High-throughput sequencing method for distinguishing and specifically detecting single-stranded DNA and double-stranded DNA in sample, product and application

Abstract

The invention discloses a high-throughput sequencing method for distinguishing and specifically detecting single-stranded DNA and double-stranded DNA in a sample, a product and application, and specific separation of the single-stranded DNA and the double-stranded DNA in the sample is realized through specific binding of streptavidin agarose gel and a biotin labeled linker-single-stranded DNA connection product. According to the present invention, the single-stranded DNA and the double-stranded DNA in the sample are distinguished and specifically detected, and the information of the single-stranded DNA and the double-stranded DNA in the sample can be simultaneously obtained.

Description

Technical Field

[0001] The present invention belongs to the field of DNA separation and sequencing, and specifically relates to a high-throughput sequencing method, product and application for distinguishing and specifically detecting single-stranded and double-stranded DNA in a sample. Background Art

[0002] Entering the 21st century, with the rise of large-scale genomic research, traditional sequencing methods can no longer meet the needs. Against this background, NGS technology came into being. NGS, also known as high-throughput sequencing, is a DNA sequencing technology developed based on PCR and gene chips, with the characteristics of short read length and high throughput. The process of NGS technology can generally be divided into four main steps: DNA library preparation, DNA cluster generation, DNA sequencing, and data analysis. Among them, library preparation is the process of extracting DNA from biological samples (such as blood, tissue, etc.), introducing sequencing adapters at both ends of the target DNA after a series of processing steps, and converting it into a library that can be sequenced by a high-throughput sequencer. The library contains a large number of DNA fragments, each of which carries a specific sequence tag so that it can be identified and distinguished during the sequencing process.

[0003] Library preparation generally includes steps such as nucleic acid extraction, nucleic acid fragmentation, end-repair, adapter ligation, amplification, purification, and quality control. Adapter ligation typically involves attaching specific adapters to the ends of DNA fragments after end-repair. These adapters contain base sequences that are used for fixed sequences, primer recognition, and library differentiation during sequencing. Amplification involves amplifying DNA fragments through PCR to increase library yield. After amplification, the target DNA fragments are extracted and purified using methods such as magnetic beads. After quality control, sequencing can be performed. Library preparation determines the overall sequencing process and results, and is the root cause of the differences between various sequencing methods.

[0004] In existing technologies, single-stranded DNA is generated in various DNA-related biological processes, and recent studies have also discovered the presence of free single-stranded DNA in human plasma. To explore the biological significance of single-stranded DNA and further explore its application value in clinical testing, DNA library construction requires not only high-throughput sequencing of double-stranded DNA but also specific analysis of single-stranded DNA sequences to obtain more comprehensive DNA information. Summary of the Invention

[0005] Currently, all NGS DNA library construction methods fall into two main categories. The first category of methods cannot construct libraries specifically for sequencing single-stranded DNA; the second category of methods only construct libraries specifically for single-stranded DNA and cannot obtain double-stranded DNA information. These methods are unable to simultaneously prepare single-stranded DNA-specific and double-stranded DNA-specific libraries from a sample, and therefore cannot obtain comprehensive DNA information.

[0006] Therefore, the present invention aims to develop a high-throughput sequencing method capable of distinguishing and specifically detecting true single-stranded and double-stranded DNA in a sample, thereby obtaining more comprehensive, accurate, and sensitive information on both double-stranded and single-stranded DNA in the sample. To address the shortcomings of the existing technology, the present invention provides the following technical solutions.

[0007] The present invention provides a method for constructing a high-throughput sequencing library for single-stranded DNA molecules and double-stranded DNA molecules in the same sample, comprising the following steps:

[0008] 1) Perform dsDNA end filling on the sample;

[0009] 2) Dephosphorylation of excess dNTPs and removal of RNA;

[0010] 3) Phenol-chloroform extraction and purification;

[0011] 4) DNA 3' end tailed with poly (dA);

[0012] 5) DNA 5' end phosphorylation;

[0013] 6) Column purification;

[0014] 7) Biotin-labeled linker specifically connects to single-stranded DNA;

[0015] 8) Streptavidin agarose binds to the ligation product of the single-stranded DNA and the adapter, separating the single-stranded DNA from the double-stranded DNA;

[0016] 9) The separated single-stranded DNA is amplified by PCR and sorted by magnetic beads to construct a single-stranded DNA library; the separated double-stranded DNA is purified by double-stranded DNA column, and then the double-stranded DNA ends are repaired, specifically by adding dA, and then ligating the double-stranded DNA with TA linkers, and then purified by magnetic beads. After purification, PCR amplification is performed, and then magnetic bead sorting is performed to construct a double-stranded DNA library. The single-stranded DNA library and the double-stranded DNA library are high-throughput sequencing libraries of single-stranded DNA molecules and double-stranded DNA molecules in the same sample.

[0017] Furthermore, the samples were subjected to PCI extraction method to extract DNA.

[0018] Furthermore, the sample includes cells, plasma, serum, and blood.

[0019] Furthermore, the sample is derived from a human or a non-human mammal.

[0020] Furthermore, the dsDNA end-finishing specifically includes the following steps:

[0021] The dsDNA ends were filled using the following system: DNA sample, 10× rCutsmart buffer, Klenow fragment (3′→5′exo-), 10 mM dNTP, ddH2O, and a reaction program with a heated lid at 105°C, 37°C, 30 min, and storage at 4°C.

[0022] Furthermore, the ratio of the DNA sample: 10×rCutsmart buffer: Klenow fragment (3′→5′exo-): 10 mM dNTP is 20-50 ng: 5 μL: 3 μL: 0.5 μL, and then the total volume of the system is supplemented with ddH2O to 10 times the added volume of 10×rCutsmart buffer.

[0023] Furthermore, the excess dNTPs were dephosphorylated using shrimp alkaline phosphatase SAP, and the RNA was removed using RNase A.

[0024] Furthermore, the concentration of the shrimp alkaline phosphatase is 1 U / μL, and the concentration of the RNaseA is 1 ng / μL.

[0025] Furthermore, the phenol-chloroform extraction and purification uses a PCI purification method to purify DNA.

[0026] Furthermore, the PCI purification method refers to a method of extracting DNA using phenol, chloroform, and isoamyl alcohol, and the PCI purification method is followed by mixing and precipitation using ddH2O.

[0027] Furthermore, in the dsDNA end-filling system, the volume ratio of shrimp alkaline phosphatase SAP:RNaseA:ddH2O used after PCI purification was 50:2:1:40.

[0028] Furthermore, the DNA 3' end tailing with poly (dA) comprises the following steps:

[0029] The DNA 3' end was tailed with poly(dA) through the following system: DNA product purified by phenol-chloroform extraction, 10× Terminal Transferase Reaction Buffer, Terminal Transferase, 1mM dATP, and a reaction procedure with a heated lid at 105°C, 37°C, 30min, and storage at 4°C.

[0030] Furthermore, the DNA product purified by phenol-chloroform extraction was mixed with ddH2O.

[0031] Furthermore, the volume ratio of the DNA product purified by phenol-chloroform extraction, 10× Terminal Transferase Reaction Buffer, Terminal Transferase, and 250 μM dATP is 40:5:4:1.

