Methods for generating double-stranded adapters

Abstract

The present application relates to sample preparation methods. The present disclosure provides single amplification methods and double amplification methods for preparing nucleic acid samples for sequencing.

Description

[0001] This application is a divisional application of an application filed on September 24, 2015, with application number 201580063859.X and invention name “Sample Preparation Method”.

[0002] Cross-references

[0003] This application claims priority to U.S. Provisional Application Serial No. 62 / 054,886, filed on September 24, 2014, and U.S. Patent Application No. 14 / 691,457, filed on April 20, 2015, which are incorporated herein by reference in their entirety. Background Art

[0004] Although the cost of nucleic acid sequencing has decreased in recent years, sample preparation is still expensive. Only a few sample preparation methods are compatible with the most popular sequencing platforms. These methods can be expensive and time-consuming. In particular, library preparation may require the generation of prominent bases on the nucleic acid fragments. This step is a relatively inefficient step in preparing sequencing libraries. Improving the efficiency of this step will reduce the amount of starting nucleic acid required for preparing sequencing libraries. New methods for sample preparation for sequencing are needed, particularly methods that improve the efficiency of generating nucleic acid fragments with prominent bases. Summary of the invention

[0005] Provided herein are methods for generating an adaptor-ligated library of nucleic acid fragments. In particular, disclosed are methods for preparing partially complementary double-stranded nucleic acid fragments such that one strand extends beyond the other to produce an overhang. Such an overhang can improve the efficiency of enzymatic and biochemical reactions. For example, such an overhang can be annealed to its complementary sequence to allow targeted pairing of nucleic acid fragments. In another example, the connection of nucleic acid fragments with complementary overhangs is more efficient than the connection of blunt-ended nucleic acid fragments.

[0006] In one aspect, the present disclosure provides a method for generating an adapter-ligated library of double-stranded nucleic acid molecules, comprising: in a single reaction vessel or reaction mixture, contacting a plurality of single-stranded polynucleotides with a first set of random primers, each random primer comprising a cleavable 5' end; extending the first set of random primers to generate a first extension product that hybridizes with the single-stranded polynucleotide, thereby forming a first double-stranded duplex; denaturing the first double-stranded duplex to a single strand; hybridizing the first polynucleotide extension product with a second set of random primers (the first set of primers and the second set of primers may be the same or different); extending the second set of random primers to generate a second extension product that hybridizes with the first extension product, thereby forming a second double-stranded duplex; cleaving the cleavable 5' end of the first extension product and / or the second extension product, thereby forming a second double-stranded duplex having one or more overhangs; and ligating one or more double-stranded adapters comprising a 3' overhang to the second double-stranded duplex of the previous step, thereby forming the adapter-ligated library. The double-stranded adapter may comprise a nucleic acid barcode in some instances, such as a sample barcode, a molecular barcode, or a randomized barcode. The random primers may be completely degenerate or partially degenerate. A single cleanup step may be used to perform the entire library preparation. No more than two cleanup steps may be used to perform the entire library preparation. Only three cleanup steps may be used to perform the entire library preparation.

[0007] In some embodiments, the single-stranded polynucleotide is ssDNA. In some embodiments, the single-stranded polynucleotide is RNA. In some embodiments, the primer is random, such as a random hexamer. In some embodiments, the length of the primer is 4 to 30 nucleotides in length. In some embodiments, the cleavable 5' end is cleavable by photocleavage. In some embodiments, the cleavable 5' end is cleavable by a nuclease. In some embodiments, the nuclease is an exonuclease. In some embodiments, the nuclease is an endonuclease. In some embodiments, the nuclease is an RNA nuclease. In some embodiments, the nuclease is a DNA nuclease. In some embodiments, the nuclease is a nuclease that can cut a DNA-RNA hybrid molecule. In some embodiments, the nuclease is a nuclease that cuts the RNA component of the DNA-RNA hybrid molecule, such as RNaseH. In some cases, the exonuclease activity is provided by the 5'-3' exonuclease activity of a polymerase such as DNA polymerase I. In some cases, the nuclease specifically cuts dU, such as uracil-DNA glycosylase. In some embodiments, the cleavable 5' end comprises a modification selected from: a fluorophore, a dideoxynucleotide, a deoxynucleotide, a radiolabel, a dye, a dUTP, a rA, a rU, a rG, a rC, a rUTP and a restriction enzyme target sequence or any combination thereof. In some embodiments, the cleavable 5' end comprises dUTP. In some embodiments, the cleavable 5' end comprises rUTP. In some embodiments, the cleavable 5' end is 1-5 nucleotides. In some embodiments, the cleavable 5' end is 1 nucleotide. In some embodiments, the cleavage removes 1-5 nucleotides of the primer. In some embodiments, the cleavage removes one or more nucleotides from the primer. In some embodiments, the cleavage includes cleaving one or more bases. In some embodiments, the one or more overhangs comprise thymine. In some embodiments, the one or more overhangs comprise adenine. In some embodiments, the one or more overhangs comprise guanine. In some embodiments, the one or more overhangs comprise cytosine. In some embodiments, the one or more overhangs comprise thymine and the 3' overhang comprises adenine. In some embodiments, the one or more overhangs comprise 1-5 nucleotides. In some embodiments, the one or more overhangs comprise 1-5 nucleotides selected from the following modifications: fluorophores, dideoxynucleotides, deoxynucleotides, radioactive labels, dyes, dUTP, rA, rU, rG, rC, rUTP and restriction enzyme target sequences or any combination thereof.In some embodiments, the extension step (e.g., using dNTPs, containing dUTP, rA, rU, rG, rC, rUTP, fluorophores, dideoxynucleotides, deoxynucleotides, radioactive labels or dyes for downstream purification or degradation of dNTPs) produces a blunt end. In some embodiments, the linker comprises a sequencing linker. In some embodiments, the linker comprises a nucleic acid barcode. In some embodiments, the linker comprises a nucleic acid barcode sequence. In some cases, the library to which the linker is connected can be sequenced. In some embodiments, the polynucleotide is an ancient polynucleotide. In some embodiments, the polynucleotide comprises less than 10 nucleic acid copies. In some embodiments, the method is performed twice or more. A single cleanup step can be used to perform the entire library preparation. No more than two cleanup steps can be used to perform the entire library preparation. Only three cleanup steps can be used to perform the entire library preparation.

[0008] In one aspect, the present disclosure provides a method for generating an adapter-ligated library of double-stranded nucleic acid molecules, comprising: in a single reaction vessel or reaction mixture, contacting a plurality of single-stranded polynucleotides with a first set of target-specific primers, each target-specific primer comprising a cleavable 5' end; extending the first set of primers (e.g., using dNTPs, dNTPs containing dUTP, rA, rU, rG, rC, rUTP, fluorophores, dideoxynucleotides, deoxynucleotides, radioactive labels or dyes for downstream purification or degradation) to generate a first extension product that hybridizes with the single-stranded polynucleotide, thereby forming a first double-stranded duplex; denaturing the first double-stranded duplex to a single strand; and ligating the first polynucleotide extension product to a single strand. A second set of target-specific primers are hybridized (the first and second sets of primers may be the same or different; for example, the first set of primers may specifically hybridize to the target nucleic acid (or upstream thereof). The second set of primers may specifically hybridize to a sequence complementary to the target nucleic acid (or upstream thereof); the second set of primers are extended to generate a second extension product that hybridizes to the first extension product, thereby forming a second double-stranded duplex; the cleavable 5' end of the first extension product and / or the second extension product is cut to form a second double-stranded duplex having one or more overhangs; and one or more double-stranded adapters comprising 3' overhangs are connected to the second double-stranded duplex of the previous step to form an adapter-connected library, wherein the method comprises only two extension steps. The double-stranded adapter may comprise, for example, a nucleic acid barcode, such as a sample barcode, a molecular barcode, or a randomized barcode. A single cleanup step may be used to perform the entire library preparation. No more than two cleanup steps may be used to perform the entire library preparation. Only three cleanup steps may be used to perform the entire library preparation.

[0009] In some embodiments, the target-specific primer comprises a 5' randomized tail. In some embodiments, the randomized tail comprises a cleavable 5' end. In some embodiments, the single-stranded polynucleotide is ssDNA. In some embodiments, the single-stranded polynucleotide is RNA. In some embodiments, the primer is random, such as a random hexamer. In some embodiments, the length of the primer is 4 to 30 nucleotides in length. In some embodiments, the cleavable 5' end can be cleaved by photocleavage. In some embodiments, the cleavable 5' end can be cleaved by a nuclease. In some embodiments, the nuclease is an exonuclease. In some embodiments, the nuclease is an endonuclease. In some embodiments, the nuclease is an RNA nuclease. In some embodiments, the nuclease is a DNA nuclease. In some embodiments, the nuclease is a nuclease that can cleave a DNA-RNA hybrid molecule. In some embodiments, the nuclease is a nuclease that cleaves the RNA component of a DNA-RNA hybrid molecule, such as RNaseH. In some cases, the exonuclease activity is provided by the 5'-3' exonuclease activity of a polymerase such as DNA polymerase I. In some cases, the nuclease specifically cuts dU, such as uracil-DNA glycosylase. In some embodiments, the cleavable 5' end comprises a modification selected from: a fluorophore, a dideoxynucleotide, a deoxynucleotide, a radiolabel, a dye, a dUTP, rA, rU, rG, rC, rUTP, and a restriction enzyme target sequence or any combination thereof. In some embodiments, the cleavable 5' end comprises dUTP. In some embodiments, the cleavable 5' end comprises rUTP. In some embodiments, the cleavable 5' end is 1-5 nucleotides. In some embodiments, the cleavable 5' end is 1 nucleotide. In some embodiments, the cleavage removes 1-5 nucleotides of the primer. In some embodiments, the cleavage removes one or more nucleotides from the primer. In some embodiments, the cleavage includes cleaving one or more bases. In some embodiments, the one or more overhangs comprise thymine. In some embodiments, the one or more overhangs comprise adenine. In some embodiments, the one or more overhangs comprise guanine. In some embodiments, the one or more overhangs comprise cytosine. In some embodiments, the one or more overhangs comprise thymine, and the 3' overhang comprises adenine. In some embodiments, the one or more overhangs comprise 1-5 nucleotides. In some embodiments, the one or more overhangs comprise 1-5 nucleotides selected from the following modifications: fluorophores, dideoxynucleotides, deoxynucleotides, radioactive labels, dyes, dUTP, rA, rU, rG, rC, rUTP and restriction enzyme target sequences or any combination thereof.In some embodiments, the extension step (e.g., using dNTPs, containing dUTP, rA, rU, rG, rC, rUTP, fluorophores, dideoxynucleotides, deoxynucleotides, radioactive labels or dyes for downstream purification or degradation of dNTPs) produces a blunt end. In some embodiments, the linker comprises a sequencing linker. In some embodiments, the linker comprises a nucleic acid barcode. In some embodiments, the linker comprises a nucleic acid barcode sequence. In some cases, the library to which the linker is connected can be sequenced. In some embodiments, the polynucleotide is an ancient polynucleotide. In some embodiments, the polynucleotide comprises less than 10 nucleic acid copies. In some embodiments, the method is performed twice or more. A single cleaning step can be used to perform the entire library preparation. No more than two cleaning steps can be used to perform the entire library preparation. Only three cleaning steps can be used to perform the entire library preparation.

