Short-chain ssDNA sequencing library construction method, sequencing method and application

Abstract

The invention relates to the field of DNA sequencing, in particular to a short-chain ssDNA sequencing library construction method, a sequencing method and application. The construction method of the sequencing library of the short-chain ssDNA comprises the following steps: a carrier connection step: connecting the 5'end of a first ssDNA fragment to a fixed carrier; adding a polynucleotide tail: taking the first ssDNA fragment as a template, carrying out enzyme reaction by utilizing terminal deoxynucleotidyl transferase, and adding the polynucleotide tail at the 3'terminal of the first ssDNA fragment to obtain tailed ssDNA; a PCR amplification step of the tailed ssDNA: taking the tailed ssDNA as a template, designing a first primer to carry out a PCR amplification reaction, and purifying an amplification product; and a sequencing library construction step: constructing the sequencing library by taking the purified amplification product as a template. By adopting the technical scheme provided by the invention, the defects in the prior art are overcome, the construction method is not limited by the ssDNA fixation state any more, the construction of the special ssDNA library is realized, and the sequencing accuracy of the sequencing library constructed by adopting the construction method provided by the invention is high.

Description

Technical Field

[0001] This invention relates to the field of DNA sequencing, specifically to a method for constructing a sequencing library of short-chain ssDNA, a sequencing method, and its applications. Background Technology

[0002] DNA sequencing refers to the process of detecting the precise sequence of bases (such as A, T, C, G) in DNA molecules through experimental techniques. It is one of the most fundamental and core technologies in modern life science research and application, and has now developed into first-generation sequencing, second-generation sequencing, and third-generation sequencing technologies.

[0003] Among them, the second-generation sequencing (NGS) technology is currently the mainstream high-throughput sequencing technology, represented by the Illumina sequencing platform which uses SBS and related technologies; while the third-generation sequencing technology refers to single-molecule sequencing technology, which does not require PCR amplification during DNA sequencing, and achieves individual sequencing of each DNA molecule.

[0004] In high-throughput DNA sequencing, library preparation is an essential and crucial step. It is not the sequencing itself, but rather the sample preprocessing work done to ensure that the sequencer can effectively and accurately read the DNA information. The core of library preparation is transforming the raw DNA sample into a standardized and recognizable form that the sequencer can process.

[0005] During library construction, DNA samples are typically physically or enzyme-digested into fragments of appropriate size (e.g., 150-800 bp). This ensures that the sequencer can effectively read each small fragment. During library construction, specific synthetic DNA fragments, called adapters, are added to both ends of each fragmented DNA. However, existing DNA library construction methods struggle with the construction of libraries for short-chain DNA.

[0006] In view of the above, this application is hereby submitted. Summary of the Invention

[0007] The purpose of this invention is to provide a method for constructing a sequencing library of short-chain ssDNA, a sequencing method, and its application, so as to solve at least one technical problem in the background art.

[0008] Specifically, the first aspect of this application provides a method for constructing a sequencing library of short-chain ssDNA, comprising the steps of:

[0009] Vector ligation steps: The 5' end of the first ssDNA fragment is ligated to the immobilization vector;

[0010] Adding a polynucleotide tail: Using the first ssDNA fragment as a template, an enzymatic reaction is performed using terminal deoxynucleotidyl transferase to add a polynucleotide tail to the 3' end of the first ssDNA fragment to obtain tailed ssDNA.

[0011] PCR amplification steps for tailed ssDNA: Using tailed ssDNA as a template, design the first primer to perform PCR amplification reaction, and purify the amplification product.

[0012] Sequencing library construction steps: Use the purified amplification product as a template to construct a sequencing library.

[0013] Preferably, the sequencing library construction step includes:

[0014] End repair step: Using the purified amplification product as a template, 5' end repair and 3' end tailing reaction are performed to obtain the repair product;

[0015] Adapter ligation step: Using the repair product as a template, a ligase is used to react and obtain a template with adapters, which is then purified;

[0016] PCR enrichment and purification steps for dsDNA: Using the purified product obtained from the adapter ligation step as a template, PCR enrichment and purification are performed to obtain a sequencing library.

[0017] Preferably, the PCR enrichment and purification step of the dsDNA includes:

[0018] Steps to obtain circularized single-stranded DNA: The tailed short-stranded ssDNA is treated and ligated into a circular form, followed by purification of the circular DNA product, and amplification is performed using the purified product as a template;

[0019] DNA strand fragmentation step: The product of the step of obtaining circular single-stranded DNA is fragmented using endonuclease and a sequencing library is constructed.

[0020] Preferably, the short-chain ssDNA has a length of 20-100 nt.