[0032] Furthermore, the DNA 5' end phosphorylation comprises the following steps:

[0033] The DNA 5' end is phosphorylated through the system: DNA 3' end tailed with poly (dA) product, 10× Terminal Transferase Reaction Buffer, 10mM ATP, 50mM DTT, T4 PNK enzyme, ddH2O, and a reaction program with a hot cover at 105°C, 37°C, 30min, and storage at 4°C.

[0034] Furthermore, the volume ratio of the DNA 3' end tailed with poly (dA), 10× Terminal Transferase Reaction Buffer, 10 mM ATP, 50 mM DTT, T4 PNK enzyme, and ddH2O is 50:3:8:8:1:10.

[0035] Furthermore, the biotin-labeled adapter specifically ligates the single-stranded DNA by specifically ligating the single-stranded DNA in the DNA product after column purification in step 6, specifically by ligating the single-stranded DNA with SEQ ID NO: 1 using T4 DNA ligase.

[0036] Furthermore, the SEQ ID NO: 1 is a 5' stem-loop linker labeled with biotin.

[0037] Furthermore, the SEQ ID NO: 1 needs to be annealed before being connected to the single-stranded DNA, specifically:

[0038] The annealed SEQ ID NO: 1 was obtained by using the following system: 100 μM SEQ ID NO: 1, 10× annealing buffer, ddH2O, and a PCR program with a heated lid at 105°C, 95°C for 5 minutes, then decreasing the temperature to 85°C at a rate of 2°C per second, and finally decreasing the temperature to 25°C at a rate of 0.1°C per second.

[0039] Furthermore, the step of ligating the single-stranded DNA with SEQ ID NO: 1 by T4 DNA ligase comprises:

[0040] The single-stranded DNA was ligated to SEQ ID NO: 1 using the following system: DNA product purified by column in step 6, 10×T4 DNA ligase buffer, 400 U / μL T4 DNA ligase, 50% PEG 8000, 50 μM SEQ ID NO: 1, and a reaction procedure of 105°C, 16°C for 2h, 75°C for 20min, and storage at 4°C with a heated lid.

[0041] Furthermore, the volume ratio of the column-purified DNA product in step 6, 10×T4 DNA ligase buffer, 400 U / μL T4 DNA ligase, 50% PEG 8000, and 50 μM SEQ ID NO: 1 is 36:8:3:32:1.

[0042] Furthermore, the step of separating the single-stranded DNA from the double-stranded DNA comprises:

[0043] Add streptavidin agarose gel to the product from step 7, mix well, incubate at 4°C for 2 hours or more, centrifuge, and separate the supernatant and precipitate. The supernatant is used for double-stranded DNA library construction, and the precipitate is used for single-stranded DNA library construction.

[0044] Furthermore, the streptavidin agarose gel needs to be used after removing the preservation solution.

[0045] Furthermore, the specific steps of amplifying the single-stranded DNA by PCR in step 9 are:

[0046] Resuspend the precipitate obtained in step 8 with ddH2O and add it to the following system: the precipitate obtained in step 8 resuspended in ddH2O, 2×Phanta UniFi Master Mix, 5 μM primer F, and 5 μM primer R, wherein primer F is SEQ ID NO: 2 and primer R is any one of SEQ ID NOs: 3-6.

[0047] Furthermore, the volume ratio of the precipitate obtained in step 8 resuspended in ddH2O, 2×Phanta UniFi MasterMix, 5 μM primer F, and 5 μM primer R is 48:50:1:1.

[0048] The present invention provides a high-throughput sequencing library prepared by the above-described method, wherein the high-throughput sequencing library comprises a single-stranded DNA library and a double-stranded DNA library of the same sample.

[0049] The present invention provides an application based on the high-throughput sequencing library described above, and sequencing is performed on the high-throughput sequencing library. The high-throughput sequencing library is suitable for Illumina sequencing platform, Roche sequencing platform, TermoFisher sequencing platform, BGI sequencing platform and MGI sequencing platform.

[0050] The present invention provides a sequence product used in the above method, wherein the sequence product comprises the nucleotide sequence described in SEQ ID NO: 1-6.

[0051] Furthermore, the sequence product also includes other reagents required for PCR, DNA end repair, and single-stranded DNA specific adapter ligation.

[0052] Advantages and beneficial effects of the present invention:

[0053] The method of constructing a high-throughput sequencing library using single-stranded DNA molecules and double-stranded DNA molecules of the present invention can simultaneously distinguish and specifically detect single-stranded and double-stranded DNA in a sample, significantly improving high-throughput sequencing. The present invention specifically connects a 5' stem-loop linker to single-stranded DNA, and after the biotin labeled on the linker binds to streptavidin agarose gel, the linker-single-stranded DNA ligation product is separated from the remaining double-stranded DNA in the system. The present invention names this new method of constructing a high-throughput sequencing library using single-stranded DNA molecules and double-stranded DNA molecules the nSDS-seq method. The nSDS-seq method separates single-stranded DNA and double-stranded DNA that are naturally present in the sample without any high-temperature treatment. The present invention achieves specific separation of single-stranded DNA and double-stranded DNA in a sample through the specific binding of streptavidin agarose gel and the biotin-labeled linker-single-stranded DNA ligation product. The present invention distinguishes and specifically detects single-stranded and double-stranded DNA in a sample, and can simultaneously obtain information on single-stranded DNA and double-stranded DNA in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The double-stranded DNA with 5' protruding ends is filled into blunt ends by KF enzyme.

[0055] Figure 2 It is a single-stranded DNA specific ligation achieved through a 5' protruding stem-loop linker.

[0056] Figure 3 This is a schematic diagram of the principle of separating single-stranded DNA and double-stranded DNA using biotin-streptavidin agarose gel.

[0057] Figure 4 This is the nSDS-seq flow chart.

[0058] Figure 5 It is a 5' protruding stem-loop linker that specifically connects to single-stranded DNA.

[0059] Figure 6 Single-stranded DNA library and double-stranded DNA library established by nSDS-seq.

[0060] Figure 7 The supernatant obtained from agarose gel separation can hardly amplify single-stranded DNA.

[0061] Figure 8 Single-stranded DNA libraries were constructed using cfDNA from SK-BR-3 cell culture media (n=3) using nSDS-seq. For PCR amplification, primer F was UMI primer-F, and primer R was T-IAP-R1, T-IAP-R2, and T-IAP-R3, respectively. PCR cycles were 22 for all assays. RFU (Relative fluorescent unit) is relative fluorescence signal.

[0062] Figure 9 Double-stranded DNA libraries were constructed using cfDNA from SK-BR-3 cell culture media (n=3) using nSDS-seq. For PCR amplification, primer F was Index Primer P501, and primer R was Index Primer P701, Index Primer P702, Index Primer P703, and Index Primer P704, respectively. Index Primers P501, P701, P702, P703, and P704 were from Hieff 384CDIPrimer for RFU (Relative fluorescent unit) is the relative fluorescence signal value.

[0063] Figure 10 A single-stranded DNA library was established using cfDNA from the culture medium of MDA-MB-468 cells using nSDS-seq.

[0064] Figure 11 A double-stranded DNA library was established using cfDNA from the culture medium of MDA-MB-468 cells using nSDS-seq.