[0010] In one aspect, the present disclosure provides a method for preparing a polynucleotide library, comprising: contacting a plurality of single-stranded polynucleotides with a first set of random primers comprising a cleavable 5' end; extending the primers to generate a first extension product that hybridizes to the polynucleotide, thereby forming a double-stranded duplex; cleaving the cleavable 5' end, thereby generating a double-stranded duplex having a first overhang; and ligating one or more adapters to the double-stranded duplex of the previous step, wherein the one or more adapters comprise an overhang complementary to the first overhang, and wherein the method comprises only one round of extension. The entire library preparation may be performed with a single cleanup step. The entire library preparation may be performed with no more than two cleanup steps. The entire library preparation may be performed with only three cleanup steps.

[0011] In some embodiments, the polynucleotide is ssDNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the primer is random, such as a random hexamer. In some embodiments, the length of the primer is 4 to 30 nucleotides in length. In some embodiments, the cleavable 5' end can be cleaved by photocleavage. In some embodiments, the cleavable 5' end can be cleaved by a nuclease. In some embodiments, the nuclease is an exonuclease. In some embodiments, the nuclease is an endonuclease. In some embodiments, the nuclease is an RNA nuclease. In some embodiments, the nuclease is a DNA nuclease. In some embodiments, the nuclease is a nuclease that can cleave a DNA-RNA hybrid molecule. In some embodiments, the nuclease is a nuclease that cleaves the RNA component of a DNA-RNA hybrid molecule, such as RNaseH. In some cases, the exonuclease activity is provided by a polymerase such as the 5'-3' exonuclease activity of DNA polymerase I. In some cases, the nuclease specifically cleaves dU, such as uracil-DNA glycosylase. In some embodiments, the cleavable 5' end comprises a modification selected from the group consisting of: a fluorophore, a dideoxynucleotide, a deoxynucleotide, a radioactive label, a dye, a dUTP, rA, rU, rG, rC, rUTP, and a restriction enzyme target sequence or any combination thereof. In some embodiments, the cleavable 5' end comprises dUTP. In some embodiments, the cleavable 5' end comprises rUTP. In some embodiments, the cleavable 5' end is from 1-5 nucleotides. In some embodiments, the cleavable 5' end is 1 nucleotide. In some embodiments, the cleavage removes 1-5 nucleotides of the primer. In some embodiments, the cleavage removes one or more nucleotides from the primer. In some embodiments, the cleavage includes cleaving one or more bases. In some embodiments, the one or more overhangs comprise thymine. In some embodiments, the one or more overhangs comprise adenine. In some embodiments, the one or more overhangs comprise guanine. In some embodiments, the one or more overhangs comprise cytosine. In some embodiments, the one or more overhangs comprise thymine, and the 3' overhang comprises adenine. In some embodiments, the one or more overhangs comprise 1-5 nucleotides. In some embodiments, the one or more overhangs comprise 1-5 nucleotides selected from the group consisting of fluorophores, dideoxynucleotides, deoxynucleotides, radiolabels, dyes, dUTP, rA, rU, rG, rC, rUTP, and restriction enzyme target sequences, or any combination thereof.In some embodiments, the extension step (e.g., using dNTPs, containing dUTP, rA, rU, rG, rC, rUTP, fluorophores, dideoxynucleotides, deoxynucleotides, radioactive labels or dyes for downstream purification or degradation of dNTPs) produces a blunt end. In some embodiments, the linker comprises a sequencing linker. In some embodiments, the linker comprises a nucleic acid barcode. In some embodiments, the linker comprises a nucleic acid randomization barcode generated by any linker oligonucleotide containing a cleavable 3' or 5' end. In some embodiments, the method further comprises sequencing the library connected to the linker. In some embodiments, the polynucleotide is an ancient polynucleotide. In some embodiments, the starting single-stranded polynucleotide comprises less than 10 copies of nucleic acid molecules. In some embodiments, the method is performed twice or more. A single cleaning step can be used to perform the entire library preparation. No more than two cleaning steps can be used to perform the entire library preparation. Only three cleaning steps can be used to perform the entire library preparation.

[0012] In one aspect, the present disclosure provides a method for generating an adapter-ligated library of double-stranded nucleic acid molecules, comprising: in a single reaction vessel or reaction mixture, contacting a plurality of single-stranded polynucleotides with a random primer library, each random primer comprising a cleavable 5' end; extending the primer to generate a first extension product that hybridizes with the single-stranded polynucleotide, thereby forming a first double-stranded duplex; denaturing the first double-stranded duplex to a single strand; hybridizing the first polynucleotide extension product with a primer from a target-specific primer library; extending the primer to generate a second extension product that hybridizes with the first extension product, thereby forming a second double-stranded duplex; cleaving the cleavable 5' end of the first extension product or the second extension product, thereby forming a second double-stranded duplex having one or more overhangs; and ligating one or more double-stranded adapters comprising a 3' overhang to the second double-stranded duplex of the previous step, thereby forming the adapter-ligated library, wherein the method comprises only two extension steps. A single cleanup step may be used to perform the entire library preparation. No more than two cleanup steps may be used to perform the entire library preparation. The entire library preparation can be performed with only three clean-up steps.

[0013] In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is RNA. In some embodiments, the primer is random, such as a random hexamer. In some embodiments, the length of the primer is 4 to 30 nucleotides in length. In some embodiments, the cleavable 5' end can be cleaved by photocleavage. In some embodiments, the cleavable 5' end can be cleaved by a nuclease. In some embodiments, the nuclease is an exonuclease. In some embodiments, the nuclease is an endonuclease. In some embodiments, the nuclease is an RNA nuclease. In some embodiments, the nuclease is a DNA nuclease. In some embodiments, the nuclease is a nuclease that can cleave a DNA-RNA hybrid molecule. In some cases, the nuclease can be a nuclease that cleaves the RNA component of a DNA-RNA hybrid molecule, such as RNaseH. In some cases, the exonuclease activity is provided by a polymerase such as the 5'-3' exonuclease activity of DNA polymerase I. In some cases, the nuclease specifically cleaves dU, such as uracil-DNA glycosylase. In some embodiments, the cleavable 5' end comprises a modification selected from the group consisting of: a fluorophore, a dideoxynucleotide, a deoxynucleotide, a radiolabel, a dye, a dUTP, rA, rU, rG, rC, rUTP, and a restriction enzyme target sequence or any combination thereof. In some embodiments, the cleavable 5' end comprises dUTP. In some embodiments, the cleavable 5' end comprises rUTP. In some embodiments, the cleavable 5' end is 1-5 nucleotides. In some embodiments, the cleavable 5' end is 1 nucleotide. In some embodiments, the cleavage removes 1-5 nucleotides of the primer. In some embodiments, the cleavage removes one or more nucleotides from the primer. In some embodiments, the cleavage includes cleaving one or more bases. In some embodiments, the one or more overhangs comprise thymine. In some embodiments, the one or more overhangs comprise adenine. In some embodiments, the one or more overhangs comprise guanine. In some embodiments, the one or more overhangs comprise cytosine. In some embodiments, the one or more overhangs comprise thymine, and the 3' overhang comprises adenine. In some embodiments, the one or more overhangs comprise 1-5 nucleotides. In some embodiments, the one or more overhangs comprise 1-5 nucleotides selected from the group consisting of fluorophores, dideoxynucleotides, deoxynucleotides, radiolabels, dyes, dUTP, rA, rU, rG, rC, rUTP, and restriction enzyme target sequences, or any combination thereof.In some embodiments, the extension step (e.g., using dNTPs, containing dUTP, rA, rU, rG, rC, rUTP, fluorophores, dideoxynucleotides, deoxynucleotides, radioactive labels or dyes for downstream purification or degradation of dNTPs) produces a blunt end. In some embodiments, the linker comprises a sequencing linker. In some embodiments, the linker comprises a nucleic acid barcode. In some embodiments, the linker comprises a nucleic acid barcode. In some embodiments, the linker comprises a nucleic acid barcode sequence. In some cases, the library to which the linker is connected can be sequenced. In some embodiments, the polynucleotide is an ancient polynucleotide. In some embodiments, the polynucleotide comprises less than 10 nucleic acid copies. In some embodiments, the method is performed twice or more. A single cleanup step can be used to perform the entire library preparation. No more than two cleanup steps can be used to perform the entire library preparation. Only three cleanup steps can be used to perform the entire library preparation.