[0021] Preferably, in the step of adding the polynucleotide tail, the polynucleotide is polyadenine deoxyribonucleotide or polyguanine deoxyribonucleotide.

[0022] Preferably, in the step of adding the polynucleotide tail, the tailing reaction rate is controlled at 30-40 nt / min.

[0023] Preferably, in the step of adding the polynucleotide tail, the enzyme reaction is carried out at a temperature of 29-31°C for 8-10 minutes, and finally inactivated at a high temperature of 96-99°C. More preferably, the enzyme reaction is carried out at a temperature of 30°C for 8-10 minutes, and finally inactivated at a high temperature of 98°C.

[0024] Preferably, the step of adding the polynucleotide tail includes, by volume, the following:

[0025] 15-25 portions of 5× terminal deoxynucleotidyl transferase reaction buffer (terminal deoxynucleotidyl transferase buffer);

[0026] 4-6 portions of terminal deoxynucleotidyl transferase (TdT enzyme);

[0027] 70-80 parts enzyme-free water;

[0028] 10μM dNTP, 1-3 times.

[0029] More preferably, the step of adding the polynucleotide tail includes, by volume, the following:

[0030] 20 portions of 5× terminal deoxynucleotidyl transferase reaction buffer (terminal deoxynucleotidyl transferase buffer);

[0031] Five portions of terminal deoxynucleotidyl transferase (TdT enzyme);

[0032] 73 portions of enzyme-free water;

[0033] Two 10μM dNTP samples.

[0034] Preferably, the terminal deoxynucleotidyl transferase is a mouse-derived muTdT enzyme.

[0035] Preferably, the dNTP is dATP or dGTP.

[0036] Preferably, the PCR amplification step of the tailed ssDNA includes, by volume, the following:

[0037] 40-45 parts enzyme-free water;

[0038] 45-55 copies of 2×PCR buffer;

[0039] 1-3 parts of dNTP mix;

[0040] 1-3 portions of DNA polymerase;

[0041] 3-5 portions of the first primer mixture.

[0042] More preferably, the PCR amplification step of the tailed ssDNA includes, by volume, the following:

[0043] 42 portions of enzyme-free water;

[0044] 50 copies of 2×PCR buffer;

[0045] Two portions of dNTP mix;

[0046] Two portions of DNA polymerase;

[0047] Four portions of the first primer mixture.

[0048] Preferably, in the PCR amplification step of the tailed ssDNA, the reaction conditions include the following steps:

[0049] Place the sample in a PCR instrument and perform the PCR amplification reaction under the following conditions:

[0050] Pre-denaturation stage: The reaction temperature is 94-96℃, preferably 95℃, and the reaction time is 2-4 min, preferably 3 min;

[0051] Denaturation stage: The reaction temperature is 94-96℃, preferably 95℃, and the reaction time is 10-20s, preferably 15s;

[0052] Annealing stage: The reaction temperature is 45-55℃, preferably 50℃, and the reaction time is 10-20s, preferably 15s;

[0053] Extension stage: The reaction temperature is 70-75℃, preferably 72℃, and the reaction time is 8-12s, preferably 10s;

[0054] Final extension stage: The reaction temperature is 70-75℃, preferably 72℃, and the reaction time is 4-6 min, preferably 5 min;

[0055] Storage stage: The temperature is 3-5℃, preferably 4℃.

[0056] The denaturation and extension stages are performed for no less than 30 cycles, and the pre-denaturation and preservation stages are each performed once to obtain the library-constructed dsDNA sample.

[0057] Preferably, in the end-repair step, the 5' end-repair reaction, by volume, comprises:

[0058] x portions of purified amplification product,

[0059] 10-20 parts End prep Mix

[0060] Replenish ultrapure water (MQ) to 60-70 parts;

[0061] Among them, the x portions contain at least 0.02 parts by weight of amplification product.

[0062] Preferably, in the end-repair step, the 5' end-repair reaction, by volume, comprises:

[0063] x portions of purified amplification product,

[0064] 15 parts End prep Mix

[0065] Ultrapure water (MQ) was replenished to 65 portions.

[0066] Preferably, the reaction conditions in the 5' end repair reaction include:

[0067] The phosphorylation reaction of the terminal repair enzyme is carried out at a temperature of 18-22℃ and a reaction time of 12-18 min; the reaction of adding the A tail is carried out at a temperature of 64-66℃ and a reaction time of 12-16 min.

[0068] Preferably, the reaction conditions in the 5' end repair reaction include:

[0069] The phosphorylation reaction of the terminal repair enzyme was carried out at a temperature of 20℃ for 15 min; the reaction of adding the A tail was carried out at a temperature of 65℃ for 15 min.