[0065] Figure 12 Single-stranded DNA libraries were established using mouse plasma and human umbilical cord blood serum cfDNA by nSDS-seq.

[0066] Figure 13Double-stranded DNA libraries were established using mouse plasma and human umbilical cord blood serum cfDNA by nSDS-seq. DETAILED DESCRIPTION

[0067] The present invention will be further described below with reference to specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. The main features of the present invention may be applied to various embodiments without departing from the scope of the present invention.

[0068] The method provided in the present invention for constructing a high-throughput sequencing library for single-stranded DNA molecules and double-stranded DNA molecules in the same sample is based on the following experimental principles:

[0069] like Figure 1 As shown, there are three 5'-end configurations for double-stranded DNA: 3'-end overhang, blunt end, and 5'-end overhang. Due to the directionality of DNA synthesis, double-stranded DNA with 5'-end overhangs can be trimmed to blunt ends. After trimming, the 5'-end configurations of the double-stranded DNA in the system are reduced to two, facilitating subsequent specific ligation. Conventional DNA polymerases possess exonuclease activity. To ensure the integrity of the single-stranded DNA in the system during the trimming process, KF enzyme (Klenow fragment 3'→5' exo-, KF exo-) is selected. KF exo- is an N-terminally truncated fragment of Escherichia coli DNA polymerase I that lacks either 5'→3' or 3'→5' exonuclease activity. After trimming with KF enzyme, the double-stranded DNA in the sample contains no 5'-end overhangs. Stem-loop linkers with 5'-end overhangs can create steric hindrance with blunt-end and 3'-end overhang double-stranded DNA, preventing the linker from ligating to the double-stranded DNA. The 5' protruding stem-loop adapter complements the 5' end of the single-stranded DNA and specifically ligates the single-stranded DNA present in the sample.

[0070] like Figure 2As shown, the 5' stem-loop adapter used in this method consists of a 5' terminal sequence, six random bases (N6), a UMI (Unique Molecular Identifier), and a 3' terminal sequence. After annealing, the adapter forms a stem-loop structure (containing an 11-bp complementary pairing region (stem) and a 38-nt non-complementary pairing region (loop)). The 5' end of the stem-loop adapter is an 18-nt overhang containing six random deoxynucleotides (NNNNNN), which allow for unbiased complementary pairing with the 5' end of single-stranded DNA while preventing ligation between the adapter and double-stranded DNA. To prevent the stem-loop adapter from ligating itself, both the 5' and 3' ends of the adapter are hydroxyl groups. Furthermore, the loop region of the 5' end adapter contains 10 random deoxynucleotides (N10) that serve as a molecular tag (UMI) to accurately distinguish DNA templates from different sources. The 5' end of the adapter is modified with biotin to distinguish single-stranded and double-stranded DNA.

[0071] like Figure 3 As shown, streptavidin agarose gel is covalently coupled to streptavidin and highly cross-linked 6% agarose. It allows for rapid, efficient, sensitive, and specific binding to biotin-labeled antibodies, nucleic acids, proteins, peptides, lectins, and other molecules. Single-stranded DNA is specifically ligated to a biotin-labeled 5' stem-loop linker, and the ligation product binds to the streptavidin agarose gel via the biotin. Double-stranded DNA, after end-padding, cannot ligate to the 5' stem-loop linker and lacks the biotin label, thus not binding to the streptavidin agarose gel. Therefore, after incubation and centrifugation of the suspension of the ligation reaction system and agarose gel, double-stranded DNA remains in the supernatant, while the single-stranded DNA-linker ligation product is bound to the gel precipitate. The separated single-stranded DNA product is then used in subsequent steps to generate a single-stranded-specific library, and the separated double-stranded DNA is then used to generate a double-stranded DNA-specific library.

[0072] Example nSDS-seq complete technical solution

[0073] nSDS-seq (Native single-stranded and double-stranded DNA sequencing) is a high-throughput sequencing method that can simultaneously distinguish and specifically detect single-stranded and double-stranded DNA in a sample. After DNA ends are filled, single-stranded DNA is specifically ligated using a 5' stem-loop adapter with biotin. The ligation product of single-stranded DNA and adapter specifically binds to streptavidin agarose gel, thereby distinguishing it from double-stranded DNA. Specific components of single-stranded DNA and double-stranded DNA are then generated and libraries are created for high-throughput sequencing. The specific flow chart is shown below. Figure 4 shown.

[0074] 1. Experimental Materials

[0075] 1.1 Synthetic oligonucleotides

[0076] Table 1. Synthetic oligonucleotides

[0077]

[0078]

[0079] 1.2 Cell culture

[0080] MDA-MB-468 cell line (purchased from the National Cell Experiment Resource Sharing Platform); SK-BR-3 cell line (purchased from the National Cell Experiment Resource Sharing Platform); culture medium: DMEM high glucose, containing GlutaPlus, sodium pyruvate, and HEPES (Sevier, G4515-500ML); fetal bovine serum: Fetal Bovine Serum (HyCloneTM, SH30396.03).

[0081] 1.3 Enzymes, buffers, and other reagents

[0082] Klenow fragment (3′→5′exo-): Klenow fragment (3′→5′exo-) (NEB, M0212L);

[0083] rCutSmart TM Buffer: rCutSmart TM Buffer (NEB, B6004V);

[0084] dNTP Mix (10 mM each) (Novozymes, P031-01);

[0085] Shrimp Alkaline Phosphatase (rSAP) (NEB, M0371L);

[0086] RNase: 20mg / ml RNase A (NEB, T3018L);

[0087] Terminal transferase: Terminal Transferase (NEB, M0315L);

[0088] Terminal transferase reaction buffer: 10× Terminal Transferase Reaction Buffer (NEB, B0315S);

[0089] dATP solution: 100 mM dATP Solution (NEB, N0440S);

[0090] Adenosine 5'-Triphosphate (ATP) (NEB, P0756S);

[0091] DTT: 0.5 M DTT (DNase, RNase & Protease free) (Biyuntian, ST041-2 ml);

[0092] T4 polynucleotide kinase (T4 PNK): T4 Polynucleotide Kinase (NEB, M0201L);

[0093] Annealing buffer: 10× annealing buffer, prepared with ultrapure water containing 100 mM Tris-HCl (pH 8.0) and 500 mM NaCl and stored at 4°C.

[0094] T4 DNA ligase: T4 DNA ligase (NEB, M0202L);

[0095] T4 DNA Ligase Reaction Buffer: T4 DNA Ligase Reaction Buffer (NEB, B0202S);

[0096] 50% PEG 8000: PEG 8000 (MCE, HY-Y0873J) powder was prepared with ultrapure water to 50% PEG 8000 and then aliquoted and frozen at -20°C.

[0097] 0.5×TBE buffer: TBE powder is prepared into 0.5×TBE buffer with ultrapure water;

[0098] 10% Acr-Bis (29:1) PAGE gel containing 7 M urea: Prepare a 200 mL solution by weighing 20 g Acr acrylamide, 0.69 g Bis bisacrylamide, and 84 g urea. Dissolve them in 0.5× TBE buffer (heat in a water bath) and dilute to 200 mL for later use. Add appropriate amounts of 10% APS (ammonium persulfate) and TEMED (tetramethylethylenediamine) to prepare the gel.