[0014] On the one hand, the present disclosure provides a method for generating a double-stranded adapter with one or more overhangs. The double-stranded adapter can be generated in a single reaction mixture or reaction vessel. The double-stranded adapter can be generated by annealing two oligonucleotide libraries. Each oligonucleotide in the first oligonucleotide library optionally includes a 5' cleavable nucleotide. Each oligonucleotide in the second oligonucleotide library can include a sequence complementary to a portion of the oligonucleotide in the first library and a 5' cleavable nucleotide. In some cases, each oligonucleotide from the second oligonucleotide library can include a barcode sequence between a sequence complementary to a portion of the oligonucleotide in the first library and the 5' cleavable nucleotide. The barcode sequence can be random. The barcode sequence can be non-random. The barcode sequence can be semi-random, such as, for example, by preparing oligonucleotides using semi-degenerate primers for synthesis. Oligonucleotides from the first library can hybridize with oligonucleotides from the second library to form a duplex. The oligonucleotide from the first library in the duplex can be extended so that the oligonucleotide from the first library is covalently linked to a sequence that is complementary to the optional barcode sequence and complementary to the cleavable nucleotide of the oligonucleotide from the second library. One or more cleavable nucleotides located at one or more ends of the linker can be cleaved. The cleaved linker can be used in any of the methods herein for library preparation and sequencing. In some cases, the second oligonucleotide pool comprises more than three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, 12 or more, 14 or more, 16 or more, 18 or more, 20 or more, 22 or more, 24 or more, 26 or more, 28 or more, 30 or more, 32 or more, 36 or more, 42 or more, 48 or more, 54 or more, 60 or more, 64 or more, 66 or more, 72 or more, 78 or more, 84 or more, 90 or more, 98 or more, 120 or more, 128 or more, 160 or more, 200 or more, 240 or more, 256 or more, 267 or more, 271 or more, 280 or more, 291 or more, 300 or more, 312 or more, 324 or more, 330 or more, 345 or more, 356 or more, 368 or more, 371 or more, 380 or more, 390 or more, 400 or more, 410 or more, 420 or more, 430 or more, 440 or more, 450 or more, 460 or more, 470 or more, 480 or more, 490 or more, 500 or more, 510 or more, 520 or more, 530 or more, 540 or more, 550 or more, 560 or more, 570 or more, 580 or more, 590 or more, 600 or more, 610 or more, 610 or more, 610 or more, 610 or more, 610 or more, 610 or more, 280 or more, 512 or more, 1024 or more, 2048 or more, or 4096 or more different sequence barcodes.

[0015] The oligonucleotide can be a length of 4 to 150 nucleotides. Except for random barcodes and 5' cleavable nucleotides, the oligonucleotide can be completely complementary. In some embodiments, the oligonucleotide is only partially complementary. In some examples, the cleavable 5' end can be cleaved by photocleavage. In some embodiments, the cleavable 5' end can be cleaved by a nuclease. In some embodiments, the nuclease is an exonuclease. In some embodiments, the nuclease is an endonuclease. In some embodiments, the nuclease is an RNA nuclease. In some embodiments, the nuclease is a DNA nuclease. In some embodiments, the nuclease is a nuclease that can cut a DNA-RNA hybrid molecule. In some embodiments, the nuclease is a nuclease that cuts the RNA component of the DNA-RNA hybrid molecule, such as RNaseH. In some cases, the exonuclease activity is provided by the 5'-3' exonuclease activity of a polymerase such as DNA polymerase I. In some cases, the nuclease specifically cuts dU, such as uracil-DNA glycosylase. In some embodiments, the cleavable 5' end comprises a modification selected from the group consisting of: a fluorophore, a dideoxynucleotide, a deoxynucleotide, a radiolabel, a dye, a dUTP, rA, rU, rG, rC, rUTP, and a restriction enzyme target sequence or any combination thereof. In some embodiments, the cleavable 5' end comprises dUTP. In some embodiments, the cleavable 5' end comprises rUTP. In some embodiments, the cleavable 5' end is 1-5 nucleotides. In some embodiments, the cleavable 5' end is 1 nucleotide. In some embodiments, the cleavage removes 1-5 nucleotides of the primer. In some embodiments, the cleavage removes one or more nucleotides from the primer. In some embodiments, the cleavage includes cleaving one or more bases. In some embodiments, the one or more overhangs comprise thymine. In some embodiments, the one or more overhangs comprise adenine. In some embodiments, the one or more overhangs comprise guanine. In some embodiments, the one or more overhangs comprise cytosine. In some embodiments, the adapter comprises a nucleic acid randomized barcode generated from either adapter oligonucleotide comprising a cleavable 3' or 5' end. In some embodiments, the method further comprises sequencing the adapter-ligated library.

[0016] In one aspect, the present disclosure provides a kit comprising: a first container comprising a set of random primers, each random primer comprising a cleavable 5' end; a second container comprising a polymerase; a third container comprising an adaptor, wherein each adaptor comprises an overhang complementary to the cleavable 5' end; and instructions for use, wherein the instructions indicate the use of the random primers for only two rounds of extension. In some embodiments, the kit further comprises a container comprising a ligase. In some embodiments, the kit further comprises a container comprising a buffer.

[0017] In the specific example of generating a double-stranded nucleic acid fragment with an overhang or a double-stranded adapter with an overhang, the 5' cleavable end comprises one or more RNA bases or one or more dU. If the 5' cleavable end comprises an RNA base, the cleavage can be mediated by RNaseH or a polymerase, such as the 5'→3' exonuclease activity of DNA polymerase I in some cases. If the cleavable 5' end comprises dU, the cleavage can be mediated by uracil-DNA glycosylase. After cutting the 5' cleavable end, the double-stranded nucleic acid fragment or the double-stranded adapter can have one or more overhangs. These overhangs can improve the efficiency of the connection reaction of the sequence with complementary overhangs. Then the nucleic acid fragment with a 5' overhang can be connected to the adapter with a complementary 3' overhang to generate a sequencing library connected by adapters.

[0018] In one aspect, the present disclosure provides a kit comprising: a first container comprising a set of random primers, each random primer comprising a cleavable 5' end; a second container comprising a polymerase; a third container comprising an adapter, wherein each adapter comprises an overhang complementary to the cleavable 5' end; and instructions for use, wherein the instructions indicate the use of the random primer library for only one round of extension. In some embodiments, the kit further comprises a container containing a ligase. In some embodiments, the kit further comprises a container containing a buffer.

[0019] This application provides the following:

[0020] 1. A method for generating an adaptor-ligated library of double-stranded nucleic acid molecules, comprising in a single reaction vessel or reaction mixture:

[0021] (a) generating a plurality of double-stranded DNA molecules comprising a cleavable 5' end of one or both strands from a plurality of single-stranded polynucleotides;

[0022] (b) cleaving the cleavable 5' end with a cleavage agent to generate a double-stranded DNA molecule comprising one or more overhangs; and

[0023] (c) ligating one or more double-stranded adapters comprising a 3' overhang to the double-stranded DNA molecule having one or more overhangs, thereby forming the adapter-ligated library; and, optionally,

[0024] (d) sequencing the adaptor-ligated library,

[0025] wherein the plurality of single-stranded polynucleotides are present in an amount less than about 5 nanograms.

[0026] 2. The method according to item 1, wherein the cleavable 5' end is selected from: rA, rC, rG, rU or dU.

[0027] 3. The method according to item 1, wherein the connecting is performed with an efficiency higher than 90%.

[0028] 4. The method according to item 1, wherein said generating comprises:

[0029] (a) contacting the plurality of single-stranded polynucleotides with a first primer library, wherein each primer from the first library comprises a cleavable 5' end;

[0030] (b) extending a primer from the first library to generate a first extension product that hybridizes to the single-stranded polynucleotide, thereby forming a first double-stranded duplex;

[0031] (c) denaturing the first double-stranded duplex into a single strand;

[0032] (d) hybridizing the first polynucleotide extension product to a second primer pool, wherein each primer from the second pool optionally comprises a cleavable 5' end;

[0033] (e) extending the primers from the second library to generate a second extension product that hybridizes to the first extension product, thereby forming a second double-stranded duplex.

[0034] 5. The method according to item 4, wherein the first primer library and the second primer library are random primer libraries comprising 5' cleavable ends.

[0035] 6. The method according to item 4, wherein the first primer library comprises random primers and the second library comprises target-specific primers.

[0036] 7. The method according to item 4, wherein the first primer library comprises target-specific primers and the second library comprises random primers.

[0037] 8. The method according to item 4, wherein the first primer library comprises target-specific primers and the second library comprises target-specific primers.

[0038] 9. The method according to item 6, wherein the target-specific primer comprises a randomized barcode sequence located upstream of the target-specific sequence.

[0039] 10. The method according to item 7, wherein the target-specific primer comprises a randomized barcode sequence located upstream of the target-specific sequence.

[0040] 11. The method according to item 8, wherein the first target-specific primer library and the second target-specific primer library comprise a barcode sequence located upstream of the target-specific sequence.

[0041] 12. The method according to item 1, wherein said generating comprises:

[0042] (a) contacting a plurality of polynucleotides with a primer pool comprising a cleavable 5' end;

[0043] (b) extending the primer to generate a first extension product that hybridizes to the polynucleotide, thereby forming a double-stranded duplex;

[0044] (c) cleaving the cleavable 5' end, thereby generating a double-stranded duplex having a first overhang; and

[0045] (d) ligating one or more adaptors to the double-stranded duplex of (c), wherein the one or more adaptors comprise an overhang that is complementary to the first overhang,

[0046] Therein the method comprises only one round of extension.

[0047] 13. The method according to item 12, wherein the primer library comprises random primers.

[0048] 14. The method according to item 12, wherein the primer library comprises target-specific primers.

[0049] 15. The method according to item 14, wherein the target-specific primer comprises a barcode sequence located upstream of the target-specific sequence.

[0050] 16. A method for generating an adaptor-ligated library of double-stranded nucleic acid molecules comprising in a single reaction vessel or reaction mixture:

[0051] (a) contacting the plurality of single-stranded polynucleotides with a first primer library, wherein each primer from the first library comprises a cleavable 5' end;

[0052] (b) extending a primer from the first library to generate a first extension product that hybridizes to the single-stranded polynucleotide, thereby forming a first double-stranded duplex;

[0053] (c) denaturing the first double-stranded duplex into a single strand;

[0054] (d) hybridizing the first polynucleotide extension product to a second primer pool, wherein each primer from the second pool optionally comprises a cleavable 5' end;

[0055] (e) extending the primers from the second library to generate a second extension product that hybridizes to the first extension product, thereby forming a second double-stranded duplex.

[0056] (f) cleaving the cleavable 5' end with a cleavage agent to generate a double-stranded DNA molecule comprising one or more overhangs; and

[0057] (g) ligating one or more double-stranded adapters comprising a 3' overhang to the double-stranded DNA molecule having one or more overhangs, thereby forming the adapter-ligated library; and, optionally,

[0058] (h) sequencing the adaptor-ligated library.

[0059] 17. The method according to item 16, wherein the cleavable 5' end is selected from: rA, rC, rG, rU or dU.

[0060] 18. The method according to item 16, wherein the first primer library and the second primer library are random primer libraries comprising a 5' cleavable end.

