specification, "adjacent to one or both ends" means within 10 nucleotides of the ends. In some embodiments, an amplification primer binding sequence is included at one or both ends of each standard. In some embodiments, an amplification primer binding sequence includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides at one or both ends of each standard. In some embodiments, a standard includes an amplification primer binding sequence at both its 3' end and its 5' end. In some embodiments, a standard includes a different amplification primer binding sequence at its 3' end relative to its 3' end. In some embodiments, a standard includes one or more oligonucleotides on the 5' side of a UMI. In some embodiments, a standard includes one or more oligonucleotides on the 3' side of a UMI. In some embodiments, a standard includes one or more oligonucleotides on the 5' side of a UMI and one or more oligonucleotides on the 3' side of a UMI.

[0135] A.UMI In some embodiments, each standard in a pool of standards comprises a UMI.

[0136] In some embodiments, the UMI is not at the 5' and / or 3' end of the standard or is not adjacent to the 5' and / or 3' end of the standard. In some embodiments, a centrally located UMI within a standard increases the probability that fragmentation of the standard (e.g., by tagmentation) will result in fragments that contain the UMI and all or a portion of the sequence from the remainder of the standard (either 5' and / or 3' of the UMI). As used herein, a "centrally" located feature refers to the center of a feature that is located within 10 nucleotides from the center of the standard. In some embodiments, a centrally located UMI within a standard has the center of the UMI within 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides of the center of the standard.

[0137] In contrast, placing the UMI proximal to the 5' and / or 3' ends of the sequence of interest may result in a higher percentage of fragments that contain only the UMI and no additional sequence from the remainder of the standard.

[0138] In some embodiments, the UMI is used to identify amplicons generated from the same LongAmp standard. In other words, sequencing of the standard containing the UMI and the upstream / downstream insertion junction bases can provide the unique molecular number and control identity of the standard, respectively. This is because each amplicon generated from the same standard has the same unique UMI, and other amplicons generated from the LongAmp standard have different UMIs.

[0139] In some embodiments, the UMIs comprise random base pairs, such that each unique UMI comprises a sequence that is different from other UMIs in the pool. In some embodiments, the UMIs comprise 10 (N10) or more, 12 (N12) or more, 14 (N14) or more, 16 (N16) or more, 18 (N18) or more, 20 (N20) or more, or 22 (N22) or more random base pairs. In some embodiments, the UMIs comprise 18 base pairs (N18). In some embodiments, the UMIs included in the pool of standards are a random set of sequences that comprise between 16 and 20 base pairs.

[0140] The use of a UMI pool with a large number of UMIs can help avoid UMI collisions. Having longer UMIs (i.e., N18 instead of N10) also reduces the chances of UMI collisions.

[0141] As used herein, "UMI collision" refers to the event of observing two reads that have the same sequence and the same UMI barcode, but originate from two different genome molecules. In amplicon sequencing, a specific position in the genome is sequenced many times, resulting in a much greater sequence depth than whole genome sequencing (see Clement et al., Bioinformatics, 34, 2018, i202-i210). Based on this sequence depth, many alleles from different genome molecules may share the same sequence, and the probability of UMI collision is much higher for amplicon sequencing compared to whole genome sequencing.

[0142] In some embodiments, the pool of standards is 1×10 10 That's it, 10 x 10 10 or more, or 100 x 10 10 7 shows the amount of synthetic double-stranded DNA containing a UMI required, 6.87 x 10 10 We present the calculations for preparing an experiment involving UMIs.

[0143] In some embodiments, as described herein, the UMI in the standard may be derived from a relatively inexpensive commercially available reagent. In some embodiments, the double-stranded oligonucleotide containing the UMI also contains one or more restriction enzyme cleavage sites for use in preparing the standard.

[0144] For example, as described below, representative synthetic dsDNA oligonucleotides are shown for preparing insertion standards (FIG. 2A) and deletion standards (FIG. 6A). In some embodiments, the synthetic dsDNA oligonucleotides contain a UMI and a restriction enzyme cleavage site (or restriction sites such as RS3 and RS4 as shown in FIG. 2A and FIG. 6A). In some embodiments, the restriction enzyme cleavage site can be used to cleave the oligonucleotide and then ligated to other oligonucleotides to prepare the final standard. Sources of UMI dsDNA oligonucleotides include gBlock gene fragments (Integrated DNA Technologies).

[0145] B. Sequence of interest As used herein, a "sequence of interest" can be any sequence that a user wishes to investigate. In some embodiments, the sequence of interest has been subjected to gene editing. For example, a user may have performed gene editing or other methods of mutagenesis (such as chemical mutagenesis) and wish to evaluate different mutations (along with the wild type sequence) in the sequence of interest.

[0146] In some embodiments, gene editing is performed using CRISPR Cas method. In some embodiments, CRISPR Cas cleavage site is present in the sequence of interest. In some embodiments, insertion or deletion mutation is likely to occur near the cleavage site in the sequence of interest. For example, Figure 5 shows the cleavage site present in the sequence of interest that is introduced using a gene editing method such as CRISPR Cas. Some sequences of interest, such as sequences of oncological samples from patients that are being evaluated for indel mutations, do not have a cleavage site introduced by gene editing method.

[0147] In some embodiments, a sequence of interest includes a restriction enzyme cleavage site that is not at the 5' and / or 3' end of the sequence of interest or that is not proximal to the 5' and / or 3' end of the sequence of interest, hi some embodiments, such cleavage sites may be useful in generating standards or may be used to evaluate a sequence of interest.

[0148] In some embodiments, the sequence of interest comprises a primer binding sequence capable of binding to long amplification primers (i.e., LA-fwd and LA-rev primers). In some embodiments, the user may evaluate the sequence of interest in order to prepare appropriate LA-fwd and LA-rev primers.

[0149] In some embodiments, the sequence of interest may contain an insertion mutation or a deletion mutation, for example, the sequence of interest may contain an insertion mutation or may be a deletion mutation (i.e., does not contain the full length sequence of the sequence of interest).

[0150] As used herein, a "wild type" sequence of interest refers to a sequence of interest that does not contain an indel mutation. In other words, a wild type sequence refers to a sequence that does not contain an insertion mutation and does not contain a deletion mutation. As used herein, a "wild type amplicon" is an amplicon that contains a wild type sequence of interest.

[0151] The sequence of interest can be any type of nucleic acid sequence. In some embodiments, the sequence of interest has been subjected to a gene editing method (such as CRISPR) and the user wishes to analyze the unique gene editing events. In some embodiments, the sequence of interest that has been subjected to gene editing can include a "cut site" as shown in the representative examples of Figures 3, 5, and 6B. Such gene editing methods can result in a variety of different types of indel mutations that the user may wish to characterize.

[0152] In some embodiments, sequences of interest that contain cancer and germline indel mutations, as well as insertions from transposable elements, can be evaluated by this method, in such embodiments, the sequence of interest may not contain a gene editing cleavage site.

[0153] In some embodiments, the sequence of interest may be all or part of a gene of interest, such as a gene known to be associated with cancer. One skilled in the art may wish to characterize the indels that a patient may have in the gene that contains the sequence of interest, and / or characterize the relative amount of different mutations. For example, one skilled in the art may wish to characterize the number of large insertion mutations present in the sequence of interest from a patient sample.

[0154] C. Standards containing universal oligonucleotides In some embodiments, all or some standards in a pool of nucleic acid standards include a 5' universal oligonucleotide and a 3' universal oligonucleotide. As used herein, "universal oligonucleotide" refers to an oligonucleotide contained in all standards in the pool. As used herein, a "5' universal oligonucleotide" is an oligonucleotide that is 5' to a UMI contained in a standard (represented as 100 in FIG. 8). As used herein, a "3' universal oligonucleotide" is an oligonucleotide that is 3' to a UMI contained in a standard (represented as 101 in FIG. 8).

[0155] In some embodiments, at least a first portion of the standards are from one pool of standards and at least a second portion of the standards are from another pool of standards. In other words, a pool of standards, each of which includes a 5' universal oligonucleotide and a 3' universal oligonucleotide, can be combined with a different pool of standards that do not include a 5' universal oligonucleotide and / or a 3' universal oligonucleotide.

[0156] In some embodiments, the pool of nucleic acid standards includes standards of different lengths, the nucleic acid standards including a unique molecular identifier (UMI), a 5' universal oligonucleotide, where the 5' universal oligonucleotide is the same for all standards, a 3' universal oligonucleotide, where the 3' universal oligonucleotide is the same for all standards, and at least one region between the UMI and the 5' universal oligonucleotide and / or between the UMI and the 3' universal oligonucleotide, where the length of the at least one region determines the length of the standard. The region between the UMI and the 5' universal oligonucleotide is shown in FIG. 8B as 102, and the region between the UMI and the 3' universal oligonucleotide is shown in FIG. 8B as 103.

[0157] In some embodiments, standards that include a 5' universal oligonucleotide and a 3' universal oligonucleotide, and also include additional sequences (e.g., the region between the UMI and the 5' universal oligonucleotide and / or the region between the UMI and the 3' universal oligonucleotide) can be referred to as "insertion standards". This is because the insertion standards can be longer in length than the wild-type sequence of interest. In this way, the insertion standards can be controlled to normalize the amplicon size bias of insertion mutations in the wild-type sequence of interest, since these insertion mutations are larger than the wild-type sequence of interest.

[0158] In some embodiments, the pool further comprises a nucleic acid standard comprising a UMI and a 5' universal oligonucleotide, the 5' universal oligonucleotide being the same for all standards, and a 3' universal oligonucleotide, the 3' universal oligonucleotide being the same for all standards, the further nucleic acid standard not comprising at least one region between the UMI and the 5' universal oligonucleotide or between the UMI and the 3' universal oligonucleotide. A standard comprising the 5' universal oligonucleotide (100) and the 3' universal oligonucleotide (101) may be referred to as a full-length standard, as shown in FIG. 8A. A full-length standard may have a similar length to a wild-type sequence of interest that does not have any insertion or deletion mutations (i.e., a wild-type sequence that does not have indels).

[0159] In some embodiments, at least one region between the UMI and the 5' universal oligonucleotide and / or between the UMI and the 3' universal oligonucleotide determines the length of the insertion standard. In some embodiments, at least one region between the UMI and the 5' universal oligonucleotide and / or between the UMI and the 3' universal oligonucleotide comprises a number of kilobases (kb) corresponding to the potential length of the insertion mutation of interest. In some embodiments, at least one region between the UMI and the 5' universal oligonucleotide and / or between the UMI and the 3' universal oligonucleotide comprises between 0.2 kb and 10 kb.

[0160] The 5' universal oligonucleotide and / or the 3' universal oligonucleotide may comprise a sequence contained in the sequence of interest. In some embodiments, the 5' universal oligonucleotide and / or the 3' universal oligonucleotide each comprise an amplicon amplified from the sequence of interest. In other words, the 5' universal oligonucleotide and / or the 3' universal oligonucleotide may be prepared by amplification, as shown in FIG. 3.

[0161] When a 5' universal oligonucleotide is prepared by amplification, it may be referred to as a "5' universal PCR adapter amplicon" or an "upstream universal PCR adapter amplicon." Figure 3 shows how a representative upstream universal PCR adapter amplicon may be generated using a long amplification forward primer (LA-fwd) and a primer that binds to a sequence of interest and contains a restriction enzyme cleavage site (RS1).

[0162] When a 3' universal oligonucleotide is prepared by amplification, it may be referred to as a "3' universal PCR adapter amplicon" or a "downstream universal PCR adapter amplicon." Figure 3 shows how a representative downstream universal PCR adapter amplicon may be generated using a long amplification reverse primer (LA-rev) and a primer that binds to a sequence of interest and contains a restriction enzyme cleavage site (RS2).