[0099] DNA loading buffer: 10× DNA Loading Buffer (Novozymes, P022-01);

[0100] Blocking solution: Blocking solution (special for D3308) (Biyuntian, D3308B);

[0101] Detergent: Detergent (5×, special for D3308) (Biyuntian, D3308W);

[0102] HRP-labeled Streptavidin: horseradish peroxidase-labeled streptavidin (Biyuntian, A0303).

[0103] 1× TBS buffer: Tris Buffered Saline (TBS, Powder) (Seville, G0001-2L) powder was prepared into 1× TBS with ultrapure water;

[0104] Binding & Washing Buffer I (2×): Prepare a solution containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl, and 0.01%-0.1% Tween-20 in ultrapure water. Dilute it twofold with ultrapure water to make Binding & Washing Buffer I (1×).

[0105] 2×PhantaUniFi Master Mix universal ultra-high-fidelity enzyme premix (Novozymes, P516-01).

[0106] 1.4 Nucleic acid purification and sorting

[0107] Phenol / chloroform / isoamyl alcohol = 25:24:1 (pH>7.8) (Baishengyue, BR1003926);

[0108] GeneJET Purification Kit (Thermo Scientific, K0702);

[0109] Streptavidin Agarose (Streptavidin Agarose Gel) (Biyuntian, P2159-5ml);

[0110] VAHTSDNAClean Beads (Novozymes, N411-03).

[0111] 1.5 Double-stranded DNA library construction kit

[0112] Universal DNA library construction kit (YEASEN, 12201);

[0113] Hieff 384CDIPrimer for (YEASEN, 12412).

[0114] 1.6 Library quality control

[0115] Qubit 1×dsDNA HS Quantification Kit (Invitrogen TM , Q33230).

[0116] N1 Cartridge (High Sensitivity Cartridge) (Bioptic, C405105).

[0117] 2. Experimental methods

[0118] 2.1 DNA extraction

[0119] Cell culture medium / plasma / serum were digested with proteinase K, and DNA was extracted by PCI method. The concentration was determined using Qubit 1× dsDNA HS quantification kit.

[0120] 2.2 DNA end filling

[0121] Thaw all reagents by inversion and mix thoroughly, then place on ice until ready to use. Prepare the reaction system as shown in Table 2 in a sterile PCR tube.

[0122] Table 2

[0123] name Volume (μL) Dosage DNA samples 20-50ng 10xrCutsmartbuffer 5 Klenowfragment(3′→5′exo-) 3 15U 10 mM dNTPs 0.5 5nmol <![CDATA[ddH2O]]> Upto50

[0124] Use a pipette to gently pipette or vortex to mix, and briefly centrifuge the reaction mixture to the bottom of the tube. Place the PCR tube in a thermal cycler and set the reaction program as shown in Table 3.

[0125] Table 3

[0126] temperature time Heated cover 105℃ On 37℃ 30min 4℃ Hold

[0127] 2.3 Removal of dNTPs and RNA

[0128] Add 2 μL of rSAP (1 U / μL) and 1 μL of RNase A (1 ng / μL) to the PCR tube from step 2.2. Gently pipette or vortex to mix, and briefly centrifuge the reaction mixture to the bottom of the tube. Place the PCR tube in a thermal cycler and set the reaction program as shown in Table 4.

[0129] Table 4

[0130] temperature time Heated cover 105℃ On 37℃ 60min 4℃ Hold

[0131] 2.4PCI (phenol-chloroform-isoamyl alcohol) extraction method to purify DNA

[0132] DNA was purified by PCI. After the DNA precipitate was dried, 40 μL of ddH2O was added to the precipitate at the bottom of the centrifuge tube, and the mixture was allowed to stand at room temperature for 15 min and then vortexed to mix.

[0133] 2.5 Adding poly(dA) tail

[0134] Thaw all reagents by inversion and mix thoroughly, then place on ice until ready to use. Prepare the reaction system as shown in Table 5 in a sterile PCR tube.

[0135] Table 5

[0136] name Volume (μL) Dosage Step 2.4 Product 40 10×TerminalTransferaseReactionBuffer 5 Terminal Transferase 4 80U 1mM dATP (1:500) 1 250 pmol

[0137] Use a pipette to gently pipette or vortex to mix, and briefly centrifuge the reaction mixture to the bottom of the tube. Place the PCR tube in a thermal cycler and set the reaction program as shown in Table 6.

[0138] Table 6

[0139] temperature time Heated cover 105℃ On 37℃ 15min 4℃ Hold

[0140] 2.6 DNA 5' end phosphorylation

[0141] Thaw all reagents by inversion and mix thoroughly. Place on ice until ready to use. Prepare the reaction mixture in the PCR tube from step 2.5 as shown in Table 7.

[0142] Table 7

[0143] name Volume (μL) Step 2.5 Product 50 10×TerminalTransferaseReactionBuffer 3 10mM ATP 8 50mM DTT 8 T4PNK enzyme (T4PolynucleotideKinase) 1 <![CDATA[ddH2O]]> 10

[0144] Use a pipette to gently pipette or vortex to mix, and briefly centrifuge to collect the reaction solution at the bottom of the tube. Place the PCR tube in a thermal cycler and set the reaction program as shown in Table 8.

[0145] Table 8

[0146]

[0147]

[0148] 2.7 Column purification

[0149] The reaction products were purified and recovered using the GeneJET Purification Kit to remove the enzymes and buffers in the reaction system.

[0150] 2.8 Single-stranded DNA-specific adapter ligation

[0151] Use T4 DNA ligase to ligate the single-stranded DNA to the biotinylated 5' stem-loop linker (UMI-LM-5'biotin). First, renature the UMI-LM-5'biotin and prepare the reaction mixture in a sterile PCR tube as shown in Table 9.

[0152] Table 9

[0153] name Volume (μL) UMI-LM-5'biotin (100 μM) 10 10xannealingbuffer 2 <![CDATA[ddH2O]]> 8

[0154] Use a pipette to gently pipette or vortex to mix, and briefly centrifuge the reaction mixture to the bottom of the tube. Place the PCR tube in a thermal cycler and set the reaction program as shown in Table 10.

[0155] Table 10

[0156] temperature Programmed cooling rate time Heated cover 105℃ On 95℃ 5min 85℃ -2℃ / s 25℃ -0.1℃ / s 4℃ Hold

[0157] After adapter annealing, use T4 DNA ligase to specifically ligate the single-stranded DNA to the adapter. Thaw all reagents, mix thoroughly by inversion, and place on ice until ready to use. Prepare the reaction mixture in a sterile PCR tube as shown in Table 11.

[0158] Table 11

[0159] name Volume (μL) Dosage Step 2.7 Product 36 10×T4 DNA ligase buffer 8 T4 DNA Ligase (400 U / μL) 3 1200U 50% PEG8000 32 UMI-LM-5'biotin (50 μM) 1 50 pmol

[0160] Use a pipette to gently pipette or vortex to mix, and briefly centrifuge the reaction mixture to the bottom of the tube. Place the PCR tube in a thermal cycler and set the reaction program as shown in Table 12.