[0061] 19. The method according to item 16, wherein the first primer library comprises random primers and the second library comprises target-specific primers.

[0062] 20. The method according to item 16, wherein the first primer library comprises target-specific primers and the second library comprises random primers.

[0063] 21. The method according to item 16, wherein the first primer library comprises target-specific primers and the second library comprises target-specific primers.

[0064] 22. A method according to item 19, wherein the target-specific primer comprises a randomized barcode sequence located upstream of the target-specific sequence.

[0065] 23. A method according to item 20, wherein the target-specific primer comprises a randomized barcode sequence located upstream of the target-specific sequence.

[0066] 24. A method according to item 21, wherein the first target-specific primer library and the second target-specific primer library comprise a barcode sequence located upstream of the target-specific sequence.

[0067] 25. A method for generating double-stranded adapters optionally comprising a sequence barcode, comprising, in a single reaction vessel or reaction mixture:

[0068] (a) combining a first oligonucleotide pool with a second oligonucleotide pool, wherein each oligonucleotide in the first oligonucleotide pool optionally comprises a cleavable 5' end, wherein each oligonucleotide in the second pool comprises (i) a sequence complementary to a portion of an oligonucleotide in the first pool; (ii) an optional barcode sequence upstream of the sequence complementary to the portion of an oligonucleotide in the first pool; and (iii) a cleavable 5' end upstream of the barcode;

[0069] (b) hybridizing an oligonucleotide from the first pool with an oligonucleotide from the second pool to form a duplex;

[0070] (c) extending the oligonucleotides from the first library such that the oligonucleotides from the first library are covalently linked to a sequence that is complementary to the selectable barcode sequence and to the cleavable 5' end of the oligonucleotides from the second library;

[0071] (d) cleaving the cleavable 5' end to generate a double-stranded adapter comprising the selectable barcode sequence and comprising one or more overhangs; and, optionally,

[0072] (e) ligating the double-stranded adaptor to a plurality of double-stranded nucleic acid molecules comprising one or more overhangs.

[0073] 26. The method according to item 25, wherein the second oligonucleotide library comprises twelve or more different barcode sequences.

[0074] 27. The method according to item 25, wherein the 5' cleavable end is selected from: rA, rC, rG, rU or dU.

[0075] 28. A method according to item 25, wherein the barcode sequence is random.

[0076] 29. A method according to item 25, wherein the barcode sequence is non-random.

[0077] 30. The method according to item 25, wherein the barcode sequence is semi-degenerate.

[0078] Incorporation by reference

[0079] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description of illustrative embodiments utilizing the principles of the present invention and the accompanying drawings, in which:

[0081] Figure 1 An exemplary embodiment of the dual amplification method of the present disclosure is depicted.

[0082] Figure 2 An exemplary embodiment of a single amplification method of the present disclosure is depicted.

[0083] Figure 3 Depicted is a 1.5% agarose gel showing the non-optimized dual amplification method of the present disclosure.

[0084] Figure 4 Explained from Figure 3 Re-amplification products of samples on 1.5% agarose gel.

[0085] Figure 5 Depicted is a 1.5% agarose gel depicting a second repeat attempt of the double amplification process with appropriate controls.

[0086] Figure 6 Replication of the dual amplification method of the present disclosure is depicted on a 1.5% agarose gel.

[0087] Figure 7 Replication of the dual amplification method of the present disclosure is shown on a 1.5% agarose gel.

[0088] Figure 8 Depicted from Figure 7 The x-axis is the DNA input and the y-axis is the DNA output concentration from the replicate experiments. The circles and squares represent the mean values, while the bars correspond to the minimum and maximum output ranges. The circles represent 150 ng of DNA input and the squares represent 75 ng of DNA input.

[0089] Fig. 9 A single amplification method of the present disclosure is illustrated.

[0090] Fig.10 The dual amplification method of the present disclosure is illustrated.

[0091] Fig.11 An exemplary primer extension method of the present disclosure is illustrated.

[0092] Fig.12 An exemplary targeted primer extension method of the present disclosure is depicted.

[0093] Fig.13The generation of linkers by annealing of two single-stranded DNA molecules is described.

[0094] Fig.14 Depicted is the generation of adapters containing random barcodes from imperfectly complementary oligonucleotides.

[0095] Fig.15 Depicted is the generation of adapters containing random barcodes from fully complementary oligonucleotides. DETAILED DESCRIPTION

[0096] definition

[0097] As used herein, "cleavable end" may refer to a nucleotide at or near the 3' or 5' end of a cleavable nucleic acid. The cleavable end may be cleaved by non-enzymatic or enzymatic means including chemical, thermal or photolysis to enable the cleavable end to be released. The cleavable end may refer to either a functional group that can be selectively cleaved or a protected form thereof. For example, the cleavable end may be located in the polymer backbone (i.e., a modified 3'-5' nucleotide connection replaces one of the phosphodiester groups), as a substituent or substitute for one of the bases or sugars in an oligonucleotide primer, and / or as a 3' terminal residue (e.g., a ribonucleotide at the 3' end of an oligodeoxyribonucleotide primer). During primer fixation, hybridization, primer extension and washing conditions, the cleavable end may be stable under standard solid phase DNA synthesis conditions. The cleavable end may also include nucleotides that can be cleaved by enzymes such as nucleases. For example, the cleavable end may be a restriction endonuclease site. Exemplary restriction endonucleases for cleaving at the cleavable ends may include BpmI, BsgI, BseRI, BsmFI, and FokI.

[0098] The cleavable end may also be a nucleotide or series of nucleotides that can block or terminate 5' to 3' enzymatic digestion by an enzyme having 5' to 3' exonuclease activity, such as T7 gene 6 exonuclease. Representative blocking nucleotides may be nucleotides containing thiophosphate, boric acid-phosphate or peptide groups. Blocking nucleotides and / or cleavable ends may be nucleotides that do not inhibit enzymatic extension of the primer.

[0099] As used herein, the terms "adapter" or "adapter" are used interchangeably and may refer to an oligonucleotide that can be attached to a nucleic acid terminus. The adaptor sequence may include, for example, a priming site, a complement of the priming site, a recognition site for an endonuclease, a common sequence, and a promoter. The adaptor may also incorporate modified nucleotides that modify the properties of the adaptor sequence. For example, a phosphorothioate group may be incorporated into one of the adaptor strands.

[0100] As used herein, the terms "library" or "group" may be used interchangeably and may refer to a collection of molecules. The molecules in the group may be identical or different from each other.

[0101] As used herein, the term "complementary" can refer to hybridization or base pairing between nucleotides or nucleic acids, such as, for example, between the two chains of a double-stranded DNA molecule or between a primer binding site on an oligonucleotide primer and a single-stranded nucleic acid to be sequenced or to be amplified. Complementary nucleotides can generally be A (adenine) and T (thymine) (or A and U) or C (cytosine) and G (guanine). Two single-stranded RNA or DNA molecules can be complementary when the nucleotides of one chain are optimally compared and compared and paired with the nucleotides of another chain through appropriate nucleotide insertion or deletion. Complementarity can exist when an RNA or DNA chain hybridizes with its complement under selective hybridization conditions.

[0102] As used herein, the term "primer" may refer to a single-stranded oligonucleotide that can serve as a template-guided DNA synthesis starting point under appropriate conditions, such as buffer and temperature, in the presence of four different nucleoside triphosphates and reagents for polymerization, such as, for example, DNA or RNA polymerase or reverse transcriptase. Primers can be fully complementary to hybridize with such a template. Primers can include a primer site that can direct the primer region of the target nucleic acid to hybridize. Primers can be primers in a primer library. Primers can be a part of a primer pair. Primer pairs (such as target-specific primers) can include a 5' upstream primer that hybridizes with the 5' end of the sequence to be amplified and a 3' downstream primer that hybridizes with the 3' end complement of the sequence to be amplified.

[0103] General Overview

[0104] In some embodiments, the present disclosure provides methods of ligating one or more adaptors to a double-stranded nucleic acid. Figure 1An exemplary embodiment of a method of the present disclosure is depicted. A single-stranded nucleic acid 105 can be contacted with a primer or random primer library 110. The primer can include a cleavable 5' end 115. The primer can be extended 120 to form a first double-stranded duplex 125. The first double-stranded duplex 125 can be denatured 130 to become a single strand 135 of the first double-stranded duplex 125. The same (or different) primer used in the first contacting step can then be contacted 140 with the denatured single strand 135. The primer can be extended 145 to generate a second double-stranded duplex 150. One or more 5' cleavable ends of the second double-stranded duplex 150 can be contacted with a cleavage agent 155, which can cleave 160 the one or more 5' cleavable ends, thereby generating one or more overhangs 165. One or more overhangs 165 can be connected 170 to a double-stranded nucleic acid 175 (e.g., a sequencing adapter), and the double-stranded nucleic acid 175 comprises an overhang complementary to one or more overhangs of the second double-stranded duplex 150. All of the above steps are preferably performed in a single reaction vessel or reaction mixture. A single cleaning step can be used to perform the method before connection. A single cleaning step can be used to perform the entire library preparation. No more than two cleaning steps can be used to perform the entire library preparation. Only three cleaning steps can be used to perform the entire library preparation. Cleaning can refer to a method for separating target nucleic acid molecules from impurities, buffers, or unwanted nucleic acid molecules such as unconnected adapters. Cleaning methods can include columns, such as Qiagen DNA columns or Qiagen MinElute reaction cleaning kits, or bead-based methods, such as Ampure XP beads.

[0105] In some embodiments, the methods of the present disclosure provide methods of ligating one or more adapters to a double-stranded nucleic acid, wherein the method uses only one round of extension (eg, amplification). Figure 2 An exemplary embodiment of a method of the present disclosure in which only one round of extension is used is depicted. A single-stranded nucleic acid 205 can be contacted with a primer (e.g., a primer of a random primer library) 210. The primer can include a cleavable 5' end 215. The primer can be extended 220 to form a first double-stranded duplex 225. One or more 5' cleavable ends of the second double-stranded duplex 225 can be contacted with a cleavage agent 230, which can cleave 235 the one or more 5' cleavable ends, thereby generating one or more overhangs 240. The one or more overhangs 240 can be connected 245 to a double-stranded nucleic acid 250 (e.g., a sequencing adapter), which includes an overhang complementary to the one or more overhangs of the first double-stranded duplex 225.