[0163] In some embodiments, the upstream and downstream universal PCR adapter amplicons can be cleaved with appropriate restriction enzymes (RS1 and RS2 can be cleaved in the example shown in FIG. 3) to prepare standards that include a UMI and a 5' universal oligonucleotide, where the 5' universal oligonucleotide is the same for all standards, and a 3' universal oligonucleotide, where the 3' universal oligonucleotide is the same for all standards. This cleavage can generate ends that are compatible for ligating these amplicons to other portions of the standards (e.g., the region between the UMI and the 5' universal oligonucleotide and / or the region between the UMI and the 3' universal oligonucleotide), as discussed below in the description of how to make the standards.

[0164] In some embodiments, at least one region between the UMI and the 5' universal oligonucleotide and / or between the UMI and the 3' universal oligonucleotide each comprises an arbitrary sequence. As used herein, "any sequence" refers to any sequence that comprises nucleotides, and does not require any particular nucleic acid sequence to be included in this arbitrary sequence. For example, one skilled in the art may wish to prepare an insertion standard in which the arbitrary sequence is random and not related to the sequence of interest. In another embodiment, the arbitrary sequence may be a known sequence that is not random, but is also not related to the sequence of interest (e.g., an unrelated gene sequence). A standard containing an arbitrary sequence can be used to normalize for the amplicon size bias of insertion mutations, since much of this bias is related to amplicon size and not related to the exact sequence contained in the insertion sequence. In some embodiments, the arbitrary sequence is double-stranded.

[0165] In some embodiments, at least one region between the UMI and the 5' universal oligonucleotide and / or between the UMI and the 3' universal oligonucleotide comprises an amplicon amplified from a sequence of interest, respectively. In other words, the region between the UMI and the 5' universal oligonucleotide and / or between the UMI and the 3' universal oligonucleotide can be prepared by amplification. In some embodiments, the amplification is from a sequence of interest, as shown in FIG. 4.

[0166] 1. Insertion Amplicon As used herein, the region between the UMI and the 5' universal oligonucleotide, when prepared by amplification, may be referred to as the "5' insertion amplicon" or the "upstream insertion amplicon." Figure 4 shows how a representative upstream insertion amplicon may be generated using a primer that binds to a sequence of interest and contains restriction enzyme cleavage sites (RS1 and RS3).

[0167] As used herein, the region between the UMI and the 3' universal oligonucleotide, when prepared by amplification, may be referred to as the "3' insertion amplicon" or the "downstream insertion amplicon." Figure 4 shows how a representative upstream insertion amplicon may be generated using restriction enzyme cleavage sites (RS2 and RS4).

[0168] In some embodiments, the reverse and forward primers used to prepare the insert amplicon determine the size of the insert amplicon, in some embodiments, a single primer pair generates an insert amplicon of a desired size.

[0169] As used herein, an "insertion amplicon" can refer to an amplicon that is either a 5' insertion amplicon or a 3' insertion amplicon. In general, an "insertion amplicon" is not limited by its location in a standard.

[0170] In some embodiments, the standards include both the upstream and downstream insert amplicons (as shown in FIG. 4). These may be referred to as "insert amplicon pairs." However, the standards may also include only either the upstream insert amplicon or the downstream insert amplicon.

[0171] 2B shows a representative pool of standards including a pool of nucleic acid standards including a 5' universal oligonucleotide and a 3' universal oligonucleotide. As shown in FIG. 2B, the pool of standards can include upstream and downstream insert amplicons prepared as shown in FIG.

[0172] D. Standards containing partially overlapping oligonucleotides In some embodiments, the pool of nucleic acid standards of different lengths includes UMIs and nucleic acid standards that include 5' partially overlapping oligonucleotides, where the 5' partially overlapping oligonucleotides are identical over at least a portion of their sequence for all standards, and / or 3' partially overlapping oligonucleotides, where the 3' partially overlapping oligonucleotides are identical over at least a portion of their sequence for all standards, and the length of the 5' partially overlapping oligonucleotides and / or the 3' partially overlapping oligonucleotides determines the length of the standards.

[0173] As used herein, "partially overlapping oligonucleotides" refers to oligonucleotides that are identical over at least a portion of their sequence for all standards. In some embodiments, standards include both 5' and 3' partially overlapping oligonucleotides.

[0174] As used herein, a "5' partially overlapping oligonucleotide" is an oligonucleotide that is 5' to a UMI contained in a standard, such as represented by 104 in Figure 8C. As used herein, a "3' partially overlapping oligonucleotide" is an oligonucleotide that is 3' to a UMI contained in a standard, such as represented by 105 in Figure 8C. In some embodiments, the 5' partially overlapping oligonucleotide and the 3' partially overlapping oligonucleotide are different. In some embodiments, the 5' partially overlapping oligonucleotide and the 3' partially overlapping oligonucleotide contain different numbers of nucleotides.

[0175] In some embodiments, the 5' partially overlapping oligonucleotides comprise at least a first portion of the sequence of interest, and the 3' partially overlapping oligonucleotides comprise at least a second portion of the sequence of interest. In other words, the 5' partially overlapping oligonucleotides comprise at least a first portion of the sequence of interest, and the 3' partially overlapping oligonucleotides may correspond to different portions of the sequence of interest.

[0176] In some embodiments, the standards include only 5' partially overlapping oligonucleotides (and no 3' partially overlapping oligonucleotides). In some embodiments, the standards include only 3' partially overlapping oligonucleotides (and no 5' partially overlapping oligonucleotides). Standards that include only 5' or 3' partially overlapping oligonucleotides can be useful to control for deletion mutations that result in the loss of large regions in the sequence of interest.

[0177] In some embodiments, the 5' partially overlapping oligonucleotides and / or the 3' partially overlapping oligonucleotides each comprise an amplicon amplified from a sequence of interest, as shown in FIG.

[0178] 1. Deletion amplicon 5' partially overlapping oligonucleotides may be referred to as 5' deletion amplicons or upstream deletion amplicons when generated by amplification from a sequence of interest. 3' partially overlapping oligonucleotides may be referred to as 3' deletion amplicons or downstream deletion amplicons when generated by amplification from a sequence of interest. For example, as shown in FIG. 5, each of the upstream deletion amplicons contains a portion of the sequence of interest (shown in black), and each of the downstream deletion amplicons also contains a portion of the sequence of interest (shown in black). In some embodiments, the portions of the sequence of interest contained in the upstream deletion amplicons and the downstream deletion amplicons may be different. FIG. 5 shows how representative upstream deletion amplicons and downstream deletion amplicons can be generated using primers that contain restriction enzyme cleavage sites (e.g., RS3 and RS4) and bind to LA-fwd and LA-rev primer binding sequences and other sequences contained in the sequence of interest.

[0179] As used herein, a "deletion amplicon" can refer to an amplicon that is either a 5' deletion amplicon or a 3' deletion amplicon. In general, a "deletion amplicon" is not limited by its location in a standard.

[0180] In some embodiments, the reverse and forward primers used to prepare the deletion amplicon determine the size of the deletion amplicon, hi some embodiments, a single primer pair generates a deletion amplicon of a desired size.

[0181] In some embodiments, the standards include both the upstream deletion amplicon and the downstream deletion amplicon (as shown in FIG. 5). These may be referred to as "deletion amplicon pairs." However, the standards may also include only either the upstream deletion amplicon or the downstream deletion amplicon.

[0182] In some embodiments, the 5' and / or 3' partially overlapping oligonucleotides each comprise a sequence that is 20 bp to 1 kb smaller than the sequence of interest. In other words, the 5' and / or 3' partially overlapping oligonucleotides can correspond to sequences found in deletion mutations of the sequence of interest.

[0183] FIG. 6B shows a representative pool of standards prepared as shown in FIG. 5, including a pool of nucleic acid standards containing upstream and downstream deletion amplicons.

[0184] II. How to prepare standards The standards and methods of use of the present invention are not limited by the means of generating the standards, in some embodiments, the standards are generated by ligating oligonucleotides together to prepare the standards.

[0185] Described herein is a method for generating a pool of nucleic acid standards, the method comprising providing multiple copies of at least one sequence of interest comprising a nucleic acid, providing a series of oligonucleotides each comprising a UMI, providing a series of insertion oligonucleotides of varying lengths, and ligating the at least one sequence of interest, at least one oligonucleotide comprising a UMI, and at least one insertion amplicon to generate a plurality of nucleic acid standards of the pool of nucleic acid standards.

[0186] In some embodiments, at least one sequence of interest and / or insertion oligonucleotide is prepared by amplification.

[0187] In some embodiments, the sequence of interest, the respective UMI-containing oligonucleotide, and / or the insertion oligonucleotide comprises a restriction enzyme cleavage site, hi some embodiments, the restriction enzyme cleavage site is proximal to the 5' and / or 3' end of the sequence of interest, the respective UMI-containing oligonucleotide, and / or the insertion oligonucleotide.

[0188] In some embodiments, the method further comprises cleaving the sequence of interest, each UMI-containing oligonucleotide, and / or the insertion oligonucleotide with a restriction enzyme prior to ligation. In some embodiments, cleaving with a restriction enzyme generates sticky ends for ligation. In some embodiments, the UMI-containing oligonucleotide is designed to contain a desired restriction enzyme cleavage site that is also contained in the sequence of interest.

[0189] Further described herein is a method for generating a pool of nucleic acid standards, the method comprising providing multiple copies of at least one sequence of interest comprising a nucleic acid, providing a set of oligonucleotides, each of which comprises a UMI, and ligating the at least one sequence of interest to at least one oligonucleotide comprising a UMI.

[0190] In some embodiments, at least one sequence of interest is prepared by amplification. In some embodiments, the sequence of interest and / or the oligonucleotide containing the UMI, respectively, comprises a restriction enzyme cleavage site. In some embodiments, the restriction enzyme cleavage site is proximal to the 5'-end and / or the 3'-end of the sequence of interest and / or the oligonucleotide containing the UMI, respectively.

[0191] In some embodiments, the method further comprises cleaving the oligonucleotides containing the sequence of interest and / or each UMI with a restriction enzyme prior to ligation.

[0192] In some embodiments, cleaving with a restriction enzyme generates sticky ends for ligation.

[0193] In some embodiments, a larger number of UMIs are available compared to the number of LongAmp standards being used, thus the number of UMIs is greater than the number of standards being created and duplication of UMIs is minimized.

[0194] III. Methods for normalizing amplicon size bias The pools of standards described herein can be used in methods to normalize amplicon size bias.

[0195] A method for normalizing amplicon size bias is described herein, the method including combining a sample containing a target nucleic acid with a pool of nucleic acid standards of different lengths, each standard containing a UMI, amplifying amplicons of a sequence of interest contained in the standards and the target nucleic acid, sequencing the amplicons of the standards and the sequence of interest to generate sequencing data, using the sequencing data from the standards to determine a bias profile based on amplicon size, and using the bias profile to normalize the amplicon size bias.

[0196] As used herein, "amplicon size bias" refers to the fact that amplicons of different sizes are amplified differently. In some embodiments, fewer large amplicons are produced in a given amplification reaction compared to shorter amplicons. In some embodiments, the amplification comprises PCR amplification. In some embodiments, the amplification is LongAmp PCR.

[0197] LongAmp PCR involves the amplification of DNA lengths that typically cannot be amplified using conventional PCR methods or reagents. Enzymes optimized for LongAmp PCR can be referred to as long-range polymerases. The results of LongAmp PCR are improved when complete amplicons are generated, so the generation of incomplete amplicons in a cycle leads to the generation of further incomplete amplicons in later PCR cycles. In some embodiments, the long-range polymerase has high processivity (i.e., incorporation of a relatively large number of nucleotides during a single binding event by DNA polymerase) and / or high extension rate.