[0161] Table 12

[0162] temperature time Heated cover 105℃ On 16℃ 2h 75℃ 20min 4℃ Hold

[0163] 2.9 Separation of single-stranded DNA and double-stranded DNA by streptavidin agarose gel

[0164] A. Take the agarose gel and remove the supernatant

[0165] Gently resuspend the streptavidin agarose gel with a pipette to create a uniform gel suspension. Place 40 μL of each aliquot into a 1.5 mL centrifuge tube for later use. Note: Using a wide-aperture pipette tip will facilitate aspirating the gel suspension. Do not vortex. Centrifuge at 600 g for 5 minutes at 4°C. Carefully remove the supernatant, avoiding the gel.

[0166] B. Washing agarose gel

[0167] Add 1× TBS buffer to a final volume of approximately 1 mL and gently resuspend the streptavidin agarose gel. Centrifuge at 600g for 5 minutes at 4°C. Carefully remove the supernatant, avoiding aspirating the gel. This completes one wash step. Repeat the washing steps twice as described above. Finally, remove the supernatant and resuspend each portion of the streptavidin agarose gel in 80 μL Binding & Washing Buffer I (2×).

[0168] C. Agarose gel binding to biotin-labeled nucleic acids

[0169] Add 80 μL of the ligation reaction product, mix thoroughly, and incubate slowly at 4°C for 2 hours on a rotating mixer. Centrifuge at 600 g for 5 minutes at 4°C. Double-stranded DNA will remain in the supernatant, which should be aspirated into a new 1.5 mL centrifuge tube. This supernatant will be used for double-stranded DNA library construction in step 2.11. The single-stranded DNA will bind to the agarose beads, which will be used for single-stranded DNA library construction in step 2.10.

[0170] 2.10 Single-stranded DNA library construction

[0171] Before library construction, the inventors experimentally compared the effects of different agarose gel washing and elution conditions on the concentration of the single-stranded DNA library. 100 ng of cfDNA from the culture medium of MDA-MB-468 cells was used for nSDS-seq library construction. The ligation system was incubated with streptavidin agarose gel at 4°C for 2 hours and then centrifuged to obtain the supernatant and agarose gel precipitate, and subsequent condition optimization experiments were performed.

[0172] First, the inventors directly eluted the agarose gel precipitate with ddH2O according to conventional knowledge.

[0173] Results Table 13 The concentration of the single-stranded library amplified by PCR using the eluate was low.

[0174] Table 13

[0175]

[0176] The inventors used both the eluate and agarose gel precipitation for amplification. The eluate-based amplification yielded a very low library concentration (samples 1 and 3), while the agarose gel after elution yielded a higher library concentration (samples 2 and 4). Samples 3 and 4 showed that, under the same conditions, two elutions had little effect. The low library concentrations in samples 1 and 3 may be due to residual 1× B&W buffer, which affected amplification, the inventors speculate.

[0177] In order to explore better conditions, the inventors planned to pretreat the agarose gel precipitate with ddH2O before testing.

[0178] Results Table 14 Removal of residual buffer before elution is the key to obtaining high-quality libraries.

[0179] Table 14

[0180]

[0181]

[0182] The results for samples 5-7 show that after treatment with ddH2O before elution, high-concentration libraries were obtained in all eluates, precipitates, and suspensions. This indicates that removing residual buffer before elution is key to obtaining high-quality libraries.

[0183] The high concentration of sample 6 also indicates that a significant amount of unreleased single-stranded DNA-adapter products remain in the precipitate, suggesting that release of these products is difficult. This is because the conventional release conditions are 95°C for 5 minutes, which the inventors have extended to 10 minutes. The concentration of sample 7 is similar to that of samples 5 and 6, indicating that suspension amplification does not significantly affect library yield.

[0184] Because amplification using a suspension eliminates the risk of single-stranded DNA sample loss, the inventors ultimately opted to use the suspension directly in the PCR amplification system. Other methods are also possible, but the most important consideration is removing any residual buffer from the agarose gel pellet before elution.

[0185] Finally, the inventors further optimized the suspension amplification method.

[0186] Results Table 15 Optimization of suspension amplification conditions.

[0187] Table 15

[0188]

[0189] Because removing the residual buffer in the agarose gel precipitation before elution is key, the inventors find that eluting with ddH2O elution once is enough before elution, and eluting twice will not increase output (samples 8, 9). In order to control the volume of the reaction system during PCR amplification, the inventors attempted to reduce the ddH2O volume used when resuspending the agarose gel to reduce the volume of the suspension. The results show that the single-stranded DNA library concentration is the highest when using 40 μL of ddH2O (samples 10, 11). Finally, the inventors have also tested the release temperature. The single-stranded DNA library concentration obtained by treating the agarose gel suspension at room temperature does not differ compared with high temperature elution (samples 12, 13), which shows that the mode of suspension amplification does not even need high temperature treatment to release DNA.

[0190] Based on the above experimental results, the inventors ultimately determined the nSDS-seq process for agarose gel washing and single-stranded DNA library PCR amplification: washing twice with 1× B&W buffer, washing the pellet once with 100 μL ddH₂O (to remove residual buffer), resuspending the agarose gel in 40 μL ddH₂O, and then using the suspension directly in the PCR amplification system. This process ensures high yield and simplifies the operation.

[0191] Therefore, the specific steps for constructing a single-stranded DNA library are as follows.

[0192] A. Agarose gel washing

[0193] Add 1 mL of Binding & Washing Buffer I (1×) to the agarose gel and gently resuspend the agarose gel. Centrifuge at 600g for 5 minutes at 4°C. Remove the supernatant, avoiding the gel. Repeat the wash cycle once more. Wash once more with 500 μL of ddH2O and resuspend the agarose gel in 30 μL of ddH2O.

[0194] B. Single-stranded DNA amplification

[0195] Perform PCR amplification on the sorted single-stranded DNA ligated with the biotin-labeled adapter. Thaw the reagents by inversion and mix thoroughly. Place on ice until ready to use. Prepare the reaction system as shown in Table 16 in a sterile PCR tube.

[0196] Table 16

[0197] name Volume (μL) Step 2.10.A Product 48 2 × Phanta UniFi Master Mix 50 primerF (5 μM) 1 primerR (5 μM) 1

[0198] Note: Primer F is UMI primer-F; primer R can be T-IAP-R1 / T-IAP-R2 / T-IAP-R3 / T-IAP-R4.

[0199] Gently pipette or vortex to mix, and briefly centrifuge to collect the reaction solution at the bottom of the tube. Place the PCR tube in a thermal cycler and set the reaction program as shown in Table 17 to perform PCR amplification.

[0200] Table 17

[0201] temperature time Step 1 95℃ 5min Step 2 95℃ 15sec Step 3 58℃ 15sec Step 4 72℃ 45sec (gotostep2, 22 cycles) Step 5 72℃ 5min 4℃ Hold

[0202] C. Magnetic bead separation of amplified products

[0203] Remove the VAHTS DNA Clean Beads from the refrigerator and equilibrate at room temperature for at least 30 minutes. Vortex or invert the beads thoroughly to ensure thorough mixing. Add 70 μL of the first-round sorting beads to the product from step 2.10.B. Vortex or pipette 10 times to mix thoroughly. Incubate at room temperature for 5 minutes. Briefly centrifuge the PCR tube and place it on a magnetic rack. Once the solution has cleared (approximately 5 minutes), carefully transfer the supernatant to a clean centrifuge tube.