[0070] Preferably, the connecting joint step includes:

[0071] The 3' A-tail of the product obtained from the end-repair step was complementaryly ligated to the 5' protruding thymine deoxyribonucleotide tail of the linker sequence. The reaction temperature was 18-22℃, and the reaction time was 13-16 min. The reaction solution, by volume, comprised:

[0072] 60-70 parts of end-of-life repair product,

[0073] 20-30 portions of rapid ligation buffer.

[0074] 4-6 parts of rapid DNA ligase.

[0075] 4-6 copies of DNA adapter.

[0076] Preferably, the reaction conditions in the connecting joint step are: a reaction temperature of 20°C and a reaction time of 15 min.

[0077] Preferably, the reaction solution in the connecting joint step includes:

[0078] 65 samples of end-of-life repair products,

[0079] 25 portions of Rapid Ligation Buffer.

[0080] Five portions of rapid DNA ligase.

[0081] Five DNA adapters.

[0082] Preferably, in the PCR enrichment step, the reaction solution, by volume, comprises:

[0083] 18-22 parts of the connector product,

[0084] 4-6 portions of PCR Primer Mix

[0085] 20-30 parts of VAHTS HiFi Amplification Mix

[0086] The amplification mixture contains a second primer.

[0087] Preferably, in the PCR enrichment step of the dsDNA, the reaction solution, by volume, comprises:

[0088] 20 sets of connector products,

[0089] Five portions of PCR Primer Mix.

[0090] 25 portions of VAHTS HiFi Amplification Mix.

[0091] Preferably, in the PCR enrichment step of the dsDNA, the reaction conditions include the following steps:

[0092] Place the sample in a PCR instrument and perform the PCR amplification reaction under the following conditions:

[0093] Pre-denaturation stage: The reaction temperature is 94-96℃, preferably 95℃, and the reaction time is 2-4 min, preferably 3 min;

[0094] Denaturation stage: The reaction temperature is 97-99℃, preferably 98℃, and the reaction time is 15-25s, preferably 20s;

[0095] Annealing stage: The reaction temperature is 55-65℃, preferably 60℃, and the reaction time is 10-20s, preferably 15s;

[0096] Extension stage: The reaction temperature is 70-75℃, preferably 72℃, and the reaction time is 25-35s, preferably 30s;

[0097] Final extension stage: The reaction temperature is 70-75℃, preferably 72℃, and the reaction time is 4-6 min, preferably 5 min;

[0098] Storage stage: The temperature is 3-5℃, preferably 4℃.

[0099] The denaturation and extension stages are performed for no less than 7 cycles, and the pre-denaturation and preservation stages are each performed once to obtain the target sequencing library.

[0100] Preferably, in the purification step of the library-constructed dsDNA sample,

[0101] 100 dsDNA samples were recovered for library preparation, 180 magnetic beads were added, and the mixture was mixed by pipetting. The mixture was then centrifuged at low speed to remove the droplets on the reaction tube wall, so that the magnetic beads were evenly dispersed in the solution. The mixture was reacted at room temperature for 10 minutes.

[0102] Preferably, in the purification step of the library-constructed dsDNA sample,

[0103] Place the reaction tube into the magnetic rack. After the magnetic beads are adsorbed onto the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution. After the surface of the magnetic beads is dry and without cracks, add 30 μl of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 min.

[0104] Optionally, the step of treating the tailed short-chain ssDNA and then ligating it into a circular form includes:

[0105] PCR amplification steps for short-chain ssDNA: Using tailed short-chain ssDNA as a template, design the first primer to perform PCR amplification reaction, and purify the amplification product.

[0106] End repair step: Using the purified amplification product as a template, 5' end repair and 3' end tailing reaction are performed;

[0107] Adapter ligation step: Using the product obtained from the end repair step as a template, a ligase is used to react and obtain a template with adapters, which is then purified.

[0108] PCR enrichment and purification steps for dsDNA: Using the purified product obtained from the adapter ligation step as a template, a second primer was designed for PCR enrichment and purification.

[0109] DNA circularization steps: denature dsDNA into single strands and link ssDNA into a circular structure using clips.

[0110] Optionally, the step of treating the tailed short-chain ssDNA and then ligating it into a circular form includes:

[0111] 5' phosphorylation step: Phosphorylate the 5' end of the tailed short ssDNA;

[0112] ssDNA circularization step: The phosphorylated ssDNA is circularized so that its 5' end is connected to its 3' end.

[0113] Furthermore, the PCR amplification step of the short-chain ssDNA includes a purification step of the first dsDNA sample, specifically including:

[0114] By volume, recover 50-150 dsDNA samples for library construction, add 150-200 magnetic beads, mix by pipetting, centrifuge at low speed to remove droplets from the reaction tube wall, so that the magnetic beads are evenly dispersed in the solution, and react at room temperature for 5-15 minutes.