[0198] Long-range polymerases with high processivity and high extension rates ensure efficient DNA synthesis of long templates and help shorten cycle times. A wide variety of protocols and long-range polymerases for use in LongAmp PCR are known, such as LongAmp Taq DNA polymerase and Phusion DNA polymerase (New England Biolabs). In some embodiments, the long-range polymerase is PrimeSTAR GXL DNA polymerase (Takara).

[0199] In some embodiments, amplicon size bias in LongAmp PCR can be normalized using the method described herein using nucleic acid standards. In some embodiments, standards are used to generate bias profiles, which can be used to normalize data for amplicons generated from sequences of interest. In some embodiments, the effect of amplicon size on the amplification of amplicons from sequences of interest can be normalized using data generated using standards described herein.

[0200] In some embodiments, amplifying an amplicon of the sequence of interest comprises amplifying an amplicon from the target nucleic acid using a pair of PCR primers that bind to primer binding sequences at the ends of the sequence of interest. In some embodiments, the standard comprises the same primer binding sequences as those at the ends of the sequence of interest.

[0201] In some embodiments, the method further comprises generating a library of fragments after amplification and prior to sequencing.

[0202] In some embodiments, generating a library of fragments is by tagmentation. Such a method is shown in FIG. 1, where fragments are generated by the Nextera fragmentation protocol. Such a method generates, for example, fragments containing different insertion mutations (indicated by arrows in FIG. 1). In this "long amp" PCR and fragmentation step, a pool of standards as described herein can be added to normalize amplicon size bias during PCR. In this way, the pool of standards is subjected to the same amplification and fragmentation conditions as the sequences of interest.

[0203] In some embodiments, the sequencing data from the standard used to determine the bias profile is the unique molecular number of UMIs contained in the standard. In other words, one skilled in the art can use standard analysis of the sequencing data to determine the number of UMIs replicated from different standards. Because these UMIs are derived from standards of different lengths, the number of different UMIs can provide a measure of the amplification efficiency of amplicons of different sizes to generate a bias profile. In this way, the number of amplicons generated for sequences different from the sequence of interest (including amplicons generated from the wild-type sequence of interest and also from the sequence of interest containing indels) can be compared to the bias profile. In other words, the comparison of data generated from the sequence of interest compared to the standard can be used to normalize the sequencing data for amplicon size bias. For example, if a similarly sized insertion standard to a large insertion mutation of the sequence of interest is amplified at a rate three times lower than a similarly sized standard to the wild-type sequence of interest, the user can normalize the copy number of these large insertion mutations compared to the wild-type sequence. Similarly, one skilled in the art can normalize for a large number of large deletion mutations (i.e., where a large amount of sequence is lost) compared to the wild-type sequence using a deletion standard.

[0204] A. Long Amplification PCR and Sequencing Long amplification PCR (LongAmp) refers to a PCR reaction optimized for long amplicons. Such a LongAmp reaction is shown in Figure 1 ("long amp" PCR). Methods for such optimized LongAmp PCR are well known in the art.

[0205] In some embodiments, the long amplicon can be greater than 5,000 kilobases, greater than 10,000 kilobases, or greater than 20,000 kilobases.

[0206] In some embodiments, long amplicons are generated from a sequence of interest that may contain large insertion mutations. For example, the long amplicon may be about 10,000 kilobases, while the wild-type amplicon from the sequence of interest is about 1,000 kilobases.

[0207] In some embodiments, LongAmp is used to optimize the identification of long insertion mutations in a sequence of interest.

[0208] LongAmp PCR may be followed by library preparation prior to sequencing of the library fragments, for example, tagmentation may be used (e.g., using Illumina's Nextera system) to prepare libraries for sequencing.

[0209] In some embodiments, standards are used to perform control assays. In some embodiments, these control assays are separate from the LongAmp PCR reactions. In some embodiments, the standards are spiked in known amounts into each LongAmp PCR reaction. By "spiked in" is meant that the standards are amplified in the same reaction solution as the LongAmp PCR reactions.

[0210] IV. Methods for Determining DNA Damage in Libraries Described herein is a quantitative PCR (qPCR) method for quality control (QC) of libraries. Such a method can allow users to determine the amount of DNA damage present in a library before carrying out further analysis of the library, such as sequencing. In some embodiments, the QC assay distinguishes between libraries with different levels of damage.

[0211] In some embodiments, these libraries can be used for sequencing. In some embodiments, the libraries are intended for long-read sequencing. In some embodiments, the libraries are prepared using tagmentation and / or bead-linked transposomes. The method of the present invention for determining DNA damage in a library can be used with libraries generated by any method.

[0212] As used herein, a "library molecule" refers to a single molecule contained within a library. In some embodiments, each library molecule may contain an insert that is different from the target nucleic acid. Library molecules may be generated using standard tagmentation or ligation protocols that are well known in the art.

[0213] Many sequencing applications require the presence of one or more adapters in the library molecule. Often these adapter sequences are at both ends of the insert. In some embodiments, sequences contained in the adapters are used in sequencing applications, for example, to allow binding of the library molecule to a flow cell or binding of a sequencing primer to the library molecule. In some embodiments, adapter sequences are required at both ends of the insert for sequencing applications, such as for binding to two different sequencing primer sequences. In such scenarios, library molecules (e.g., nicked libraries or their amplicons) lacking one adapter sequence cannot be successfully sequenced.

[0214] In some embodiments, the library includes library molecules that include long read hairpin adaptors. The insert size in the long read library molecules can be 5 kb or more, 10 kb or more, 15 kb or more, 20 kb or more, 25 kb or more, or 30 kb or more. In some embodiments, hairpin adaptors can be added to long regions of DNA contained in inserts in library molecules. In some embodiments, hairpin adaptors can be added to inserts using ligation or tagmentation protocols. For example, NEB's NEBNext Multiplex Oligos for Illumina® uses adaptor ligation with a unique hairpin loop structure that minimizes adaptor dimer formation.

[0215] In some embodiments, hairpin adaptors may be added to the inserts during the tagmentation reaction. "Tagmentation" refers to the use of a transposase to fragment and tag nucleic acids. Tagmentation involves modification of DNA with a transposome complex that includes a transposase enzyme complexed with one or more tags (such as adaptor sequences) that include transposon end sequences (referred to herein as transposons). Tagmentation can result in simultaneous fragmentation of DNA and ligation of adaptors to the 5' ends of both strands of the double-stranded fragments. However, tagmentation is only one method of generating libraries, and other methods (such as ligation) can also be used to generate libraries for use in the QC assays of the present invention.

[0216] In some embodiments, a method for determining the presence of DNA damage in a library comprising one or more library molecules, each library molecule comprising a double-stranded DNA insert having a hairpin adapter at each end of the insert, the method comprising: denaturing a first strand and a second strand of the double-stranded DNA insert comprised in the library molecule, annealing a forward primer and a reverse primer to the library molecule, amplifying to generate a library amplicon, and assessing the presence of DNA damage based on the number of library amplicons generated. An exemplary method is shown in Figure 9, which shows that a nicked library molecule does not generate a full-length amplicon.

[0217] The methods described herein can use long-range polymerases to amplify library molecules for QC. In some embodiments, the QC assay distinguishes libraries with different levels of damage, resulting in a Cq value that correlates with the damage rate in the library preparation. The methods described herein can be applied to any library that contains one or more hairpin adapters, and can be used in particular for library preparation of long inserts for long-read sequencing. In some embodiments, the use of the QC assay of the present invention avoids the use of damaged libraries, resulting in savings in time, money, and consumables.

[0218] A. DNA damage in libraries All methods of library preparation can introduce damage to the nucleic acids during the preparation process. For example, any pipetting step can result in shearing of the nucleic acids. Although users can take steps to reduce potential damage, this damage cannot be completely avoided or predicted.

[0219] The inserts in the library molecules may comprise double-stranded nucleic acids obtained as fragments from one or more larger nucleic acids. Fragmentation can be performed using any of a variety of techniques known in the art, including, for example, nebulization, sonication, chemical cleavage, enzymatic cleavage, or physical shearing. However, any of these fragmentation methods have the potential to introduce DNA damage (e.g., DNA nicking).

[0220] Therefore, it is important to be able to assess DNA damage in a library. For example, users do not want to perform further sequencing on libraries with extensive DNA damage because the sequencing quality is poor. Similarly, if much of the library is damaged, users may have difficulty determining the appropriate amount of library product to sequence. For many sequencing platforms, library molecules require adapter sequences at both ends of the fragments for use such as binding to flow cells or binding to sequencing primers. If the appropriate adapter is not present, for example, if the library molecule has DNA damage, the library molecule (and its amplicon) will not generate analyzable sequencing data.

[0221] The assessment of DNA damage can allow users to avoid further use of damaged libraries.In this way, users can save time and reagent costs for applications such as sequencing, where low library quality prevents the generation of high quality data.In some embodiments, low quality libraries are excluded from sequencing.

[0222] In some embodiments, the DNA damage is one or more nicks. In some embodiments, the one or more nicks can be converted to double-stranded breaks before performing the QC assay.

[0223] 1. Nick In some embodiments, the DNA damage comprises one or more nicks in the library molecule. As used herein, one or more nicks may be a single nick or multiple separate nicks.

[0224] In some embodiments, one or more nicks are in the insert contained in the library molecule. Since the insert can be a double-stranded insert, the nick refers to a break in one strand of the insert, where the break is not present in the other strand at that position. Thus, as used herein, a nick can refer to a discontinuity in a double-stranded DNA insert, where there is no phosphodiester bond between adjacent nucleotides in one strand. In some embodiments, one or more nicks are generated by DNA damage during library preparation. For example, shearing during pipetting can result in a nick in the library molecule.

[0225] In some embodiments, as described below, a greater proportion of library molecules in the library that contain one or more nicks produces a greater Cq value in a QC assay.

[0226] In some embodiments, the DNA damage comprises two or more nicks in the library molecule, wherein the nicks are in the same strand of the double-stranded DNA insert.

[0227] In some embodiments, the DNA damage comprises two or more nicks in the library molecule, the nicks being in both strands of the double-stranded DNA insert. If the two or more nicks are in different strands, these nicks may be in different positions to distinguish them from double-stranded DNA breaks, as described below.

[0228] When a nick is encountered during amplification, DNA polymerase may not be able to extend the amplicon beyond the nick. Thus, one or more nicks may result in the generation of an incomplete amplicon that does not have the complete sequence of the library molecule. In some embodiments, when a library molecule contains one or more nicks, the forward primer and / or reverse primer cannot generate an amplicon that corresponds to the complete sequence of the library molecule. Such an amplicon that does not contain the complete sequence of the library may not be sequenceable (due to the lack of adapter sequences that should be at one or both ends of the insert).

[0229] In some embodiments, amplicons generated from library molecules that contain nicks lack sequences for binding to the forward and / or reverse primers.

[0230] In some embodiments, library molecules containing nicks generate fewer amplicons during amplification compared to library molecules that do not contain nicks. As described below, the QC method of the present invention can estimate the Cq value of library molecules containing nicks and therefore indicate to a user that the library is of relatively low quality (having a high Cq value) or relatively high quality (having a low Cq value). In this way, the Cq value can be used to estimate the quality of a given library to evaluate whether to further evaluate the library, such as by sequencing, and to avoid the time and cost associated with sequencing a library that produces insufficient data.

[0231] 2. Double-stranded DNA breaks generated from nicks In some embodiments, the method further comprises generating a double-stranded break from the nick. In some embodiments, the double-stranded break is generated from the nick prior to annealing the forward and reverse primers in the QC method.