[0204] Add 20 μL of the second round of sorting magnetic beads to the supernatant. Vortex to mix or pipette 10 times to mix, and let it stand at room temperature for 5 minutes. Centrifuge the PCR tube briefly and place it on a magnetic rack. After the solution is clarified (about 5 minutes), carefully remove the supernatant. Keep the PCR tube in the magnetic rack, add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant. Repeat the rinse once, for a total of two rinses. Finally, use a 10 μL pipette tip to aspirate the remaining liquid. Keep the PCR tube in the magnetic rack, open the lid and dry the magnetic beads until they just crack (about 5 minutes).

[0205] Remove the PCR tube from the magnetic rack and add 21 μL of ddH2O. Vortex or gently pipette to mix thoroughly, and let it sit at room temperature for 5 minutes. Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the magnetic beads and liquid. After the solution has cleared (approximately 5 minutes), carefully transfer 20 μL of the supernatant to a new PCR tube, being careful not to touch the magnetic beads.

[0206] 2.11 Double-stranded DNA library construction

[0207] A. Column purification

[0208] The reaction products were purified and recovered using the GeneJET Purification Kit to remove enzymes and buffers from the supernatant after agarose gel bead separation of the ligation reaction system.

[0209] B. End repair / addition of dA tail

[0210] Fill the DNA ends, phosphorylate the 5' end, and add a dA tail to the 3' end. Thaw all reagents by inversion and mix thoroughly. Place on ice until ready to use. Prepare the reaction mixture in a sterile PCR tube as shown in Table 18.

[0211] Table 18

[0212]

[0213] Gently pipette or vortex to mix, and briefly centrifuge the reaction mixture to the bottom of the tube. Place the PCR tube in a thermal cycler and set the reaction program as shown in Table 19 to perform the end repair / dA tailing reaction.

[0214] Table 19

[0215] temperature time Heated cover 105℃ On 30℃ 20min 72℃ 20min 4℃ Hold

[0216] C. Connector connection

[0217] Connect the product of step 2.11.B to HieffNGS 384CD1Primer kit PEAdapter connector. Thaw all reagents by inversion and mix thoroughly. Place on ice until ready to use. Prepare the reaction mixture in the PCR tubes in step 2.11.B as shown in Table 20.

[0218] Table 20

[0219] name Volume (μL) 2.11.B step product 60 LigationEnhancer 30 PEAdapter (10μM) 5 NovelT4DNALigase 5

[0220] Gently pipette or vortex to mix, and briefly centrifuge to collect the reaction solution at the bottom of the tube. Place the PCR tube in a thermal cycler and set the reaction program as shown in Table 21 to perform the adapter ligation reaction.

[0221] Table 21

[0222] temperature time Hot cover Off 20℃ 15min 4℃ Hold

[0223] D. Magnetic bead purification of ligation products

[0224] Remove the VAHTS DNA Clean Beads from the refrigerator and equilibrate at room temperature for at least 30 minutes. Vortex or invert the beads thoroughly to ensure thorough mixing. Pipette 60 μL of VAHTS DNA Clean Beads into the product from step 2.11.C. Vortex or pipette up and down 10 times to mix thoroughly. Incubate at room temperature for 5 minutes. Briefly centrifuge the PCR tube and place it on a magnetic rack to separate the beads and liquid. Once the solution has cleared (approximately 5 minutes), carefully remove the supernatant.

[0225] Keeping the PCR tube in the magnetic rack, rinse the beads with 200 μL of freshly prepared 80% ethanol (do not disturb the beads). Incubate at room temperature for 30 seconds, then carefully remove the supernatant. Repeat the rinse once, for a total of two rinses. Finally, use a 10 μL pipette tip to remove any remaining liquid. Keeping the PCR tube in the magnetic rack, open the lid and air-dry the beads until just cracks appear (no more than 5 minutes).

[0226] Remove the PCR tube from the magnetic rack and add 21 μL of ddH2O. Vortex or gently pipette to mix thoroughly. Let stand at room temperature for 5 minutes. Briefly centrifuge the PCR tube and place it on the magnetic rack. Once the solution has cleared (approximately 5 minutes), carefully transfer 20 μL of the supernatant to a new PCR tube, being careful not to disturb the magnetic beads.

[0227] E. Double-stranded DNA amplification

[0228] Perform PCR amplification and enrichment on the purified adapter-ligated products. Thaw the reagents by inversion and mix thoroughly. Place on ice until ready to use. Prepare the reaction mixture in a sterile PCR tube as shown in Table 22.

[0229] Table 22

[0230]

[0231] Note: Index Primer P501, P701, P702, P703, P704 are from Hieff 384CDIPrimerfor

[0232] Gently pipette or vortex to mix, and briefly centrifuge to collect the reaction solution at the bottom of the tube. Place the PCR tube in a thermal cycler and set the reaction program as shown in Table 23 to perform PCR amplification.

[0233] Table 23

[0234] temperature time Step 1 98℃ 1min Step 2 98℃ 10sec Step 3 60℃ 30sec Step 4 72℃ 30sec (gotostep2, 10 cycles) Step 5 72℃ 5min 4℃ Hold

[0235] F. Magnetic bead separation of amplified products

[0236] Bring the product from step 2.11.E to 100 μL with ddH2O. Perform two rounds of separation using VAHTS DNAClean Beads. Add 70 μL of beads in the first round and 20 μL in the second round. After drying, elute with 21 μL of ddH2O. Follow the same protocol as in step 2.10.C.

[0237] 2.12 Quality Control

[0238] A. Qubit assay concentration

[0239] The concentration was determined using the Qubit 1× dsDNA HS quantification kit.

[0240] B. Capillary electrophoresis detection of library length distribution

[0241] The library length distribution was detected using the N1 Cartridge high sensitivity cartridge.

[0242] 3. Experimental results

[0243] 3.1 Specific ligation of single-stranded DNA via 5' protruding stem-loop linkers

[0244] The specific ligation of single-stranded DNA mediated by a 5' stem-loop linker was verified by polyacrylamide gel electrophoresis and biotin detection experiments. Streptavidin, with a molecular weight of 66 kDa, binds to biotin with high specificity. HRP (horseradish peroxidase) catalyzes ECL reagents to produce chemiluminescence in Western blot, EMSA, Southern blot, or Northern blot assays. The 5' end of the stem-loop linker, which specifically ligates single-stranded DNA, is labeled with biotin, and the DNA ligated to the linker can be detected using HRP-labeled streptavidin. The specificity of the linker-single-stranded DNA ligation can be verified by measuring the size of the linker ligation product.

[0245] To simulate single-stranded DNA and double-stranded DNA with two overhanging ends, the inventors designed and synthesized double-stranded DNA with a 3' overhang (ds95, constructed from oligo 95-F and oligo 95-R, hereafter referred to as ds95), a double-stranded DNA with a 5' overhang (ds80, constructed from NNN oligo 80-F and oligo 65-R, hereafter referred to as ds80), and a single-stranded DNA (ss50, constructed from NNN-oligo 50-F, hereafter referred to as ss50) for specificity verification experiments. To maintain consistency and allow for ligation to stem-loop linkers, the overhangs of ds80 and ss50 consisted of six random deoxyribonucleic acid residues. After DNA end-finishing, ds80 was blunt-ended, while ds95 retained a 3' overhang.