[0106] Primers

[0107] The present disclosure provides primers comprising one or more modifications (e.g., cleavable nucleotides). One or more modifications (e.g., cleavable nucleotides) may be at the 5' end. One or more modifications (e.g., cleavable nucleotides) may be at the 3' end. One or more modifications (e.g., cleavable nucleotides) may be between the ends of the primer. In some cases, one or more modifications (e.g., cleavable nucleotides) are at the 5' end.

[0108] One or more cleavable nucleotides can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides. One or more cleavable nucleotides can comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides. Exemplary cleavable nucleotides can include RNA, DNA, PNA or LNA, dU, dA, dT, dC and dG or any combination thereof. In some embodiments, the cleavable nucleotides are RNA nucleotides.

[0109] A cleavable nucleotide may contain a modification. A non-cleavable nucleotide may contain a modification that enables it to be cleaved. Exemplary modifications may include, but are not limited to, biotinylated nucleotides, methylated nucleotides, acetylated nucleotides, PNA nucleotides, polyadenylated, polyuridylated, and LNA nucleotides. In some cases, an exemplary modification is a 5-mer that can be added by a guanylyl transferase. ’ 7-Methylguanosine.

[0110] Primers can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length. Primers can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length. In some cases, primers are 6 nucleotides (hexamers) in length.

[0111] Primers can be part of a primer library. A primer library can contain at least 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 primers or more. A primer library can contain at most 100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 primers or more.

[0112] Primer can be random primer (for example random hexamer primer). Random primer library can comprise primers that are substantially different from each other. Random primer library can be hybridized with multiple positions in target nucleic acid. Random primer library can have multiple primers, wherein each primer is different from other primers. Primer in random primer library can have at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% or more complementarity and / or identity with another primer in random primer library. Primer in random primer library can have at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% or more complementarity and / or identity with another primer in random primer library.

[0113] Primer can be a target specific primer. Target specific primer can be a primer designed to hybridize with a specific region of a target nucleic acid. Target specific primer can hybridize with a target nucleic acid having at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% complementarity. Target specific primer can hybridize with a target nucleic acid having at most 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% complementarity. Target specific primer can hybridize with a target nucleic acid in excess of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the primer length. The target-specific primer can hybridize to the target nucleic acid over at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the primer length. The target-specific primer can hybridize to the target nucleic acid and have at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more mismatches. The target-specific primer can hybridize to the target nucleic acid and have at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more mismatches.

[0114] In some cases, the target-specific primer comprises a tail. The tail can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides in length. The tail can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides in length. The tail can be 1-5 nucleotides, 1-10 nucleotides, 1-15 nucleotides, 1-30 nucleotides, 5-10 nucleotides, 5-15 nucleotides or 5-30 nucleotides in length. The tail can comprise a random sequence. The tail can comprise a random hexamer. The tail comprising a random sequence can be any random sequence of the present disclosure. For example, a random tail sequence can comprise nucleotides, such as, for example, fluorophores, dideoxynucleotides, deoxynucleotides, radioactive labels, dyes, dUTP, rA, rU, rG, rC, rUTP, 5'7-methylguanosine added by guanylate transferase, or restriction enzyme target sequences.

[0115] method

[0116] The present disclosure provides methods for sample preparation (e.g., adapter ligation for sequencing). A sample comprising a single-stranded nucleic acid (e.g., RNA and / or DNA) can be contacted with a primer of the present disclosure. The primer can comprise a modification (e.g., a cleavable nucleotide at its 5' end). The primer (or a primer in a primer library) can bind to one or more positions within a target nucleic acid (e.g., a fragmented target nucleic acid) library. The primer (or a primer in a primer library) can bind to a specific position within a target nucleic acid library.

[0117] Primers can be extended (e.g., using dNTPs, dNTPs containing dUTP, rA, rU, rG, rC, rUTP, fluorophores, dideoxynucleotides, deoxynucleotides, radioactive labels or dyes for downstream purification or degradation). In some cases, primer extension occurs only once. The length of primer extension can depend on the type of polymerase used to perform the extension. Exemplary polymerases that can be used for extension can include, but are not limited to, DNA polymerase, RNA polymerase, reverse transcriptase, DNA polymerase I, polyA polymerase, Bst polymerase large fragment, Bst 2.0 (NEB), Bst 2.0 Warmstart polymerase, Klenow (exo-), Klenow large fragment, DNA polymerase I large (Klenow) fragment, T4 DNA polymerase, Bsu polymerase large fragment, Sulfolobus DNA polymerase, Deep Vent (exo-) DNA polymerase (NEB), Deep Vent DNA polymerase (NEB), Vent DNA polymerase, Taq, phi29, T7 RNA polymerase, transferases (e.g., guanylate transferases that add 5' 7-methylguanosine, methyltransferases, acyltransferases, selenoyltransferases, transketolases, glycosyltransferases, acyltransferases), and rTth DNA polymerase. The length of the primer extension product can be at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 or more bases. The length of the primer extension product can be at most 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 or more bases. Primer extension can produce a double-stranded duplex, wherein one strand comprises a cleavable 5' end. Extension can be performed at room temperature. The extension can be carried out without changing the reaction temperature.

[0118] In some cases, the method includes a second round of extension. The second round of extension includes denaturing the double-stranded duplex containing the cleavable 5' end into its corresponding single strand. One strand of the denatured double-stranded duplex may correspond to the original single-stranded target nucleic acid. One strand of the denatured double-stranded duplex may correspond to the primer extension product incorporated into the cleavable 5' end. In the second round of extension, primers from a primer library may hybridize with one or more regions on the primer extension product. Primers may be extended by primer extension. The length of primer extension may depend on the type of polymerase used to extend. Exemplary polymerases that can be used for extension can include, but are not limited to, DNA polymerases, RNA polymerases, reverse transcriptases, DNA polymerase I, polyA polymerases, Bst polymerase large fragment, Bst 2.0 (NEB), Bst 2.0 Warmstart polymerase, Klenow (exo-), Klenow large fragment, DNA polymerase I large (Klenow) fragment, T4 DNA polymerase, Bsu polymerase large fragment, Sulfolobus DNA polymerase, Deep Vent (exo-) DNA polymerase (NEB), Deep Vent DNA polymerase (NEB), Vent DNA polymerase, Taq, phi29, T7 RNA polymerase, transferases (e.g., guanylate transferases that add 5' 7-methylguanosine, methyltransferases, acyltransferases, selenoyltransferases, transketolases, glycosyltransferases, acyltransferases), and rTth DNA polymerase. The second primer extension product can be at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 bases or more in length. The second primer extension product can be at most 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 bases or more in length. Primer extension can produce a double-stranded duplex in which both strands comprise a cleavable 5' end.

[0119] When there are two rounds of primer extension, the first round of extension can be performed with a target-specific primer, and the second round of extension can be performed with a random primer. When there are two rounds of primer extension, the first round of extension can be performed with a random primer, and the second round of extension can be performed with a target-specific primer.

[0120] In some cases, there can be multiple rounds of primer extension. The number of rounds of primer extension can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. The number of rounds of primer extension can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. In some cases, one round of primer extension is performed. In some cases, two rounds of primer extension are performed.

[0121] Adapter ligation

[0122] A double-stranded duplex containing one or two cleavable ends can be cut with a cleavage agent to remove the cleavable end. The cleavage agent can be specific for the cleavable nucleotide on the 5' cleavable end. For example, if the nucleotide is uracil, the cleavable agent can be uracil-DNA glycosylase (UDG). Exemplary uracil-DNA glycosylases can include but are not limited to Escherichia coli (E.coli) uracil-DNA glycosylase (NEB), uracil DNA-glycosylase (Life Technologies), Afu uracil-DNA glycosylase, Antarctic thermosensitive uracil DNA-glycosylase, Epicentre HK-uracil-N- glycosylase, thermosensitive uracil-N-glycosylase, or uracil-DNA cleavage mixture, or ThermoScientific uracil-DNA glycosylase. The cleavage agent can be non-specific for a specific sequence. For example, the cleavage agent can be a prokaryotic DNA polymerase I that maintains its 5'→3' exonuclease activity. DNA polymerase I can be from different organisms (e.g., E. coli, Bacillus subtilis (B.subtilis) and Thermus aquaticus (Thermus aquaticus)). Such DNA polymerase I can be purchased from, for example, New England BioLabs, Life Technologies and Epicentre. In other examples, the cutting agent can be a nuclease that cuts the RNA portion of the DNA-RNA hybrid molecule. Such nucleases can include, but are not limited to, ribonuclease hybrid enzymes thermostable RNAse H (Epicentre), RBase HII, RNase If, ​​RNAse A, Rnase I, RnaseP, RNase T1 and Riboshredder Rnase Blend. The cutting agent can cut the phosphodiester bond between the cleavable nucleotide and the adjacent non-cleavable nucleotide. The cutting agent can cut off at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides of one or more 5' cleavable ends. The cleavage agent may cleave off up to 12, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides of one or more 5' cleavable ends. In particularly preferred embodiments, cleavage produces overhangs with identical sequences, thereby allowing ligation of adaptors to complementary overhangs.

[0123] The cleavage agent may be a nuclease. The nuclease may be an endonuclease. Examples of endonucleases include restriction enzymes, type I endonucleases, type II endonucleases, T7 endonucleases, T4 endonucleases, Bal 31 endonucleases, S1 nucleases, AP endonucleases, DNaseI and Endo R. The nuclease may be an exonuclease. Examples of exonucleases include 5'-3' exonucleases, 3'-5' exonucleases, exonucleases I, exonucleases II, exonucleases III, exonucleases IV, exonucleases V, T7 exonucleases and lambda exonucleases. The nuclease may be a DNA nuclease. The nuclease may be an RNA nuclease. Examples of RNA nucleases may include RNaseA, RNaseH and RNase T1. Nucleases may cleave DNA. Nucleases may cleave RNA. Nucleases may cleave one or more RNA-DNA hybrid chains.

[0124] The cleavage of the 5' cleavable end can produce an overhang. The overhang can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides in length. The overhang can be at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides in length. The overhang can be 1-5 nucleotides, 1-10 nucleotides, 1-15 nucleotides, 1-30 nucleotides, 5-10 nucleotides, 5-15 nucleotides or 5-30 nucleotides. The overhang can include one or more nucleotides with modification. Exemplary modifications can include, for example, fluorophores, dideoxynucleotides, deoxynucleotides, radioactive labels, dyes, dUTP, rA, rU, rG, rC, rUTP and restriction enzyme target sequences.