[0232] In some embodiments, an enzyme is used to prepare a double-stranded break from the nick. In other words, generating a double-stranded break can be performed using an enzyme reaction. In some embodiments, the enzyme reaction is performed by an endonuclease. In some embodiments, the endonuclease is T7 endonuclease.

[0233] In some embodiments, a library molecule that contains a double-stranded break does not generate an amplicon corresponding to the full sequence of the library molecule during amplification, in some embodiments, the double-stranded break cleaves the library molecule within the insert and a full-length amplicon of the library molecule cannot be generated after cleavage.

[0234] In some embodiments, the amplicon generated from the library molecule containing the double-stranded break lacks sequences for binding to the forward primer and / or reverse primer. In some embodiments, the double-stranded break cleaves the library molecule within the insert, separating the primer binding sequences contained in the two different hairpin adapters (associated with the two ends of the library insert). In some embodiments, after cleavage, neither the forward primer nor the reverse primer can generate a full-length amplicon after binding to the library molecule.

[0235] B. Hairpin adapter As used herein, "hairpin" refers to a nucleic acid that comprises a pair of nucleic acid sequences that are at least partially complementary to each other. These two nucleic acid sequences that are at least partially complementary can bind to each other and mediate folding of the nucleic acid. In some embodiments, the two nucleic acid sequences that are at least partially complementary generate a nucleic acid with a hairpin secondary structure.

[0236] As used herein, a "hairpin adaptor" refers to an adaptor that comprises at least one pair of nucleic acid sequences that are at least partially complementary to each other. In some embodiments, the hairpin adaptor has a folded secondary structure.

[0237] In some embodiments, the hairpin adaptor comprises one or more adaptor sequences. In some embodiments, the adaptor sequence comprises a primer sequence, an index tag sequence, a capture sequence, a barcode sequence, a cleavage sequence, or a sequencing-related sequence, or a combination thereof. As used herein, a sequencing-related sequence may be any sequence associated with a subsequent sequencing step. A sequencing-related sequence may act to simplify downstream sequencing steps. For example, a sequencing-related sequence may be a sequence that would otherwise be incorporated via a step of ligating an adaptor to a nucleic acid fragment. In some embodiments, the adaptor sequence comprises a P5 or P7 sequence (or their complements) to facilitate binding to a flow cell in a particular sequencing method.

[0238] In some embodiments, the hairpin adaptor comprises an amplification primer sequence (i.e., a sequence that binds to an amplification primer sequence). In some embodiments, the hairpin adaptor comprises an amplification primer sequence and all or a portion of a sequence that is at least partially complementary to the adaptor sequence. In some embodiments, the amplification primer sequence included in the hairpin is a universal primer sequence. A universal sequence is a region of nucleotide sequence that is common (i.e., shared) by two or more nucleic acid fragments.

[0239] In some embodiments, either the forward primer or the reverse primer binds to one or more sequences contained within one or both hairpin adaptors. In some embodiments, both the forward primer and the reverse primer bind to one or more sequences contained within one or both hairpin adaptors. In some embodiments, the forward primer binds to a sequence contained within the hairpin adaptor attached to a first end of the double-stranded DNA insert, and the reverse primer binds to a sequence contained within the hairpin adaptor attached to a second end of the double-stranded DNA insert.

[0240] In some embodiments, the library molecules comprise an insert that comprises a double-stranded nucleic acid and a hairpin adaptor at both ends of the insert. In some embodiments, the insert comprises a fragment from the target nucleic acid. Methods for incorporating hairpin adaptors are well known in the art, such as by ligation or tagmentation.

[0241] For example, NEBNext® Multiplex Oligos for Illumina® (New England BioLabs) provide hairpin adaptors and primers to increase the yield of library products. In some embodiments, the hairpin adaptors contain a hairpin loop structure that minimizes adaptor dimer formation. In some embodiments, the hairpin adaptors are ligated to end-repaired dA-tailed DNA. In some embodiments, the hairpin adaptors contain a loop containing uracil that is removed by treatment with the USER Reagent. In some embodiments, the USER Enzyme is a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase (such as endonuclease VIII). In some embodiments, USER treatment can open the loop of the hairpin adaptor, making it available as a substrate for amplification to incorporate index primers and subsequent sequencing.

[0242] In some embodiments, hairpin adaptors are incorporated using locus-specific primers and USER Reagents to generate overhangs for ligating hairpin adaptors. An exemplary method is SMRTbell library preparation (see Pacific Biosciences, SMRTbell Library Preparation & SMRT Sequencing Workflow Updates, 2017).

[0243] In some embodiments, hairpin adaptors are included in library molecules with relatively large inserts, and the library molecules are designed for long read sequencing.

[0244] In some embodiments, each hairpin adaptor comprises an amplification primer binding site. In some embodiments, the hairpin adaptor at a first end of the insert comprises a different amplification primer binding site than the hairpin adaptor at a second end of the insert. In some embodiments, the hairpin adaptor at the first end of the insert comprises a first amplification primer binding site and the hairpin adaptor at the second end of the insert comprises a second amplification primer binding site. In some embodiments, the first amplification primer binding site and the second amplification primer binding site mediate amplification in opposite directions.

[0245] In some embodiments, such as those shown in FIG. 9, a hairpin adapter at a first end of the insert may include a forward amplification primer binding site and a hairpin adapter at a second end of the insert may include a reverse amplification primer binding site.

[0246] C. Amplification In some embodiments, the method further comprises amplifying the library molecule using an amplification primer that binds to the amplification primer sequence, hi some embodiments, one or both hairpin adaptors included in the library molecule comprise an amplification primer.

[0247] In some embodiments, the amplifying is optimized to amplify library molecules that are 5 kb or more, 10 kb or more, 15 kb or more, 20 kb or more, 25 kb or more, or 30 kb or more.

[0248] In some embodiments, the amplifying is performed with a polymerase that is optimized for amplifying long amplicons, hi some embodiments, the polymerase is optimized for amplifying amplicons of 20 kb or more, or 30 kb or more.

[0249] Many exemplary polymerases that are optimized for amplifying long amplicons are known in the art. One exemplary polymerase is PrimeSTAR GXL DNA polymerase (Takara).

[0250] In some embodiments, the polymerase has a higher processivity and / or extension rate compared to wild-type Taq polymerase, hi some embodiments, the polymerase contains one or more mutations or fusions that increase the processivity or extension rate.

[0251] As used herein, the "processivity" of a polymerase refers to the number of nucleotides that the polymerase can incorporate into DNA during a single template-binding event before dissociating from the DNA template. Thus, a polymerase with relatively high processivity can incorporate a large number of nucleotides during a single template-binding event. Higher processivity can increase the likelihood that a complete amplicon is generated during a PCR cycle.

[0252] As used herein, the "extension rate" of a polymerase is the number of nucleotides that can be incorporated into DNA over a period of time. In some embodiments, a polymerase with a relatively high extension rate can generate a complete amplicon of a library molecule during a PCR cycle. In some embodiments, the polymerase has an extension rate of 2 kb / min or more, 3 kb / min or more, or 4 kb / min or more.

[0253] In some embodiments, the polymerase has an extension rate of 3 kb / min or greater.

[0254] In some embodiments, the amplification is exponential.

[0255] In some embodiments, 30 or more or 40 or more cycles of amplification are performed.

[0256] In some embodiments, the amplification primers may include index sequences. These index sequences may be used to identify the location in the sample and the array. In some embodiments, the index sequence includes a unique molecular identifier (UMI). UMIs are described in International Publication Nos. WO 2016 / 176091, WO 2018 / 197950, WO 2018 / 197945, WO 2018 / 200380, and WO 2018 / 204423, each of which is incorporated herein by reference in its entirety.

[0257] In some embodiments, the sample is amplified on a solid support.

[0258] For example, in some embodiments, samples are amplified using cluster amplification methodology, as exemplified by the disclosures of U.S. Patent Nos. 7,985,565 and 7,115,400, the contents of each of which are incorporated herein by reference in their entirety. The incorporated materials of U.S. Patent Nos. 7,985,565 and 7,115,400 describe a method of solid-phase nucleic acid amplification that allows the amplification products to be immobilized on a solid support to form an array consisting of clusters or "colonies" of immobilized nucleic acid molecules. Each cluster or colony on such an array is formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary polynucleotide strands. Arrays so formed are generally referred to herein as "clustered arrays." The product of a solid-phase amplification reaction, such as those described in U.S. Patent Nos. 7,985,565 and 7,115,400, is a so-called "bridged" structure formed by annealing a pair of immobilized polynucleotide strands and immobilized complementary strands, both of which are immobilized on a solid support at their 5' ends, in some embodiments, via covalent bonds. The cluster amplification methodology is an example of a method for producing immobilized amplicons using an immobilized nucleic acid template. Other suitable methodologies can also be used to produce immobilized amplicons from the immobilized DNA fragments produced according to the methods provided herein. For example, one or more clusters or colonies can be formed by solid-phase PCR, regardless of whether one or both primers of each pair of amplification primers are immobilized.

[0259] In other embodiments, the sample is amplified in solution. For example, in some embodiments, the sample is cleaved or otherwise released from the solid support, and then the amplification primer is hybridized to the released molecule in solution. In other embodiments, the amplification primer is hybridized to the desired sample for one or more initial amplification steps, followed by subsequent amplification steps in solution. In some embodiments, immobilized nucleic acid templates can be used to generate liquid phase amplicons.

[0260] It will be understood that any of the amplification methodologies described herein or generally known in the art can be used with universal or target-specific primers to amplify the desired sample. Suitable methods for amplification include, but are not limited to, polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription-mediated amplification (TMA) and nucleic acid sequence-based amplification (NASBA), as described in U.S. Pat. No. 8,003,354, the entirety of which is incorporated herein by reference. The above amplification methods can be used to amplify one or more nucleic acids of interest. For example, PCR, including multiplex PCR, SDA, TMA, NASBA, etc., can be used to amplify immobilized DNA fragments. In some embodiments, primers specifically directed to the nucleic acid of interest are included in the amplification reaction.

[0261] Other suitable methods for amplifying polynucleotides can include oligonucleotide extension and ligation, rolling circle amplification (RCA) (Lizardi et al., Nat. Genet. 19:225-232 (1998), incorporated herein by reference), and oligonucleotide ligation assay (OLA) (see generally U.S. Pat. Nos. 7,582,420, 5,185,243, 5,679,524, and 5,573,907; European Patent Nos. 0320308(B1); 0336731(B1); 0439182(B1); WO 90 / 01069; WO 89 / 12696; and WO 89 / 09835, all of which are incorporated by reference). It should be understood that these amplification methodologies can be designed to amplify immobilized DNA fragments. For example, in some embodiments, the amplification method can include a ligation probe amplification or oligonucleotide ligation assay (OLA) reaction that includes a primer specifically directed to the nucleic acid of interest. In some embodiments, the amplification method can include a primer extension ligation reaction that includes a primer specifically directed to the nucleic acid of interest. As a non-limiting example of primer extension and ligation primers that can be specifically designed to amplify the nucleic acid of interest, the amplification can include primers used in the GoldenGate assay (Illumina, Inc., San Diego, CA) as exemplified in U.S. Patent Nos. 7,582,420 and 7,611,869, each of which is incorporated herein by reference in its entirety.