[0246] ss50 was mixed with ds95 and ds80, respectively, and then ligated with a biotin-labeled 5' stem-loop linker. The linker-ss50 ligation product was smaller than the nonspecific ligation products (linker-ds80 and ds95 ligation products). The ligation reaction system was subjected to polyacrylamide gel electrophoresis, transferred to a membrane, and incubated with HRP-labeled Streptavidin. The biotin-labeled DNA specifically bound to the HRP-labeled Streptavidin, and the resulting product was visualized using chemiluminescence from an ECL reagent.

[0247] Figure 5Specific ligation of a 5'-protruding stem-loop adapter to single-stranded DNA. Lane 1 represents a control without ligase, and lane 7 represents a blank control containing only the biotinylated adapter. Lanes 2, 3, and 4, respectively, show reactions with ds80, ds95, and ss50 alone. No ligation products appear in lanes 2 and 3, while ligation products from ss50 and the adapter appear in lane 4. Lanes 5 and 6 show reactions with a 1:1 mixture of ss50, ds80, and ds95, respectively, and the adapter. In a mixed system of single- and double-stranded DNA, the 5'-stem-loop adapter ligated exclusively to ss50, with virtually no nonspecific ligation products from the adapter to ds80 or ds95. Furthermore, the amount of ligation product in lanes 4, 5, and 6 was virtually unchanged, indicating that the efficiency of adapter-ssDNA ligation was unaffected in the mixed system. These results demonstrate that 5'-stem-loop adapters can specifically and efficiently ligate to single-stranded DNA in this system.

[0248] 3.2nSDS-seq can separate single-stranded DNA and double-stranded DNA using streptavidin agarose gel

[0249] Single-stranded and double-stranded DNA libraries were generated using cfDNA from MDA-MB-468 cell culture media using nSDS-seq to verify the effectiveness of streptavidin-agarose gel separation for single-stranded and double-stranded DNA. 50 ng of cfDNA was used. PCR cycles were 22 for single-stranded DNA library construction and 10 for double-stranded DNA library construction. Capillary electrophoresis was performed on a Qsep1 instrument using the N1 high-sensitivity cartridge.

[0250] The results are as follows Figure 6 As shown, nSDS-seq can simultaneously generate single-stranded DNA and double-stranded DNA libraries from cfDNA samples cultured from MDA-MB-468 cells. The single-stranded DNA library is primarily distributed in the 200-400 bp range, while the double-stranded DNA library is primarily distributed in the 300-500 bp range. RFU (Relative fluorescent unit) represents the relative fluorescence signal.

[0251] To test whether streptavidin agarose gel can completely separate single-stranded DNA connected to the 5' stem-loop linker in cfDNA, the supernatant obtained after separation was PCR amplified using primers designed for single-stranded DNA specifically connected to the 5' stem-loop linker. The less product, the more complete the binding of single-stranded DNA to the agarose gel. In order to strictly detect the possibility of trace amounts of single-stranded DNA in the supernatant, the number of PCR amplification cycles was set to 30. Capillary electrophoresis was performed on the Qsep1 instrument using the N1 Cartridge high-sensitivity card holder, as shown in the results. Figure 7As shown, in nSDS-seq, the supernatant obtained by streptavidin-agarose gel separation yields almost no single-stranded DNA. This demonstrates that single-stranded DNA, specifically ligated to the 5' stem-loop linker, can fully bind to the streptavidin-agarose gel, ensuring both the yield of the single-stranded DNA fraction and the specificity of the double-stranded DNA fraction. RFU (Relative fluorescent unit) represents the relative fluorescence signal.

[0252] 3.3nSDS-seq can be applied to a variety of samples such as cell culture medium, mouse plasma, and human umbilical cord blood serum DNA

[0253] To confirm the applicability of nSDS-seq technology, the inventors constructed nSDS-seq libraries using cfDNA from cell culture medium, mouse plasma, and human umbilical cord blood serum. The library preparation used 50 ng of cfDNA from cell culture medium and 25 ng of cfDNA from mouse plasma and human umbilical cord blood serum. Capillary electrophoresis was performed on a Qsep1 instrument using the N1 high-sensitivity cartridge. The results demonstrated that nSDS-seq can simultaneously generate single-stranded and double-stranded DNA libraries from cfDNA samples from cell culture medium, mouse plasma, and human umbilical cord blood serum.

[0254] The length of the cell culture medium cfDNA single-stranded DNA library is mainly distributed in the range of about 200-300bp (results Figure 8 ), the length of the double-stranded DNA library is mainly distributed in the range of about 300-400bp (results Figure 9 The library length distribution was basically consistent between biological replicates, indicating the high stability of nSDS-seq.

[0255] MDA-MB-468 cell culture medium cfDNA was used to establish a single-stranded DNA library by nSDS-seq. Figure 10 Figure 2 shows cfDNA from MDA-MB-468 cell culture medium (n = 3). PCR amplification used primer F with UMI primer-F, and primer R with T-IAP-R1, T-IAP-R2, and T-IAP-R3, respectively. PCR cycles were 22 for all assays. RFU (Relative fluorescent unit) represents relative fluorescence signal.

[0256] Double-stranded DNA library was constructed by nSDS-seq using cfDNA from MDA-MB-468 cell culture medium. Figure 11As shown, cfDNA from MDA-MB-468 cell culture medium (n=3). During PCR amplification, primer F was Index Primer P501, and primer R was Index Primer P701, Index Primer P702, Index Primer P703, and Index Primer P704, respectively. Index Primers P501, P701, P702, P703, and P704 were from Hieff 384CDIPrimer for RFU (Relative fluorescent unit) is the relative fluorescence signal value.

[0257] Single-stranded DNA libraries were constructed by nSDS-seq from mouse plasma and human umbilical cord blood serum cfDNA. Figure 12 Figure 2 shows mouse plasma cfDNA and human umbilical cord blood serum cfDNA. PCR amplification used primer F with UMI primer-F and primer R with T-IAP-R1. PCR cycles were 22 for all assays. RFU (Relative fluorescent unit) represents relative fluorescence signal.

[0258] Mouse plasma and human umbilical cord blood serum cfDNA were used to construct double-stranded DNA libraries by nSDS-seq. Figure 13 As shown, mouse plasma cfDNA and human umbilical cord blood serum cfDNA. Primer F for PCR amplification was Index Primer P501, and primer R was Index Primer P701. Index Primer P501 and P701 were from Hieff 384CDIPrimer for RFU (Relative fluorescent unit) is the relative fluorescence signal value.