[0125] In some cases, the overhang is one nucleotide in length. A nucleotide overhang can be A, T, G, C, or U. In some cases, a nucleotide overhang is A. In some cases, a nucleotide overhang is T.

[0126] The adaptor to be ligated may comprise an overhang. The overhang of the adaptor may be complementary to the overhang of the cleavable end (ie, the overhang of the adaptor may hybridize to the overhang of the cleavable end by forming a sticky end).

[0127] The adapter to be connected may include a sequencing adapter (e.g., for deep sequencing). The adapter to be connected may include a nucleic acid barcode sequence (e.g., for labeling a nucleic acid sample). The adapter may include a nucleic acid random barcode generated by any adapter oligonucleotide containing a cleavable 3' or 5' end. The adapter may include any nucleic acid sequence (e.g., a restriction enzyme sequence, a modified nucleotide, a cleavable nucleotide, an uncleavable nucleotide). The adapter may be all double-stranded or substantially double-stranded. The adapter may include a secondary structure. A double-stranded adapter may include two oligonucleotides that are at least partially complementary. The adapter may be phosphorylated or non-phosphorylated on one or both chains. The adapter may include a double-stranded portion and a single-stranded overhang portion that is fully or partially complementary to the overhang. For example, when DNA is digested with the restriction enzyme EcoRI, the resulting double-stranded fragment may be flanked by a single-stranded overhang 5'-AATT-3' at either end, and the adapter carrying the single-stranded overhang 5'-AATT-3' may hybridize with the fragment by complementarity between the overhanging regions. This adapter hybridization with the "sticky end" of the fragment can facilitate the connection of the adapter to the fragment. Blunt end connection may also be possible. The exonuclease activity of the Klenow fragment can be used to convert the blunt end into a sticky end. For example, when digesting DNA with PvuII, by incubating the fragment with Klenow in the presence of dTTP and dCTP, the blunt end can be converted to a two base pair overhang. The overhang can also be converted to a blunt end by filling the overhang or removing the overhang.

[0128] The ligation can be performed by using a ligase. Exemplary ligases can include Ampligase thermostable DNA ligase, E. coli DNA ligase, Taq DNA ligase, T7 DNA ligase, T3 DNA ligase, 9°N DNA ligase, ElectroLigase (NEB), DNA ligase, RNA ligase, T4 DNA ligase, DNA ligase I, DNA ligase III, and DNA ligase IV. The ligation method may include utilizing T4 DNA ligase, which catalyzes the formation of phosphodiester bonds between juxtaposed 5' phosphate and 3' hydroxyl termini in a duplex DNA or RNA having blunt and sticky ends; Taq DNA ligase, which catalyzes the formation of phosphodiester bonds between juxtaposed 5' phosphate and 3' hydroxyl termini of two adjacent oligonucleotides hybridized to a complementary target DNA; E. coli DNA ligase, which catalyzes the formation of phosphodiester bonds between juxtaposed 5' phosphate and 3' hydroxyl termini in a duplex DNA containing sticky ends; and T4 RNA ligase, which catalyzes the ligation of a 5' phosphoryl-terminated nucleic acid donor to a 3' hydroxyl-terminated nucleic acid acceptor by forming a 3' to 5' phosphodiester bond. The ligation efficiency of any disclosed method of ligating an adaptor to a nucleic acid having an overhang generated by cleaving a cleavable 5' nucleotide may be >50%, >60%, >70%, >80%, >90%, >95%, >98%, >99%, or >99.5%.

[0129] In some embodiments, a double-stranded adapter is used, and only one strand is connected to the primer extension product. The connection of one strand of the adapter can be selectively blocked. For example, one strand of the adapter can be designed to introduce a gap of one or more nucleotides between the 5' end of the strand of the adapter and the 3' end of the target nucleic acid. The absence of a phosphate at the 5' end of the adapter can block the connection of this 5' end to the available 3' OH.

[0130] Sequencing

[0131] The double-stranded product comprising the single-stranded polynucleotide connected to the adapter can be sequenced. Exemplary sequencing techniques can include, for example, emulsion PCR (pyrophosphate sequencing from Roche 454, semiconductor sequencing from Ion Torrent, SOLiD connection sequencing from Life Technologies, synthetic sequencing from Intelligent Biosystems), bridge amplification (such as Solexa / lllumina) on the flow cell, isothermal sequencing (Life Technologies) of Wildfire technology, or rolling circle colonies (rolonies) / nanospheres (Complete Genomics, Intelligent Biosystems, Polonator) generated by rolling circle amplification. Sequencing techniques, such as Heliscope (Helicos), SMRT technology (Pacific Biosciences) or nanopore sequencing (Oxford Nanopore) that allow direct sequencing of single molecules without the need for advance clonal amplification can be suitable sequencing platforms.

[0132] sample

[0133] The sample used in the method of the present disclosure may include various sources and compositions containing nucleic acids. The sample may be a biological sample, but the term also includes other samples, for example, containing nucleic acids, such as artificial samples of PCR products or compositions containing purified nucleic acids, for example. Exemplary samples may include, but are not limited to, whole blood; blood products; red blood cells; white blood cells; buffy coat; swabs; urine; sputum; saliva; semen; lymph fluid; amniotic fluid; cerebrospinal fluid; peritoneal effusion; pleural effusion; biopsy samples; cyst fluid; synovial fluid; vitreous fluid; aqueous humor; cyst fluid; eye wash fluid; eye aspirate; plasma; serum; lung lavage fluid; lung aspirate; animals, including human or plant tissues, including but not limited to liver, spleen, kidney, lung, intestine, brain, heart, muscle, pancreas, cell culture, and lysates, extracts or substances and fractions obtained from the above samples or any cells and microorganisms and viruses that may be present on or in the samples. Materials obtained from clinical or forensic environments containing nucleic acids may also be within the meaning of the term "sample". The sample can be a biological sample derived from humans, animals, plants, bacteria or fungi. The sample can be derived from cells, tissues and / or organs. The sample can also be a processed sample, such as a preserved, fixed and / or stable sample. The sample can contain RNA. The sample can contain DNA.

[0134] Reagent test kit

[0135] The present disclosure provides a kit. The kit may include reagents for implementing the method of the present disclosure. The kit may include primers of the present disclosure (e.g., primers with 5' cleavable ends). The kit may include a library of primers (e.g., random primers). The kit may include target-specific primers. The kit may include different libraries of target-specific primers.

[0136] The kit may include one or more adapters, polymerases, cleavage agents and / or ligases. The kit may include reagents for primer extension, such as dNTPs and / or NTPs. The kit may include a buffer. The buffer may be used for dilution, activation and / or reconstitution of all items in the kit. The kit may be maintained at room temperature. The kit may be maintained at 4° C. The kit may be frozen.

[0137] The kit may include instructions for use. The instructions may be handwritten or electronic. The instructions for use may describe how to use the kit in the methods of the present disclosure. For example, the instructions for use may describe the following method: contacting a single-stranded polynucleotide sample with a random primer library from the kit; extending the primer to generate a first extension product that hybridizes with the single-stranded polynucleotide, thereby generating a first double-stranded duplex; denaturing the double-stranded duplex into its single strand; hybridizing the primer library with a single first extension product strand; extending the primer to generate a second extension product that hybridizes with the first extension product, thereby generating a second double-stranded duplex; cleaving one or more 5' cleavable ends of the second double-stranded duplex, thereby generating one or more nucleotide overhangs; and connecting one or more adapters to the one or more nucleotide overhangs.

[0138] In some cases, the instructions for use may describe the following method: contacting a single-stranded polynucleotide sample with a target-specific primer library from a kit; extending the primer to generate a first extension product that hybridizes to the single-stranded polynucleotide, thereby generating a first double-stranded duplex; denaturing the double-stranded duplex into its single strands; hybridizing the primer library to the single first extension product strand; extending the primer to generate a second extension product that hybridizes to the first extension product, thereby generating a second double-stranded duplex; cleaving one or more 5' cleavable ends of the second double-stranded duplex, thereby generating one or more nucleotide overhangs; and ligating one or more adapters to the one or more nucleotide overhangs, wherein only two extension steps are performed.

[0139] In some cases, the instructions for use may describe the following method: contacting a single-stranded polynucleotide sample with a random primer library from a kit; extending the primer to generate a first extension product that hybridizes to the single-stranded polynucleotide, thereby generating a first double-stranded duplex; cleaving one or more 5' cleavable ends of the second double-stranded duplex, thereby generating one or more nucleotide overhangs; and ligating one or more adapters to the one or more nucleotide overhangs, wherein only one round of primer extension is performed.

[0140] In some cases, the instructions for use may describe the following method: contacting a single-stranded polynucleotide sample with a random primer library from a kit; extending the primer to generate a first extension product that hybridizes to the single-stranded polynucleotide, thereby generating a first double-stranded duplex; denaturing the double-stranded duplex into its single strands; hybridizing a target-specific primer library to the single first extension product strand; extending the primer to generate a second extension product that hybridizes to the first extension product, thereby generating a second double-stranded duplex; cleaving one or more 5' cleavable ends of the second double-stranded duplex, thereby generating one or more nucleotide overhangs; and ligating one or more adapters to the one or more nucleotide overhangs, wherein only two extension steps are performed.

[0141] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Those skilled in the art will now appreciate that many variations, changes and substitutions will be appreciated without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in the process of practicing the present invention. It is intended that the scope of the present invention be limited by the appended claims, and that methods and structures and their equivalents within the scope of these claims are thus covered.