[0262] Exemplary isothermal amplification methods that can be used in the methods of the present disclosure include, but are not limited to, Multiple Displacement Amplification (MDA), as exemplified, for example, by Dean et al., Proc. Natl. Acad. Sci. USA 99:5261-66 (2002), or isothermal strand displacement nucleic acid amplification, as exemplified, for example, by U.S. Pat. No. 6,214,587, each of which is incorporated herein by reference in its entirety. Other non-PCR-based methods that can be used in the present disclosure include strand displacement amplification (SDA), as described, for example, in Walker et al., Molecular Methods for Virus Detection, Academic Press, Inc., 1995, U.S. Patent Nos. 5,455,166 and 5,130,238, and Walker et al., Nucl.Acids Res.20:1691-96 (1992), or hyperbranched strand displacement amplification, as described, for example, in Lage et al., Genome Research 13:294-307 (2003), each of which is incorporated herein by reference in its entirety. Isothermal amplification methods can be used with strand-displacing Phi 29 polymerase or large fragment, 5'→3' exo- of Bst DNA polymerase for random primer amplification of genomic DNA. The use of these polymerases takes advantage of their high processivity and strand displacement activity. High processivity allows the polymerase to produce fragments of 10-20 kb in length. As noted above, polymerases with low processivity and strand displacement activity, such as Klenow polymerase, can be used to produce smaller fragments under isothermal conditions. Further description of the amplification reaction, conditions, and components is described in detail in the disclosure of U.S. Patent No. 7,670,810, which is incorporated herein by reference in its entirety.

[0263] D. Sequencing In some embodiments, the method further comprises sequencing the library products and the amplified library products (i.e., amplicons). In some embodiments, the analysis of the library after the QC assay is sequencing.

[0264] In some embodiments, the method comprises determining conditions for analysis of the library based on the Cq value. In some embodiments, a QC assay is used to determine the conditions for sequencing the library. In some embodiments, a QC assay is used to determine that a given library should not be sequenced. For example, a QC assay may infer that there are not enough library molecules in a given library, and as a result, the sequencing data generated from the library is of low quality.

[0265] In some embodiments, the methods allow for sequencing of the entire sequence of the insert.

[0266] One exemplary sequencing methodology is sequencing-by-synthesis (SBS). In SBS, the extension of a nucleic acid primer along a nucleic acid template is monitored to determine the sequence of nucleotides in the template. The underlying chemical process can be polymerization (e.g., catalyzed by a polymerase enzyme). In certain polymer-based SBS embodiments, fluorescently labeled nucleotides are added to the primer (thereby extending the primer) in a template-dependent manner such that detection of the order and type of nucleotides added to the primer can be used to determine the sequence of the template.

[0267] A flow cell provides a convenient solid support for sequencing. For example, to initiate the first SBS cycle, one or more labeled nucleotides, DNA polymerase, etc. can be flowed / passed through a flow cell housing one or more amplified nucleic acid molecules. The site at which the primer extension incorporates the labeled nucleotide can be detected. Optionally, the nucleotide can further include a reversible termination feature that terminates further primer extension once the nucleotide is added to the primer. For example, a nucleotide analog with a reversible terminator portion can be added to the primer such that no further extension occurs until a deblocking agent is delivered to remove the portion. Thus, in an embodiment using a reversible termination, a deblocking reagent can be delivered to the flow cell (before or after detection occurs). Washing can be performed between the various delivery steps. The cycle is then repeated n times to extend the primer with n nucleotides, thereby allowing a sequence of length n to be detected. Exemplary SBS procedures, fluidics systems and detection platforms that can be readily adapted for use with amplicons generated by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497, U.S. Pat. No. 7,057,026, WO 91 / 06678, WO 07 / 123744, U.S. Pat. Nos. 7,329,492, 7,211,414, 7,315,019, 7,405,281, and U.S. Patent Application Publication No. 2008 / 0108082, each of which is incorporated herein by reference.

[0268] Other sequencing procedures using cyclic reactions can be used, such as pyrosequencing. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent nucleic acid chain (Ronaghi et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi, Genome Res. 11(1), 3-11 (2001); Ronaghi et al. Science 281(5375), 363 (1998); U.S. Patent Nos. 6,210,891, 6,258,568, and 6,274,320, each of which is incorporated herein by reference). In pyrosequencing, the released PPi can be detected by its immediate conversion to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP produced can be detected via luciferase-generated photons. Therefore, sequencing reaction can be monitored through luminescence detection system.The excitation radiation source used in fluorescence-based detection system is not necessary for pyrosequencing procedure.Useful fluid systems, detectors and procedures that can be adapted to the application of pyrosequencing to the amplicons generated according to the present disclosure are described in, for example, International Publication No. 2012058096, US Patent Application Publication No. 2005 / 0191698(A1), US Patent No. 7,595,883 and US Patent No. 7,244,559, each of which is incorporated herein by reference.

[0269] Some embodiments may utilize methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation may be detected via fluorescence resonance energy transfer (FRET) interactions between fluorophore-bearing polymerases and γ-phosphate-labeled nucleotides or using zeromode waveguides (ZMW). Techniques and reagents for FRET-based sequencing are described, for example, in Levene et al. Science 299, 682-686 (2003), Lundquist et al. Opt. Lett. 33, 1026-1028 (2008), Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference.

[0270] Some SBS embodiments include detection of protons released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can be performed using electronic detectors and related technology available from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies), or the sequencing methods and systems described in U.S. Patent Application Publication Nos. 2009 / 0026082(A1), 2009 / 0127589(A1), 2010 / 0137143(A1), or 2010 / 0282617(A1), each of which is incorporated herein by reference. The methods described herein for amplifying nucleic acids using kinetic exclusion can be readily adapted to substrates used to detect protons. More specifically, the methods described herein can be used to generate clonal populations of amplicons used to detect protons.

[0271] Another useful sequencing technique is nanopore sequencing (see, e.g., Deamer et al. Trends Biotechnol. 18, 147-151 (2000); Deamer et al. Acc. Chem. Res. 35:817-825 (2002); Li et al. Nat. Mater. 2:611-615 (2003), the disclosures of which are incorporated herein by reference). In some nanopore embodiments, the nucleic acid or individual nucleotides removed from the nucleic acid pass through the nanopore. As the nucleic acid or nucleotide passes through the nanopore, the type of each nucleotide can be identified by measuring the fluctuation in the electrical conductance of the pore. (U.S. Pat. No. 7,001,792; Soni et al. Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2, 459-481 (2007); Cockroft et al. J. Am. Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference).

[0272] Exemplary methods for array-based expression and genotyping that can be applied for detection according to the present disclosure are described in U.S. Pat. Nos. 7,582,420, 6,890,741, 6,913,884, or 6,355,431, or U.S. Patent Publication Nos. 2005 / 0053980(A1), 2009 / 0186349(A1), or 2005 / 0181440(A1), each of which is incorporated herein by reference.

[0273] An advantage of the methods described herein is that they provide rapid and efficient detection of multiple nucleic acids in parallel. Thus, the present disclosure provides an integrated system that can prepare and detect nucleic acids using techniques known in the art, such as those exemplified above. Thus, the integrated system of the present disclosure can include fluidic components that can deliver amplification and / or sequencing reagents to one or more immobilized DNA fragments, the system including components such as pumps, valves, reservoirs, fluid lines, etc. A flow cell can be configured and / or used in the integrated system for detecting nucleic acids. Exemplary flow cells are described, for example, in U.S. Patent Application Publication Nos. 2010 / 0111768(A1) and 2012 / 0270305(A1), each of which is incorporated herein by reference. As exemplified for the flow cell, one or more of the fluidic components of the integrated system can be used for amplification and detection methods. Taking the nucleic acid sequencing embodiment as an example, one or more of the fluidic components of the integrated system can be used for delivery of sequencing reagents in the amplification methods described herein and in the sequencing methods as exemplified above. Alternatively, an integrated system may include separate fluidic systems for performing the amplification method and for performing the detection method. Examples of integrated sequencing systems that can generate amplified nucleic acids and sequence the nucleic acids include, but are not limited to, the MiSeq™ platform (Illumina, Inc., San Diego, Calif.) and the devices described in U.S. Patent Application Publication No. 2012 / 0270305, which is incorporated herein by reference.

[0274] E.Cq value In some embodiments, the number of library amplicons produced is estimated by quantitative PCR (qPCR). In some embodiments, the number of library amplicons produced is estimated by measuring the cycle of quantification (Cq, also known as quantification cycle) value.

[0275] As used herein, Cq value is the PCR cycle number at which the reaction curve of a sample crosses the threshold line.Therefore, Cq value indicates the number of PCR cycles required to detect a signal above noise for a given sample.

[0276] This can be determined using fluorescent dyes and probes, which measure the number of amplification cycles required to detect fluorescence. Using this method, the Cq value is the cycle number at which the fluorescence of the PCR product can be detected above the background signal. Thus, a higher Cq value indicates that less nucleic acid is present in the sample.

[0277] As described in Bustin et al., Clinical Chemistry 55(4):611-622(2009), the terms threshold cycle (Ct), crossing point (Cp), and top (TOP) all refer to the same measurement as the Cq value, and the difference in name is simply due to different instruments. These terms (Ct, Cp, and TOP) all refer to the method of determining the PCR cycle number at which the sample's reaction curve crosses the threshold line, and therefore these values ​​are all synonyms for the Cq value.

[0278] In some embodiments, the more library amplicons there are, the lower the Cq value.In some embodiments, the library with lower Cq value has less DNA damage.In some embodiments, the library with less DNA damage produces better sequencing results.

[0279] In some embodiments, those library products that contain a nick do not produce an amplicon that corresponds to the complete sequence of the library molecule, hi some embodiments, extension during an amplification cycle (i.e., production of an amplicon) stops at the site of the nick in the library molecule.

[0280] For example, Figure 9 shows how nicked library molecules (i.e., damaged libraries) generate less signal because amplification does not produce the complete sequence of a library molecule that has both the forward and reverse amplification primer binding sites.

[0281] In some embodiments, the Cq value correlates with the percentage of the lesion in the library. In some embodiments, the lesion was introduced during library preparation.

[0282] In some embodiments, a high Cq value correlates with more DNA damage in the library molecules. In some embodiments, libraries with high Cq values ​​exhibit lower sequencing performance. In some embodiments, lower sequencing performance is measured by total power (Gb) or P1 percent.

[0283] In some embodiments, anomalously low (eg, less than 2.58) Cq values ​​may also have poorer sequencing performance.

[0284] In some embodiments, a desired Cq range that will result in a sequencing run with adequate data quality depending on the use of the next library can be determined. In some embodiments, the desired Cq range can be 2.58 to 5. The Cq range can vary based on the particular type of library used. Thus, a user can perform initial experiments to determine a desired Cq range that will result in sequencing data of sufficient quality, and then select only sequence libraries with Cq values ​​within this range. Such analysis to determine a desired Cq range is readily performed by one of skill in the art, and such a determination is not considered an undue burden.

[0285] F. Long-read sequencing Standard short-read sequencing provides accurate base-level sequences to provide short-range information, but short-read sequencing may not provide long-range genomic information. Furthermore, long-range haplotype reconstruction is difficult with standard methods because haplotype information is not retained for sequenced genomes or references with short-read data. Thus, while standard sequencing and analysis approaches can generally call single nucleotide variants (SNVs), these methods may not identify the full spectrum of structural variations found in individual genomes. Genomic "structural variations" as used herein refer to events larger than SNVs, including events of 50 base pairs or more. Representative structural variants include copy number variations, inversions, deletions, and duplications.

[0286] "Concatenated long read sequencing" or "concatenated read sequencing" refers to a sequencing method that provides long-range information about a genomic sequence.

[0287] In some embodiments, linked read sequencing uses molecular barcodes to tag reads that originate from the same long DNA fragment.If a unique barcode is added to all the reads generated from an individual DNA molecule, the reads can be linked together for that DNA molecule.In other words, the reads that share a barcode can be classified as originating from a single long input molecule, allowing long-range information to be assembled from short reads.