[0259] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing a high-throughput sequencing library for single-stranded DNA molecules and double-stranded DNA molecules in the same sample, comprising the following steps: 1) Perform dsDNA end filling on the sample; 2) Dephosphorylation of excess dNTPs and removal of RNA; 3) Phenol-chloroform extraction and purification; 4) DNA 3' end tailed with poly (dA); 5) DNA 5' end phosphorylation; 6) Column purification; 7) Biotin-labeled linker specifically connects to single-stranded DNA; 8) Streptavidin agarose binds to the ligation product of the single-stranded DNA and the adapter, separating the single-stranded DNA from the double-stranded DNA; 9) The separated single-stranded DNA is amplified by PCR and sorted by magnetic beads to construct a single-stranded DNA library; the separated double-stranded DNA is purified by double-stranded DNA column, and then the double-stranded DNA ends are repaired, specifically by adding dA, and then ligating the double-stranded DNA with TA linkers, and then purified by magnetic beads. After purification, PCR amplification is performed, and then magnetic bead sorting is performed to construct a double-stranded DNA library. The single-stranded DNA library and the double-stranded DNA library are high-throughput sequencing libraries of single-stranded DNA molecules and double-stranded DNA molecules in the same sample.

2. The method according to claim 1, wherein the sample is subjected to PCI extraction to extract DNA; preferably, the sample comprises cells, plasma, serum, or blood; Preferably, the sample is derived from a human or non-human mammal.

3. The method according to claim 1, wherein the dsDNA end-finishing step comprises the following steps: The dsDNA ends were filled using the following system: DNA sample, 10× rCutsmart buffer, Klenow fragment (3′→5′ exo-), 10 mM dNTP, ddH2O, with a heated lid at 105°C, 37°C for 30 min, and storage at 4°C. Preferably, the ratio of the DNA sample: 10×rCutsmart buffer: Klenow fragment (3′→5′exo-): 10 mM dNTP is 20-50 ng: 5 μL: 3 μL: 0.5 μL, and then the total volume of the system is supplemented with ddH2O to 10 times the volume of 10×rCutsmart buffer. Preferably, the excess dNTPs are dephosphorylated by using shrimp alkaline phosphatase SAP, and the RNA removal is performed by using RNaseA; Preferably, the concentration of the shrimp alkaline phosphatase is 1 U / μL, and the concentration of the RNase A is 1 ng / μL; Preferably, the phenol-chloroform extraction and purification uses PCI purification method to purify DNA; Preferably, the PCI purification method refers to a method of extracting DNA using phenol, chloroform, or isoamyl alcohol, and the PCI purification method is followed by mixing and precipitation using ddH2O; Preferably, the volume ratio of the system for dsDNA end-finishing: shrimp alkaline phosphatase SAP: RNase A: ddH2O used after PCI purification is 50:2:1:

40.

4. The method according to claim 1, wherein the DNA 3' end is tailed with poly (dA) comprising the following steps: The DNA 3' end was tailed with poly(dA) by using the following system: DNA product purified by phenol-chloroform extraction, 10× Terminal Transferase Reaction Buffer, Terminal Transferase, 1 mM dATP, and a reaction procedure with a heated lid at 105°C, 37°C for 30 min, and storage at 4°C. Preferably, the DNA product purified by phenol-chloroform extraction is mixed with ddH2O; Preferably, the volume ratio of the DNA product purified by phenol-chloroform extraction, 10× Terminal Transferase Reaction Buffer, Terminal Transferase, and 250 μM dATP is 40:5:4:

1.

5. The method according to claim 1, wherein the DNA 5' end phosphorylation comprises the following steps: phosphorylating the DNA 5' end by using a system comprising: a product obtained by poly(dA) tailing of the DNA 3' end, 10× Terminal Transferase Reaction Buffer, 10 mM ATP, 50 mM DTT, T4 PNK enzyme, and ddH2O, with a reaction procedure of 105°C, 37°C, 30 min, and storage at 4°C with a heated lid; Preferably, the volume ratio of the DNA 3' end tailed with poly (dA), 10× Terminal Transferase Reaction Buffer, 10 mM ATP, 50 mM DTT, T4 PNK enzyme, and ddH2O is 50:3:8:8:1:10; Preferably, the biotin-labeled adapter specifically ligates the single-stranded DNA by specifically ligating the single-stranded DNA in the DNA product after column purification in step 6, specifically by ligating the single-stranded DNA with SEQ ID NO: 1 using T4 DNA ligase; Preferably, the SEQ ID NO: 1 is a 5' stem-loop linker with a biotin label; Preferably, the SEQ ID NO: 1 needs to be annealed before being ligated to the single-stranded DNA, specifically: The annealed SEQ ID NO: 1 was obtained by using the following system: 100 μM SEQ ID NO: 1, 10× annealing buffer, ddH2O, and a PCR program with a heated lid at 105°C, 95°C for 5 min, followed by a temperature decrease of 2°C per second to 85°C, and finally to 25°C at a rate of 0.1°C per second. Preferably, the step of ligating the single-stranded DNA with SEQ ID NO: 1 by T4 DNA ligase comprises: The single-stranded DNA was ligated to SEQ ID NO: 1 using the following system: DNA product purified by column in step 6, 10× T4 DNA ligase buffer, 400 U / μL T4 DNA ligase, 50% PEG 8000, 50 μM SEQ ID NO: 1, with a heating lid at 105°C, 16°C for 2h, 75°C for 20min, and storage at 4°C; Preferably, the volume ratio of the column-purified DNA product in step 6, 10×T4 DNA ligase buffer, 400 U / μL T4 DNA ligase, 50% PEG 8000, and 50 μM SEQ ID NO: 1 is 36:8:3:32:

1.

6. The method according to claim 1, wherein the step of separating single-stranded DNA from double-stranded DNA comprises: Add streptavidin agarose gel to the product from step 7, mix well, incubate at 4°C for 2 hours or more, centrifuge, and separate the supernatant and precipitate. The supernatant is used for double-stranded DNA library construction, and the precipitate is used for single-stranded DNA library construction; Preferably, the streptavidin agarose gel needs to be used after removing the preservation solution.

7. The method according to claim 1, wherein the specific steps of amplifying the single-stranded DNA by PCR in step 9 are: Resuspend the precipitate obtained in step 8 in ddH2O and add it to the following system: the precipitate obtained in step 8 resuspended in ddH2O, 2×Phanta UniFiMaster Mix, 5 μM primer F, and 5 μM primer R, wherein primer F is SEQ ID NO: 2 and primer R is any one of SEQ ID NOs: 3-6; Preferably, the volume ratio of the precipitated product obtained in step 8 of the ddH2O resuspension, 2×Phanta UniFi Master Mix, 5 μM primer F, and 5 μM primer R is 48:50:1:

1.

8. A high-throughput sequencing library prepared by the method according to any one of claims 1 to 7, wherein the high-throughput sequencing library comprises a single-stranded DNA library and a double-stranded DNA library of the same sample.

9. A use of the high-throughput sequencing library according to claim 8, wherein the high-throughput sequencing library is sequenced, wherein the high-throughput sequencing library is suitable for Illumina sequencing platform, Roche sequencing platform, TermoFisher sequencing platform, BGI sequencing platform and MGI sequencing platform.

10. A sequence product used in the method according to any one of claims 1 to 7, wherein the sequence product comprises the nucleotide sequence of SEQ ID NO: 1 to 6; Preferably, the sequence product also includes other reagents required for PCR, DNA end repair, and single-stranded DNA specific adapter ligation.

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