[0142] Example

[0143] Example 1: Single amplification of DNA

[0144] The genomic DNA is fragmented according to the standard sample preparation procedure for next-generation sequencing. In the presence of reversibly modified random hexamers, the sample is denatured immediately. A mixture of dNTP (or containing dUTP, rA, rU, rG, rC, rUTP, fluorophore, dideoxynucleotide, deoxynucleotide, radiolabeled or dye for downstream purification or degradation of dNTP), polymerase and buffer is added to the sample. According to the incubation time and conditions applicable to the polymerase, a single extension is performed by the modified hexamer. DNA polymerase I with 5'-3' nuclease activity is used to remove the overhang that exceeds the template molecule due to extension. After the extension is completed, the sample is purified using a standard purification procedure. The sample is then treated to remove the modification (different treatments are performed according to the modification type), and the sample is added to the TA / flat-end ligation reaction containing a modified adapter (e.g., Illumina adapter). An adapter comprises the sequence of a second paired end adapter, and the second paired end adapter comprises an overhang complementary to the overhang on the extension product. The second adapter comprises the sequence of the first paired end adapter, which comprises a blunt end that will be connected to the blunt end of one of the two ends of the extension product. Then, optionally, the size of the reaction product is selected and the reaction is directly placed in an indexing PCR reaction that can be selected based on the adapter used. After the optional PCR, the size of the sample is selected to fit the size range of the next generation sequencing. Fig. 9 An exemplary diagram of a single amplification method of the present disclosure is shown in FIG.

[0145] Example 2: Double amplification of DNA

[0146] Fig.10An exemplary diagram of the single amplification method of the present disclosure is shown in FIG. The genomic DNA is fragmented according to the standard sample preparation procedure for next-generation sequencing. In the presence of a reversibly modified random hexamer, the sample is denatured immediately. A mixture of dNTPs (or, for example, containing dUTP, rA, rU, rG, rC, rUTP, fluorophores, dideoxynucleotides, deoxynucleotides, radiolabeled or dyes for downstream purification or degradation of dNTPs), polymerase and buffer is added to the sample. The first extension starting from the modified hexamer follows the incubation time and conditions applicable to the polymerase. Once the extension is completed, the sample is denatured, and if necessary, additional polymerase is added to the sample. The second extension follows the same conditions as the first extension. After the second extension is completed, the sample is purified using a standard purification procedure. The sample is then treated to remove the modification (different treatments are performed according to the modification type), and the sample is added to a TA ligation reaction containing an adapter (e.g., an Illumina adapter). Optionally, the size of the reaction product is then selected and the reaction product is directly placed in an indexed PCR reaction according to a standard indexed PCR procedure. Alternatively, altered adapters can be used with non-PCR based methods.After PCR, the size of the sample is selected to fit the size range of next generation sequencing.

[0147] For example, Figure 3 Depicted is the sample preparation prepared by the dual amplification method. The first lane contains O'Gene Ruler 1kb plus DNA ladder. The second brightest band is 500bp. The lower bands correspond to 400, 300 and 200bp. The second lane shows a test sample starting with 150ng DNA. The sample is then treated with the dual amplification method to produce linker-connected nucleic acid fragments. The third lane starts with 75ng DNA and is then treated with the dual amplification method to produce linker-connected nucleic acid fragments. The fourth lane starts with 150ng DNA and is then treated with the dual amplification method to produce linker-connected nucleic acid fragments. The fifth lane starts with 75ng DNA and is then treated with the dual amplification method to produce linker-connected nucleic acid fragments. The fifth lane is the same molecular weight standard ladder used in the first lane. The concentration of each final product after PCR is: Lane 2: 21ng / μl; Lane 3: 34.3ng / μl; Lane 4: 25.8ng / μl; Lane 5: 27.5ng / μl.

[0148] Figure 4 The diagram shows Figure 3 The re-amplification products of the samples were analyzed on a 1.5% agarose gel. Figure 3The adapter-linked nucleic acid library fragments were removed from the adapter-adapter products shown below 200 bp in the sample, and the sample was size selected by gel excision of about 220-350 bp, which corresponds to the minimum and maximum of the brightest size range. The gel excised samples were column purified and used as template for reamplification PCR reactions. The first lane contains O'GeneRuler 1 kb plus DNA ladder. The brightest band is 500 bp below. The lower bands correspond to 400, 300 and 200 bp. The second lane shows the Figure 3 Lane 2, re-amplification of size selected product starting with 150 ng template processed according to the dual amplification method. Lane 3 shows the product from Figure 3 Lane 3, re-amplification of size selected product starting with 75 ng template processed according to the dual amplification method. Lane 4 shows the product from Figure 3 Lane 4, re-amplification product of size selected product, starting with 150 ng template processed according to the double amplification method. Lane 5 shows the Figure 3 Lane 5, reamplification product of size selected product starting with 75 ng template processed according to the dual amplification method. Lane 6 is the same molecular weight standard ladder used in lane 1. Qualitatively, the samples appear to be amplified after size selection.

[0149] Figure 5 A 1.5% agarose gel describing the second repeat attempt of the double amplification procedure with appropriate controls is depicted. The first lane contains O'GeneRuler 1kb plus DNA ladder. The brightest band corresponds to 500bp below. The bands below correspond to 400, 300 and 200bp. The second lane shows a test sample starting with 150ng DNA. The sample is then treated with a double amplification method to produce nucleic acid fragments connected by adapters. The third lane is 150ng DNA, which is fragmented but not subjected to the double amplification method and immediately flows out on the agarose gel. The fourth lane undergoes the double amplification method in the absence of DNA and modified hexamer, but contains Illumina adapters. The fifth lane undergoes the double amplification protocol in the absence of DNA, but contains modified hexamer and Illumina adapters. The sixth lane undergoes the double amplification process, but lacks DNA input and only contains adapters during the ligation reaction. The seventh lane is a negative control reaction, which undergoes the double amplification protocol in the absence of any DNA, modified hexamer and adapters. The eighth lane is the same as the first lane.

[0150] Figure 6 Depicted Figure 31.5% agarose gel. The first lane contains O'GeneRuler 1kb plus DNA ladder. The brightest band below corresponds to 500bp. The bands below correspond to 400, 300 and 200bp. The second lane shows a sample with 150ng starting DNA. The third lane shows a sample with 150bp starting DNA. The fourth lane shows a sample with 75ng starting DNA. The fifth lane shows a sample with 75ng DNA, which was fragmented but not subjected to the dual amplification protocol and immediately flowed out of the agarose gel. The sixth lane is a negative control without DNA. The seventh lane is the same as the first lane.

[0151] Figure 7 Shows Figure 3 The conditions of the procedure in are replicated on a 1.5% agarose gel. The first lane contains O'GeneRuler 1kb plus DNA ladder. The brightest band below corresponds to 500bp. The bands below correspond to 400, 300, and 200bp. Lanes 2-7 are products of 150ng of starting DNA processed using the dual amplification protocol. Lane eight is the same as lane one. Lanes 9-14 are products of 75ng of starting DNA processed using the dual amplification protocol. Lane 15 is the same as lane one. The concentration of dsDNA product in each lane was quantified using the Qubit HS dsDNA kit and Figure 8 Shown in.

[0152] Figure 8 Depicted from Figure 7 Figure 2 shows the results of replicates of 150 ng of input DNA processed according to the dual amplification protocol. The x-axis is the DNA input and the y-axis is the DNA output concentration from the replicate dual amplification protocol. The circles and squares represent the mean values, while the bars correspond to the minimum and maximum output ranges. The circles represent the products of 150 ng of input DNA processed according to the dual amplification protocol, and the squares represent the products of 75 ng of input DNA processed according to the dual amplification protocol.

[0153] To determine the amount of DNA that can successfully generate a library, 15ng, 5ng, and 500pg of DNA were processed according to the single amplification method. As measured by Agilent Bioanalyzer and qPCR, both 15ng and 5ng samples produced successful libraries. The results of the 500pg sample were inconclusive.

[0154] Example 3: RNA Dual Amplification in a Single Reaction Mix or Container

[0155] According to the standard sample preparation procedure of the next generation sequencing, mRNA is fragmented. In the presence of a random hexamer of reversible modification, the sample is denatured immediately. A mixture of dNTP (or, for example, containing dUTP, rA, rU, rG, rC, rUTP, fluorophore, dideoxynucleotide, deoxynucleotide, radiolabeled or dye for downstream purification or degraded dNTP), reverse transcriptase (RT), RNAse inhibitor and buffer is added to the sample. The first extension starting from the modified hexamer follows the incubation time and conditions applicable to RT. Once the extension is completed, the sample is denatured and quenched, and polymerase is added to the sample. The second extension follows the conditions applicable to polymerase. After the second extension is completed, the sample is purified using a standard purification procedure. The sample is then processed to remove the modification (different treatments are performed according to the modification type), and the sample is added to the TA ligation reaction containing an adapter (e.g., Illumina). Then optionally, the size of the reaction product is selected and the reaction product is directly placed in the indexed PCR reaction according to the standard indexed PCR procedure. Alternatively, the adapter that changes can be used together with a non-PCR-based method. After PCR, the size of the sample is selected to fit the size range for next-generation sequencing.

[0156] Example 4: Double purification and double amplification of RNA

[0157] According to the standard sample preparation procedure of the next generation sequencing, mRNA is fragmented. In the presence of random hexamers, the sample is denatured immediately. A mixture of dNTP, reverse transcriptase (RT) and buffer is added to the sample. The first extension starting from the hexamer follows the incubation time and conditions suitable for RT. Once the extension is completed, the sample is purified according to the standard procedure. Next, the reversibly modified random hexamer is added to the sample and denatured, and the polymerase is added to the sample. The second extension follows the conditions suitable for using dNTP, containing dUTP, rA, rU, rG, rC, rUTP, fluorophore, dideoxynucleotide, deoxynucleotide, radioactive label or dye for downstream purification or degradation of dNTP polymerase. After the second extension is completed, the sample is purified using the standard purification procedure. The sample is then treated to remove the modification (different treatments are performed according to the modification type), and the sample is added to the TA / blunt end connection reaction containing a modified adapter (e.g., from Illumina). An adapter comprises the sequence of the second paired end adapter, and the second paired end adapter contains an overhang complementary to the overhang on the double extension product. The second adapter comprises the sequence of the first paired end adapter, which comprises a flat end that can be connected to the flat end on one side of the extension product. Optionally, the size of the reaction product is selected and the reaction product is directly placed in an indexed PCR reaction that can be selected based on the adapter used. After the optional PCR, the size of the sample is selected to fit the size range of the next generation sequencing.