[0288] In some embodiments, concatenated reads sequencing may be used for haplotype reconstruction. In some embodiments, concatenated reads sequencing improves calling of structural variants. In some embodiments, concatenated reads sequencing improves access to regions of the genome with limited accessibility. In some embodiments, concatenated reads sequencing is used for de novo diploid assembly. In some embodiments, concatenated reads sequencing improves sequencing of highly polymorphic sequences (such as human leukocyte antigen genes) that require de novo assembly.

[0289] In some embodiments, the sequencing is long read sequencing of library molecules that are 5 kb or more, 10 kb or more, 15 kb or more, 20 kb or more, 25 kb or more, or 30 kb or more.

[0290] G. Methods Involving Preparation of Double-Stranded DNA Breaks In some embodiments, the nick is converted into a double-stranded DNA break. The advantage of generating a double-stranded DNA break from a nick is that after the double-stranded break is generated in the library product, no amplicon corresponding to the complete library molecule can be generated. In this way, the library molecule containing a nick does not generate any amplicon corresponding to the complete sequence of the library product. In contrast, the nicked library molecule containing a nick in one strand of the double-stranded insert generates fewer amplicons, but can generate some amplicons corresponding to the complete sequence of the library product (shown in FIG. 9). This is because either the reverse primer or the forward primer can generate an amplicon corresponding to the complete sequence of the library molecule.

[0291] The advantage of generating a double-stranded break from a nick is that a library molecule with a double-stranded break cannot generate a full-length amplicon that has both binding sites for the forward and reverse primers.

[0292] In some embodiments, the nick is converted to a double-stranded break using an endonuclease. In some embodiments, the endonuclease is a mutant T7 endonuclease. In some embodiments, the mutant endonuclease is maltose binding protein (MBP)-T7 Endo I. In some embodiments, the T7 endonuclease generates a counter nick to generate a double-stranded break in the DNA where the nick was previously located in a single strand. The generation of such a double-stranded break from a nick can be referred to as a cut across the nick.

[0293] H. Method using SMRTbell template In some embodiments, the library molecule comprises two hairpin adaptors ligated to the ends of the double-stranded DNA fragments, in some embodiments, such adaptors form a closed loop.

[0294] Although the present invention is not limited to this preparation method, in some embodiments, the library molecules are SMRTbell templates. SMRTbell templates are well known in the art for use with single molecule real-time (SMRT) sequencing. In some embodiments, SMRT sequencing uses the method of Pacific Biosciences (PacBio) (see, e.g., Rhoads and Au, Genomics Proteomics Bioinformatics 13:278-289 (2015)). As used herein, SMRT sequencing and PacBio sequencing may be used interchangeably.

[0295] The SMRT sequencing approach utilizes circular consensus sequencing (CCS) to generate highly accurate, long, high-fidelity reads with >99% accuracy and ≥3 passes. To generate the highest output of HiFi reads per sequencing run, high-quality SMRTbell templates must be generated that allow for constant rolling circle amplification (RCA). For example, the PacBio Sequel system can sequence library molecules with ligated hairpin adaptors using on-platform RCA. Thus, to generate CCS reads, the polymerase should be sequenced in repeated passes to generate a long polymerase read length ≥3 times the length of the insert.

[0296] For the polymerase in the SMRT system to sequence efficiently, the input library must be of high quality. During the library preparation process, damage can be introduced into the DNA, either by pipetting, storage or other handling and / or procedural errors. When a nicked SMRTbell template is loaded into the Bio Sequel system for sequencing, the polymerase will drop out at the nick site, terminating the RCA, and as a result, the P1 percent will decrease along with the CCS output from that sequencing run.

[0297] The advantage of SMRT sequencing is its longer read lengths and faster runs than certain other sequencing methods. For example, the PacBio system is known to be capable of generating read lengths in excess of 60 kilobases. These longer read lengths can allow for precise location and sequence of repetitive regions within a single read, which may not be available on other sequencing platforms.

[0298] In summary, SMRT sequencing is known to have lower throughput, higher error rates, and higher cost per base than some other methods, and users will want to minimize these drawbacks. In some embodiments, the library quality control methods of the present invention allow users to select libraries for sequencing that are more likely to produce sequencing runs of sufficient quality using methods such as SMRT sequencing. In this way, users can avoid the expense and time spent on sequencing runs with DNA damage that limit their ability to produce quality sequencing data.

[0299] In some embodiments, the QC methods described herein maximize P1 percent and total output from an SMRT sequencing run. In some embodiments, the qPCR QC methods described herein allow customers to avoid loading damaged libraries onto the SMRT sequencing platform, thus saving time, money, reagents, and consumables. Figures 13A-15C show some representative data from QC assays with SMRT sequencing.

[0300] V. How to Determine DNA Damage Using Fluorescence The amount of DNA damage in a sample containing DNA can also be measured using fluorescence by the methods described herein. In some embodiments, DNA damage can be quantified in the sample DNA using fluorescence before libraries are prepared. Such a workflow can be very attractive because it allows the user to determine when there is too much DNA damage in the sample, which is detrimental to downstream assays such as sequencing. For example, the user can quantify DNA damage in a sample and then prepare libraries from the sample only if there is a low level (e.g., 5% or less) of DNA damage. In this way, the user can save time and resources by not preparing libraries from samples with moderate (e.g., greater than 5%) levels of DNA damage.

[0301] In some embodiments, a method for quantifying DNA damage in a sample comprising DNA using fluorescence includes: a. i. an aliquot of the sample containing DNA; ii. one or more DNA repair enzymes; iii. combining dNTPs, where one or more dNTPs are fluorescently labeled; b. preparing repaired DNA; c. Dephosphorylate the phosphate from dNTPs; d. binding the repaired DNA to carboxylate or cellulose beads; e. eluting the bound repaired DNA from the carboxylate or cellulose beads with a resuspension buffer; f. Measuring the fluorescence of the repaired DNA to determine the amount of DNA damage.

[0302] An overview of the method for quantifying DNA damage is shown in FIG. 16, and the results of representative experiments using this method are shown in FIGS.

[0303] In some embodiments, higher fluorescence of repaired DNA indicates more DNA damage, in other words, when there is a higher level of DNA damage, more fluorescently labeled dNTPs are incorporated.

[0304] In some embodiments, the fluorescence of repaired DNA is linear over a range of different amounts of DNA damage. In this way, the dynamic range of the assay (i.e., the full range of DNA damage that can be accurately measured) is improved, and thus the user can evaluate the relative differences in damage for various libraries. In some embodiments, if the user is evaluating samples for a sensitive downstream assay where relatively small amounts of DNA damage may adversely affect the results, the wide linear range can help to accurately determine the amount of DNA damage.

[0305] In some embodiments, the method can assess DNA damage in an aliquot of a sample. In other words, a user can take a small sample, quantify the DNA damage, and then potentially perform more assays (such as library preparation or sequencing) based on the results of the DNA damage quantification.

[0306] In some embodiments, the method can assess DNA damage induced by manipulation of a sample by evaluating an aliquot of the same sample before and after manipulation. In this way, the user can directly measure any DNA damage induced by the manipulation.

[0307] In some embodiments, the operation is sequencing of a sample. For example, a user may wish to evaluate the effect of different sequencing reagents on a sample containing DNA to determine whether a particular reagent induces DNA damage.

[0308] In some embodiments, measuring the fluorescence of the repaired DNA includes creating a standard curve of dilutions of the repaired DNA and measuring the fluorescence of the dilutions of the repaired DNA. In some embodiments, the use of a standard curve can increase the dynamic range of the assay to allow for quantification of small amounts of DNA damage. Such methods for quantifying small amounts of DNA damage can be useful when even small amounts of DNA damage can be detrimental to the results of downstream assays (such as sequencing).

[0309] In some embodiments, measuring the fluorescence of the repaired DNA includes comparing the fluorescence of the repaired DNA against a separate standard curve of dilutions of one or more fluorescently labeled dNTPs alone to determine the number of fluorophores contained in the repaired DNA.

[0310] In some embodiments, the method further comprises calculating a normalized number of fluorophores contained in the repaired DNA by dividing the determined number of fluorophores by the mass of the repaired DNA. Such a measure can estimate the percentage of DNA that is damaged.

[0311] In some embodiments, the DNA is a library comprising genomic DNA, cDNA, or fragmented double-stranded DNA. If the DNA is genomic DNA or cDNA, the method may be performed prior to library preparation.

[0312] In some embodiments, the DNA is genomic DNA and cDNA, and the method further comprises preparing a library after determining the amount of DNA damage.

[0313] In some embodiments, the library is prepared when the amount of DNA damage is 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of total nucleotides. In other words, the library can be prepared when DNA damage is determined to be low. The amount of DNA damage acceptable for library preparation or other downstream assays depends on the sensitivity of the downstream assay and the type of DNA damage. For example, short-read sequencing may give acceptable sequencing results even with a moderate level of DNA damage (e.g., 5% or less). In contrast, long-read sequencing may require a lower level of DNA damage (e.g., 2% or less) for acceptable results and may be more sensitive to damage induced by nicking.

[0314] In some embodiments, where an assay of the invention determines the presence of a particular type of damage (such as nicking), this damage may be repaired prior to further steps such as library preparation or sequencing.

[0315] In some embodiments, libraries are not prepared if the amount of DNA damage is 5% or more, 4% or more, 3% or more, 2% or more, or 1% or more of the total nucleotides. In this way, users avoid wasting time and resources in preparing libraries (and performing further downstream assays, such as sequencing) when there is a level of DNA damage that will adversely affect the results of the downstream assays.

[0316] In some embodiments, the repaired DNA is bound to carboxylate or cellulose beads and eluted more than once before measuring fluorescence. In some embodiments, multiple rounds of bead-based purification improve the results of the method. In some embodiments, multiple rounds of bead-based purification reduce non-specific signals. In some embodiments, multiple rounds of bead-based purification are performed, and the repaired DNA is bound to carboxylate or cellulose beads and eluted twice before measuring fluorescence.

[0317] Carboxylate beads (eg, SPRI beads) and cellulose beads are commercially available for DNA purification and size selection applications, and such beads may be used in the methods of the invention.

[0318] In some embodiments, the carboxylate or cellulose beads are magnetic, a property that can aid in washing the beads after binding of the repaired DNA.

[0319] In some embodiments, preparing the repaired DNA is performed at 37° C. In some embodiments, preparing the repaired DNA is performed for 10 minutes or more, 20 minutes or more, 30 minutes or more, 45 minutes or more, or 60 minutes or more.

[0320] In some embodiments, dephosphorylating the phosphate from the dNTP reduces non-specific binding of the dNTP and improves the assay results.

[0321] In some embodiments, dephosphorylating the phosphate from the dNTP is performed using an enzyme, hi some embodiments, the enzyme for dephosphorylating the phosphate from the dNTP is shrimp alkaline phosphatase (SAP) or calf intestinal alkaline phosphatase (CIP).

[0322] A variety of different DNA repair enzymes can be used in this method, and as used herein, "DNA damage" can refer to multiple different types of DNA modifications (e.g., nicks and thymine dimers) that can be present in the DNA contained in a single sample.

[0323] In some embodiments, the one or more DNA repair enzymes include a DNA polymerase. In some embodiments, the DNA polymerase has 5'-3' polymerase activity but lacks 5'-3' exonuclease activity. In some embodiments, the DNA polymerase is Bst DNA polymerase, large fragment. In some embodiments, the one or more DNA repair enzymes include a ligase. In some embodiments, the ligase is Taq ligase. In some embodiments, the DNA damage comprises a nick in double-stranded DNA.

[0324] In some embodiments, the one or more DNA repair enzymes include T4 pyrimidine dimer glycosylase (PDG). In some embodiments, the DNA damage includes thymine dimers. In some embodiments, the thymine dimers are induced by ultraviolet radiation.

[0325] In some embodiments, the one or more DNA repair enzymes comprise uracil DNA glycosylase (UDG) and an apurinic or apyrimidinic site lyase, hi some embodiments, the DNA damage comprises uracil.

[0326] In some embodiments, the one or more DNA repair enzymes comprise formamidopyrimidine DNA glycosylase (FPG) and an apurinic or apyrimidinic site lyase, hi some embodiments, the DNA damage comprises an oxidized base.

[0327] In some embodiments, two or more DNA repair enzymes are used. In some embodiments, one or more DNA repair enzymes are a mixture of multiple DNA repair enzymes. Such an approach can be used when the user suspects that DNA damage may include two or more types of damage modifications to DNA (i.e., a combination of thymine dimers and nicks or any other modifications).

[0328] In some embodiments, the dNTPs include dATP, dGTP, dCTP, and dTTP or dUTP. Any or all of the dNTPs may be fluorescently labeled. In some embodiments, all of the dNTPs are fluorescently labeled. In some embodiments, dUTP and dCTP are fluorescently labeled.

[0329] Any suitable fluorescent label may be included in the dNTP. In some embodiments, the fluorescent label is Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 633, fluorescein isothiocyanate (FITC), or tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC), although other fluorescent labels with a wide range of excitation spectra may be used. In some embodiments, the fluorescent label has an excitation wavelength that does not damage DNA.

[0330] Working Example Example 1. Normalizing amplicon size bias of LongAmp PCR reactions using standards Figure 1 shows a representative LongAmp PCR reaction followed by fragmentation using, for example, the Nextera product (Illumina). As described herein, a pool of nucleic acid standards of different lengths can be used to normalize for amplicon size bias in this experiment.

[0331] Long amplification PCR can be performed to generate amplicons from sequences of interest contained in target nucleic acid fragments within a sample (as shown in Figure 1). The sample may be a sample comprised of nucleic acid that has been subjected to gene editing, where the user expects that several different types of indel mutations may be present.

[0332] During this PCR reaction, as described herein, a pool of nucleic acid standards of different lengths can be included in the reaction. The pool can include full-length standards (e.g., those shown in FIG. 8A), insertion standards (e.g., those shown in FIG. 8B), and deletion standards (e.g., those shown in FIG. 8C). In this way, the standards are amplified under the same conditions as the sequence of interest.

[0333] A typical method for preparing an intercalation standard is as follows. Step 1) The oligonucleotide shown in FIG. 2A containing the N18 UMI is digested with restriction enzymes that cut at restriction site 3 (RS3) and restriction site 4 (RS4). Step 2) The PCR product of FIG. 3 is digested with restriction enzymes that cut at restriction site 1 (RS1) and restriction site 2 (RS2). Step 3) Digest the PCR product of FIG. 4 with RS1 and RS2. Step 4) Ligate the products of steps 2 and 3. Step 5) Digest the PCR product of FIG. 4 with RS3 and RS4. Step 6) Ligate the product of step 5 with the product of step 1.

[0334] These steps for preparing the insertion standard are expected to produce the product shown in Figure 2B. The order of RS digestion is not fixed. Furthermore, if all restriction enzymes digesting with RS are buffer compatible, all digestion steps may be combined. Alternatively, the digestion steps may be performed in separate steps. The ligation steps (steps 4 and 6) may also be combined as the final step in the method for preparing the insertion standard.

[0335] A typical method for making deletion standards is as follows. Step 1) The oligonucleotide shown in Figure 6A (which is identical to the oligonucleotide shown in Figure 2A and contains an N18 UMI) is digested with RS3 and RS4. Step 2) Digest the PCR product of Figure 5 with RS3 and RS4. Step 3) Ligate the product of step 2 with the product of step 1.

[0336] These steps for preparing the deletion standards are expected to produce the products shown in Figure 6B.

[0337] After amplifying the sequence of interest with the standard, the amplicons (from the standard and from the sequence of interest) can then be subjected to a method for preparing a sequencing library. Figure 1A shows that this can be Nextera fragmentation (i.e., tagmentation), where the transposase incorporates adapter sequences at both ends of the fragment. The fragments can then be sequenced using sequences (e.g., sequencing primer binding sites) contained in these adapter sequences.

[0338] The library (composed of fragments generated from the sequence of interest and the standards) can then be sequenced. The UMIs contained in the individual standards can be used to generate a bias profile. This bias profile accounts for the fact that larger standards have fewer unique replicates, since the replicates of a given standard can be identified using the UMIs of the standards. These data can be used to normalize the amplicon size bias. In this way, the user can estimate the original copy number with a given indel mutation in the sequence of interest. In other words, this method can control for the fact that a large insertion mutation of the sequence of interest (resulting in a significantly larger amplicon of the sequence of interest) generates fewer amplicons than the wild-type sequence of interest or a deletion mutation of the sequence of interest.

[0339] Example 2. Library quality control assessment A quantitative PCR (qPCR) assay was performed for quality control (QC) of the libraries. The QC qPCR assay used PrimeStar GXL DNA polymerase (Takara), a long-range polymerase known to be able to amplify long targets (e.g., >30 kb) with high fidelity, to amplify the unnicked template strand. During amplification, a forward primer specific to a hairpin adaptor contained in a library molecule extends to the opposite adaptor, generating a new template strand for the reverse primer, only if the template is not disrupted by a nick. In contrast, no signal from the new template strand is generated if the polymerase encounters a nick (shown in Figure 9).

[0340] Control experiments were performed to determine how nicks affect Cq values. The qPCR master mix consisted of 0.5 U long-range polymerase (PrimeStar GXL polymerase), forward and reverse primers each designed to bind to a specific sequence within the hairpin adapter, 1× EvaGreen, 200 μM of each dNTP, 1× PrimeStar buffer, and approximately 200 pg / μL DNA input (input can be reduced to the fg range if necessary).

[0341] 20x EvaGreen was diluted to 5x in water and then included on the reaction plate along with a standard curve (library with Nextera adapters and P5 / P7 amplification primers) generated with samples to confirm efficient amplification. The following cycling parameters were performed: initial denaturation at 95°C for 2 minutes, followed by 30 cycles of 95°C for 30 seconds, 50°C for 30 seconds, and 68°C for 15 seconds. Reactions were performed in duplicate and Cq values ​​were averaged.

[0342] Table 2 provides an overview of the qPCR master mix.

[0343] [Table 2]

[0344] EvaGreen® Dye and EvaGreen® Plus Dye are green fluorescent nucleic acid dyes that are essentially non-fluorescent by themselves but become highly fluorescent when bound to dsDNA. Thus, EvaGreen can be used in digital PCR and isothermal amplification applications.

[0345] Nickase treatment caused a dose-dependent increase in DNA damage and average Cq for both the 10 ng (FIGS. 10A and 10B) and 20 ng (FIGS. 10C and 10D) libraries. These results indicate that the qPCR results of this QC assay yield lower Cq for higher quality libraries and higher Cq for damaged libraries (e.g., those composed of library molecules containing nicks).

[0346] Similar results were seen after endonuclease treatment (FIG. 11 and FIG. 12A and FIG. 12B) using a combination of Vibrio vulnificus nuclease (VVN, a non-specific nuclease) and a T7 endonuclease mutant to generate double-stranded breaks from nicks. Thus, generating double-stranded breaks from nicked templates, resulting in separation of primer sequences required for amplification, further demonstrates that the QC assay can identify libraries of poor quality.

[0347] Example 3. Quality control of SMRTbell libraries Figures 13A-15C show further experiments with SMRTbell libraries containing hairpin adapters at both ends of the double-stranded fragments, using the method described in Example 2. These analyses of different libraries confirm that the total sequencing output is consistently increased for libraries with lower Cq values. In other words, there was a strong correlation between the qPCR results in the QC step and the measured total sequencing output (i.e., gigabases sequenced). In general, libraries with lower Cq values ​​in the QC assay had higher total sequencing output. For example, libraries with Cq values ​​of about 3 in the QC assay showed a P1 percent variation of 39% to 67%, compared to 17% when Cq values ​​exceeded 9 (Figures 13A-13C). Library 8 is noted as an outlier to this relationship.

[0348] Furthermore, the data in Figures 14A-C show that Cq values ​​in the range of 3-4 generated approximately 366 gigabases on average. In contrast, library 10 was predicted to perform poorly based on its QC value of over 6 (Figure 14A), and the sequencing results showed relatively low total power and P1 percent (Figures 14B and 14C). Thus, the QC assay could predict libraries with poor sequencing performance. In general, a lower average Cq for a library correlated with a higher P1 percent, but this was not the case for library 14 (corresponding to library 8 in Figures 13A-C).

[0349] Figures 15A-C similarly show that the best total sequencing output (gigabases) was observed for library fractions with lower Cq values ​​in the QC assay (i.e., different fractions prepared from the same library, such as F4, F5, and F6) compared to library fractions with higher Cq values.

[0350] Therefore, the QC method of the present invention is a useful tool for deciding to sequence (or not sequence) individual libraries. Such a QC method is particularly beneficial because the quality of the library may vary in a way that the user cannot predict based only on the existing QC method. For example, the pipetting force used in one sample may cause degradation that is not seen in other libraries generated by the same user. Only a method that can evaluate the quality of an already created library can control for random changes that affect the quality of the sequencing data. Thus, the skilled artisan can use initial experiments to generate a range of desired Cq values ​​based on the specific library used, which can be used to select libraries for sequencing using the QC method.

[0351] Example 4. Measurement of DNA damage using fluorescence Users may also wish to measure DNA damage using fluorescence. For example, users may wish to measure DNA damage before preparing libraries to ensure that the level of DNA damage in the sample is acceptable. For example, users may wish to use a method of quantifying DNA damage that is flexible to use on genomic DNA or cDNA before library preparation, or on libraries that have already been prepared. However, current assays that contain both fluorescently labeled nucleotides and proteins often suffer from high non-specific binding of unincorporated fluorescent nucleotides.

[0352] The assay of the present invention was developed to improve the signal-to-noise ratio of fluorimetry. The method uses both shrimp alkaline phosphatase (SAP) digestion and SPRI (carboxylate bead) binding / elution steps to significantly reduce non-specific binding. Depending on the user's preference, cellulose beads can be used in place of carboxylate beads and calf intestinal alkaline phosphate can be used in place of SAP in any of the methods described.

[0353] Figure 16 outlines a method of the invention that incorporates two steps of DNA repair (in this example using Bst polymerase and Taq ligase) in the presence of fluorescently labeled dNTPs, followed by treatment with SAP and SPRI bead-based purification. The treated sample containing the repaired DNA is then measured to determine the amount of fluorescence.

[0354] The first experiment tested different conditions to reduce non-specific binding of dNTPs. Figure 17 shows that SAP treatment of sheared genomic DNA (gDNA) with a single SPRI bead-based purification substantially reduced non-specific binding of fluorescent nucleotides compared to assays without SAP treatment. In other words, the bead-based purification step with SAP treatment reduced non-specific fluorescence.

[0355] Furthermore, Figure 18 shows that a second SPRI bead-based purification step reduced nonspecific binding of fluorescent nucleotides to levels comparable to buffer. Such low background is important for accurate measurement of low amounts of DNA damage (i.e., when a low percentage of nucleotides in the DNA are damaged).