[0158] Example 5: Targeted amplification using single extension with reversibly modified random hexamer primers

[0159] The genomic DNA is fragmented according to the standard sample preparation procedure for next-generation sequencing. In the presence of reversibly modified random hexamers, the sample is denatured immediately, and a mixture of dNTPs (or, for example, containing dUTP, rA, rU, rG, rC, rUTP, fluorophores, dideoxynucleotides, deoxynucleotides, radiolabeled or dyes for downstream purification or degradation of dNTPs), polymerase and buffer are added to the sample. According to the incubation time and conditions applicable to the polymerase, a single extension is performed from the modified hexamer. After the extension is completed, the sample is purified using a standard purification procedure. The sample is then treated to remove the modification (different treatments are performed according to the modification type), and the sample is added to a TA / blunt-end ligation reaction containing a modified adapter (e.g., an Illumina adapter). An adapter comprises a modified sequence similar to a second paired-end adapter, and the second paired-end adapter comprises an overhang complementary to an overhang on an extension product. The second adapter comprises a modified sequence of a first paired-end adapter, and the first paired-end adapter comprises a blunt end that will be connected to the blunt end of one of the two ends of the extension product.

[0160] If these adapters are not enough to bind to the flow cell for sequencing, then an indexed PCR reaction can be performed to incorporate the appropriate sequence. The indexed PCR reaction can be used to add a label or barcode to the sample. Alternatively, the size of the ligation reaction product is selected and the reaction product is directly placed in the indexed PCR reaction. The reaction comprises an indexed primer, a multiplex primer and a target-specific primer comprising an appropriate sequencing oligonucleotide sequence for binding to the flow cell. After the PCR reaction, the size of the sample is selected to be suitable for the size range of the next generation sequencing.

[0161] Example 6: Alternative Targeted Amplification Method Using Dual Amplification with Reversibly Modified Random Hexamers

[0162] The genomic DNA is fragmented according to the standard sample preparation procedure for next-generation sequencing. In the presence of reversibly modified random hexamers, the sample is immediately denatured, quenched, and a mixture of dNTP (or, for example, containing dUTP, rA, rU, rG, rC, rUTP, fluorophores, dideoxynucleotides, deoxynucleotides, radiolabeled or dyes for downstream purification or degraded dNTPs), polymerase and buffer is added to the sample. The first extension starting from the modified hexamer follows the incubation time and conditions applicable to the polymerase. Once the extension is completed, the sample is denatured, quenched, and additional polymerase is added to the sample. The second extension follows the same conditions as the first extension. After the second extension is completed, the sample is purified using a standard purification procedure. The sample is then treated to remove the modification (different treatments are performed according to the modification type), and the sample is added to the TA ligation reaction containing the modified adapter (e.g., Illumina adapter).

[0163] If these adapters are not enough to bind to the flow cell for sequencing, an indexing reaction can be performed to incorporate the appropriate sequence. Alternatively, the size of the ligation reaction product is selected and the reaction product is directly placed in the indexing PCR reaction. The reaction comprises an indexing primer, a multiplex primer, and a target-specific primer containing an appropriate oligonucleotide sequence for binding to the flow cell. After PCR, the size of the sample is selected to fit the size range of the next generation sequencing.

[0164] Example 7: Alternative method for targeted amplification - single amplification with reversibly modified targeting primers

[0165] Fig.11An exemplary diagram of a single amplification method of the present disclosure is shown in . Genomic DNA is fragmented according to the standard sample preparation procedure for next-generation sequencing. In the presence of a reversibly modified targeting primer, the sample is immediately denatured, quenched, and a mixture of dNTPs (or, for example, containing dUTP, rA, rU, rG, rC, rUTP, fluorophores, dideoxynucleotides, deoxynucleotides, radioactive labels or dyes for downstream purification or degradation of dNTPs), polymerase and buffer are added to the sample. The first extension starting from the modified targeting primer follows the incubation time and conditions applicable to the polymerase. After the polymerase extension is completed, the sample is purified using a standard purification procedure. The sample is then treated to remove the modification (different treatments are performed according to the modification type), and the sample is added to a TA / blunt end ligation reaction containing a modified adapter (e.g., an Illumina adapter). An adapter comprises a second paired end adapter, such as the sequence of a second Illumina adapter, and the second paired end adapter comprises an overhang complementary to the overhang on the extension product. The second adapter comprises a first paired end adapter, such as the sequence of a first Illumina adapter, which comprises a blunt end that will be connected to the blunt end of one of the two ends of the extension product. Then, optionally, the size of the reaction product is selected and the reaction product is directly placed in an indexed PCR reaction that can be selected based on the adapter used. After the optional PCR, the size of the sample is selected to fit the size range of the next generation sequencing.

[0166] Example 8: Alternative method for targeted amplification - dual amplification with reversibly modified targeting primers

[0167] Fig.12An exemplary diagram of the single amplification method of the present disclosure is shown in . Genomic DNA is fragmented according to the standard sample preparation procedure for next-generation sequencing. In the presence of reversibly modified targeting primers, the sample is denatured immediately, and a mixture of dNTP (or containing dUTP, rA, rU, rG, rC, rUTP, fluorophore, dideoxynucleotide, deoxynucleotide, radiolabeled or dye for downstream purification or degradation of dNTP), polymerase and buffer is added to the sample. Once the extension is completed, the modified random hexamer is added to the sample and denatured, quenched, and additional polymerase is added to the sample. The second extension follows the same conditions as the first extension. After the second extension is completed, the sample is purified using a standard purification procedure. The sample is then treated to remove the modification (different treatments are performed according to the modification type), and the sample is added to a TA ligation reaction containing an adapter (e.g., an Illumina adapter). Then optionally, the size of the reaction product is selected and the reaction product is directly placed in an indexed PCR reaction according to a standard indexed PCR procedure. Alternatively, the adapter that is changed can be used together with a non-PCR-based method. After PCR, the size of the sample is selected to fit the size range for next-generation sequencing.

[0168] Example 9: Generation of adapters containing overhangs and random barcodes

[0169] Fig.13 A standard method for forming adapters for preparing next-generation sequencing libraries is shown. Fig.13 In the process, two oligonucleotides containing complementary sequences hybridize under selective conditions to produce a double-stranded linker. Fig.14 14 is an exemplary diagram of a method for producing a linker containing a random barcode sequence using the method of the present disclosure. The first oligonucleotide library (1410) contains oligonucleotides containing a sequence complementary to the sequence in the second oligonucleotide library (1415). In addition to the complementary sequence, the second oligonucleotide also contains random nucleotides synthesized and incorporated at its 5' end, and one or more cleavable nucleotides are on its more 5' side. Such cleavable nucleotides can be RNA nucleotides. The two oligonucleotide libraries are mixed and allowed to hybridize. The double-stranded product (1420) is treated with dNTPs, buffer and polymerase to extend the first oligonucleotide so that it includes a sequence complementary to the random barcode and the cleavable 5' nucleotide (1425). Then the cleavage (1430) contains a cleavable 5' nucleotide selected from rU, rA, rC, rG or dU. By treating with RNaseH, or in the case of dU, by treating with uracil-DNA glycosylase to complete the cleavage to generate a final linker product (1430) containing at least one overhang. Fig.15 Shown with Fig.14The same procedure except that oligonucleotide 2 (1515) contains the sequence of oligonucleotide 1 (1510) except that oligonucleotide 2 only has an additional random barcode and one or more cleavable nucleotides at its 5' end.

[0170] Example 10: Preparation of a library without amplification errors

[0171] The genomic DNA is fragmented according to the standard sample preparation procedure for next-generation sequencing. In the presence of modified random hexamers, the sample is denatured immediately. A mixture of dNTPs and a variety of cleavable dNTPs, polymerases, and buffers is added to the sample. According to the incubation time and conditions suitable for the polymerase, a single extension is performed from the modified hexamer. DNA polymerase I with 5'-3' nuclease activity is used to remove the overhangs that exceed the template molecule due to extension. After the extension is completed, the sample is treated with a cutting agent that generates an overhang and cuts one or more nucleotides in the extended chain. The sample is then purified using a standard purification procedure. After treatment and purification, the library contains a single-stranded template sequence with a linker sequence at both ends, and the extended chain is cut so that the two ends of the library with the linker sequence are no longer continuous. The library prepared by this method has no extension or PCR-induced errors because only the template chain has two linker sequences necessary for bridge amplification and sequencing.

Claims

1. A method for generating a double-stranded adapter comprising a barcode sequence, comprising, in a single reaction vessel or reaction mixture: (a) combining a first single-stranded oligonucleotide pool with a second single-stranded oligonucleotide pool, wherein each oligonucleotide in the first single-stranded oligonucleotide pool comprises a cleavable 5' end, wherein each oligonucleotide in the second single-stranded oligonucleotide pool comprises: (i) a sequence complementary to a portion of an oligonucleotide in the first single-stranded oligonucleotide pool; (ii) a barcode sequence located upstream of the sequence complementary to the portion of the oligonucleotides in the first single-stranded oligonucleotide pool; and (iii) a cleavable 5' end located upstream of the barcode sequence; (b) hybridizing the first single-stranded oligonucleotide pool with complementary portions of single-stranded oligonucleotides in a second single-stranded oligonucleotide pool to form a duplex having a 5' overhang, wherein the 5' overhang of the oligonucleotide from the second single-stranded oligonucleotide pool comprises a cleavable 5' upstream and a barcode sequence; (c) extending the hybridized oligonucleotide of the first single-stranded oligonucleotide library to the 5' end of the overhang of the oligonucleotide from the second single-stranded oligonucleotide library to form a double-stranded sequence, the double-stranded sequence comprising the barcode sequence and the cleavable 5' end of the oligonucleotide from the second single-stranded oligonucleotide library; (d) One or both of the double-stranded sequences produced in step (c) may be cleaved at the 5' end to produce a double-stranded adapter comprising the barcode sequence and 3' overhangs at one or both ends.

2. The method according to claim 1, further comprising: (e) generating an adapter-ligated sequencing library by ligating the double-stranded adapter of step (d) to a double-stranded DNA molecule comprising one or more overhangs. The method according to claim 1 , wherein the second single-stranded oligonucleotide pool comprises more than twelve different barcode sequences. The method according to claim 2 , wherein the second single-stranded oligonucleotide pool comprises more than twelve different barcode sequences.

5. The method of any one of claims 1-4, wherein the cleavable 5' end is selected from the group consisting of: rA, rC, rG, rU or dU. The method of claim 5 , wherein the barcode sequence is random. The method of claim 5 , wherein the barcode sequence is non-random.

8. The method of claim 5, wherein the barcode sequence is semi-degenerate.

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