[0356] Based on the first experiment, two steps of SPRI bead-based purification were performed after SAP treatment in further experiments. Using the method of the present invention, a comparison of the effectiveness of commercial repair mixes versus our DNA repair enzyme mix was performed. PreCR Repair Mix (NEB) was compared with a custom repair mix of Taq ligase (40U), Bst polymerase large fragment (8U), and T4 PDG (1U) using this protocol. As shown in Figure 19A, PreCR mix did not show the expected fluorescence increase as the damage of the sample increased, while the custom repair mix showed these expected increases. PreCR Mix samples also had larger standard deviations and lower signals, and such discrepancies can also be found in the literature from groups optimizing DNA damage repair formulations. In contrast, the custom repair enzyme mix using the method of the present invention had lower standard deviations and higher signal-to-noise ratios (Figure 19B).

[0357] The method of the present invention using a custom mix of DNA repair enzymes determined by the user also adds flexibility to the workflow since the user can choose which repair enzymes to utilize in the assay. For example, the assay of the present invention can be designed to detect different types of damage in DNA by utilizing different DNA damage repair enzymes. Incorporation of T4 pyrimidine dimer glycosylase (T4 PDG) enzyme in the DNA repair enzyme mixture can allow for the repair and subsequent detection of damage caused by UV irradiation such as thymine dimers. As shown in Figure 20, the method using a DNA repair enzyme mixture containing Taq ligase, Bst polymerase, and T4 PDG (UV damage specific repair enzyme) was able to evaluate UV-induced DNA damage. As the amount of UV light and exposure time increased, the DNA damage measured by the assay of the present invention also increased, indicating the ability of the assay of the present invention to measure DNA damage over a wide range.

[0358] Figure 21 further shows that the fluorescent signal of the DNA damage measurement increased when the DNA samples were exposed to different amounts of nicking enzyme (Nt.BspQI), thus the assay of the present invention can sensitively measure the amount of nicked DNA over a wide range.

[0359] If the user desires, uracil DNA glycosylase (UDG) and an apurinic or apyrimidinic site lyase, and / or formamidopyrimidine DNA glycosylase (FPG) and an apurinic or apyrimidinic site lyase can be included in the enzyme repair mix to allow for the repair and subsequent detection of uracil or oxidized bases, respectively.

[0360] The modular nature of this assay makes it a flexible and customizable tool for detecting different types of damage in double-stranded DNA based on the activity and specificity of the enzyme used.

[0361] Example 5. Measurement of DNA damage using fluorescence Based on initial experiments, an exemplary assay protocol was developed for use with a DNA repair enzyme mix containing Taq ligase, Bst polymerase, and T4 PDG. Table 3 provides the reagents for use in this assay, Table 4 provides the dNTP master mix contents, and Table 5 provides the DNA damage assay contents.

[0362] [Table 3]

[0363] [Table 4]

[0364] [Table 5]

[0365] A typical assay protocol can be carried out as follows. 1. Prepare dNTP dilutions and dNTP master mix as described in Tables 4 and 5. Place on ice. 2. Quantify sample and control gDNA using Qubit. Dilute gDNA to 100ng / μL and place on ice. 3. Prepare assay mix in strip tubes on ice, two per sample, gently pipette to mix. Incubate for 30 minutes at 37° C. in a thermocycler with heated lid. 4. After 30 minutes, remove from thermocycler and add 1 μL of shrimp alkaline phosphatase (SAP) to each sample. Mix by gently pipetting and incubate for 60 minutes at 37° C. in a thermocycler with a heated lid. 5. After incubation, dilute to 100 μL with Resuspension Buffer (RSB). Vortex to mix AMPure PB (SPRI) beads and add 100 μL of SPRI beads. Pipette to mix and gently shake at room temperature for 15 minutes. 6. Magnetize the beads using a benchtop magnetic rack and, without disturbing the bead pellet, wash the samples twice with 100 μL of 80% ethanol. After the second wash, be sure to spin down and completely aspirate off all the ethanol. 7. Resuspend the beads in 100 μL of RSB. Gently rock at room temperature for 15 minutes. 8. Magnetize the beads using a benchtop magnetic rack and aspirate the supernatant into a new strip tube. 9. Optionally, repeat the SPRI wash (steps 5-8). Make 100 μL of standard curve samples using AF-546 dUTP in RSB starting at 10.5 nM and decreasing the concentration in half (5 nM, 2.5 nM, 1.25 nM, 625 pM, 312 pM, 156 pM, 78 pM, and 39 pM). 11. Pipette 45 μL of each purified sample into duplicate into a 96-well plate. Pipette 45 μL of standard curve samples into duplicate into a 96-well plate. 12. Place the plate into the plate holder of the Cytation 5 multimode reader (Agilent). Select Alexa Fluor 546 as the fluorophore and measure the fluorescence of the samples and standard curve samples in a single read. 13. Dilute remaining samples and controls 1:10 in RSB and quantitate recovered DNA on Qubit. Use the standard curve to calculate dye molecules incorporated into DNA. Divide the number of dye molecules by the mass of gDNA recovered to determine the normalized number of dye molecules.

[0366] One of skill in the art can use this representative protocol with the DNA repair enzyme mixture of their choice to assess DNA damage in a sample.

[0367] Equivalent The foregoing specification is believed to be sufficient to enable one skilled in the art to practice the embodiments. The foregoing description and examples set forth certain embodiments in detail and explain the best mode contemplated by the inventors. However, no matter how detailed the foregoing appears in the text, it will be understood that the embodiments can be embodied in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

[0368] As used herein, the term about refers to numerical values, including, for example, integers, ratios, and percentages, whether or not explicitly stated. The term generally refers to a range of numerical values ​​(e.g., ±5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When a term such as at least and about precedes a list of numerical values ​​or ranges, the term modifies all of the values ​​or ranges provided in the list. In some cases, the term about may include numerical values ​​that are rounded to the nearest significant figure.

Claims

1. A pool of nucleic acid standards of different lengths, the nucleic acid standards having a unique molecular identifier (UMI), as well as a. a 5' universal oligonucleotide, said 5' universal oligonucleotide being the same for all standards; b. a 3' universal oligonucleotide, said 3' universal oligonucleotide being the same for all standards; c. at least one region between the UMI and the 5' universal oligonucleotide and / or between the UMI and the 3' universal oligonucleotide; A pool of standards, wherein the length of said at least one region determines the length of said standard.

2. The pool comprises a UMI, and a. a 5' universal oligonucleotide, said 5' universal oligonucleotide being the same for all standards; b. a 3' universal oligonucleotide, said 3' universal oligonucleotide being the same for all standards; 2. The pool of standards of claim 1, wherein the additional nucleic acid standards do not include at least one region between the UMI and the 5' universal oligonucleotide or between the UMI and the 3' universal oligonucleotide.

3. 2. The pool of standards of claim 1, wherein the at least one region between the UMI and the 5' universal oligonucleotide and / or the UMI and the 3' universal oligonucleotide comprises between 0.2 kb and 10 kb.

4. 2. The pool of standards of claim 1, wherein the 5' universal oligonucleotide and / or the 3' universal oligonucleotide each comprise an amplicon amplified from a sequence of interest.

5. 2. The pool of standards of claim 1, wherein the at least one region between the UMI and the 5' universal oligonucleotide and / or the UMI and the 3' universal oligonucleotide each comprises an amplicon amplified from a sequence of interest.

6. 2. The pool of standards of claim 1, wherein the at least one region between the UMI and the 5' universal oligonucleotide and / or the UMI and the 3' universal oligonucleotide each comprises an arbitrary sequence.

7. A pool of nucleic acid standards of different lengths, the nucleic acid standards comprising a UMI, as well as a. 5' partially overlapping oligonucleotides, said 5' partially overlapping oligonucleotides being identical over at least a portion of their sequence for all said standards, and / or b. 3' partially overlapping oligonucleotides, said 3' partially overlapping oligonucleotides being identical over at least a portion of their sequence for all said standards; A pool of standards, wherein the length of said 5' partially overlapping oligonucleotides and / or said 3' partially overlapping oligonucleotides determines the length of said standards.

8. 2. The pool of standards of claim 1, wherein the standards are double-stranded.

9. 2. The pool of standards of claim 1 , wherein the standards comprise double-stranded DNA.

10. 2. The pool of standards of claim 1, wherein each standard comprises a different UMI.

11. 2. The pool of standards of claim 1, wherein the UMIs contained in the pool of standards are a random set of sequences comprising 16-20 base pairs.

12. 12. The pool of standards of claim 11, wherein the UMIs included in the pool of standards are a random set of sequences comprising 18 base pairs.

13. The pool of standards is 1×10 10 That's it, 10 x 10 10 or more, or 100 x 10 10 2. The pool of standards of claim 1, comprising:

14. 2. The pool of standards of claim 1, wherein the number of standards in the pool is greater than the number of amplicons produced by an amplification reaction.

15. A pool of standards, a. at least a first portion of said standard is from claim 1, b. At least a second portion of the standards is a pool of nucleic acid standards of different lengths, the nucleic acid standards comprising a UMI, as well as i. 5' partially overlapping oligonucleotides, said 5' partially overlapping oligonucleotides being identical over at least a portion of their sequence for all said standards, and / or ii. 3' partially overlapping oligonucleotides, said 3' partially overlapping oligonucleotides being identical over at least a portion of their sequence for all said standards; A pool of standards, wherein the length of said 5' partially overlapping oligonucleotides and / or said 3' partially overlapping oligonucleotides determines the length of said standards.

16. 1. A method for generating a pool of nucleic acid standards, comprising: a. providing multiple copies of at least one sequence of interest comprising a nucleic acid; b. providing a series of oligonucleotides, each of which comprises a UMI; c. providing a series of insertion oligonucleotides of various lengths; d. ligating (a) at least one sequence of interest, (b) at least one oligonucleotide comprising a UMI, and (c) at least one insertion amplicon to generate a plurality of nucleic acid standards of the pool of nucleic acid standards.

17. 1. A method for generating a pool of nucleic acid standards, comprising: a. providing multiple copies of at least one sequence of interest comprising a nucleic acid; b. providing a series of oligonucleotides, each of which comprises a UMI; c. ligating (a) at least one sequence of interest and (b) at least one oligonucleotide containing a UMI.

18. 1. A method for normalizing amplicon size bias, comprising: a. combining a sample containing a target nucleic acid with a pool of nucleic acid standards of different lengths, each standard containing a UMI; b. amplifying amplicons of sequences of interest contained in the standard and the target nucleic acid; c. sequencing the amplicons of the standards and the sequence of interest to generate sequencing data; d. determining a bias profile based on amplicon size using sequencing data from said standards; e. using the bias profile to normalize amplicon size bias.

19. 1. A method for determining the presence of DNA damage in a library comprising one or more library molecules, each library molecule comprising a double-stranded DNA insert having a hairpin adaptor at each end of the insert, the method comprising: a. denaturing the first and second strands of the double-stranded DNA inserts contained in library molecules; b. Annealing a forward primer and a reverse primer to the library molecule; c. amplifying to generate library amplicons; d. assessing the presence of DNA damage based on the number of library amplicons generated.

20. 1. A method for quantifying DNA damage in a sample containing DNA using fluorescence, comprising: a. i. an aliquot of a sample containing DNA; ii. one or more DNA repair enzymes; iii. combining dNTPs, where one or more dNTPs are fluorescently labeled; b. Preparing repaired DNA; c. dephosphorylating the phosphate from the dNTP; d. Binding the repaired DNA to carboxylate or cellulose beads; e. eluting the bound repaired DNA from the carboxylate or cellulose beads with a resuspension buffer; f) measuring the fluorescence of the repaired DNA to determine the amount of DNA damage.

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