[0115] Place the reaction tube into the magnetic rack. After the magnetic beads are adsorbed onto the tube wall, remove the solution. Rinse the tube wall with 60-85% alcohol and remove the solution. After the surface of the magnetic beads is dry and without cracks, add 20-40 μl of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 3-6 minutes.

[0116] The reaction tube was placed in a magnetic rack. After the magnetic beads were adsorbed onto the tube wall, the solution was recovered and the nucleic acid concentration was measured.

[0117] Furthermore, in the purification step of the first dsDNA sample,

[0118] 100 dsDNA samples were recovered for library preparation, 180 magnetic beads were added, and the mixture was mixed by pipetting. The mixture was then centrifuged at low speed to remove the droplets on the reaction tube wall, so that the magnetic beads were evenly dispersed in the solution. The mixture was reacted at room temperature for 10 minutes.

[0119] Furthermore, in the purification step of the first dsDNA sample,

[0120] Place the reaction tube into the magnetic rack. After the magnetic beads are adsorbed onto the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution. After the surface of the magnetic beads is dry and without cracks, add 30 μl of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 min.

[0121] Furthermore, in the step of linking ssDNA into a circular DNA ring using a clip, the reaction solution, by volume, comprises:

[0122] 30-50 copies of denatured single-stranded DNA

[0123] 10-20 portions of ligation buffer (Splint buffer)

[0124] 3-10 parts of ligase,

[0125] Place at 36-38℃ and react for 10-30 minutes.

[0126] Furthermore, in the step of linking ssDNA into a circular DNA ring using a clip, the reaction solution, by volume, comprises:

[0127] 40 samples of denatured single-stranded DNA

[0128] 15 portions of Splint buffer.

[0129] Five portions of ligase.

[0130] Place at 37°C and react for 15 minutes.

[0131] Optionally, in the 5' end phosphorylation step, the reaction solution, by volume, comprises:

[0132] Contains 0.04-0.06 parts by weight of tailed short-chain ssDNA x parts.

[0133] 1-3 portions of 10× polynucleotide kinase buffer.

[0134] 0.1mM ATP, 0.5-2 parts.

[0135] Polynucleotide kinase 0.5-2 parts,

[0136] Topping up with ultrapure water (MQ) to 10-30 parts,

[0137] The reaction temperature is 36-38℃, the reaction time is 20-40 min, followed by inactivation at 70-80℃ for 5-20 min.

[0138] Furthermore, in the 5' end phosphorylation step, the reaction solution, by volume, comprises:

[0139] Contains x portions of 0.05 parts by weight of tailed short-chain ssDNA.

[0140] Two portions of 10× polynucleotide kinase buffer.

[0141] 0.1mM ATP, 1 serving

[0142] One part of polynucleotide kinase,

[0143] Topping up with ultrapure water (MQ) to 20 parts.

[0144] The reaction temperature was 37℃, the reaction time was 30 min, and then the reaction was carried out at 75℃ for 10 min to deactivate the virus.

[0145] Further, in the step of circularizing the phosphorylated ssDNA, the reaction solution, by volume, comprises:

[0146] Phosphorylated ssDNA 15-20 parts,

[0147] 1-3 portions of 10× circligase buffer.

[0148] 0.5-2 parts of 50mM MnCl2

[0149] Single-stranded DNA circligase (ssDNA circligase) 0.5-2 parts,

[0150] The reaction temperature is 55-65℃, the reaction time is 30-90 min, followed by inactivation at 75-85℃ for 5-20 min.

[0151] Further, in the step of circularizing the phosphorylated ssDNA, the reaction solution, by volume, comprises:

[0152] 16 phosphorylated ssDNA samples

[0153] Two 10× circligase buffer solutions.

[0154] 1 part of 50mM MnCl2

[0155] One part of single-stranded DNA circligase (ssDNA circligase),

[0156] The reaction temperature was 60℃, the reaction time was 60 min, and then the reaction was carried out at 80℃ for 10 min to deactivate the virus.

[0157] Furthermore, in the step of obtaining circularized single-stranded DNA, the amplification using the purified product as a template includes the following steps:

[0158] Rolling circle amplification was performed using DNA polymerase and random hexamer primers.

[0159] A second aspect of this application provides a next-generation sequencing method, the sequencing method comprising the method for constructing a sequencing library of short-chain ssDNA as described in the first aspect of this application.

[0160] A third aspect of this application provides a third-generation sequencing method, the sequencing method comprising the short-chain ssDNA sequencing library construction method as described in the first aspect of this application.

[0161] A fourth aspect of this application provides an application of the short-chain ssDNA sequencing library construction method of the first aspect of this application in short-chain ssDNA synthesis.

[0162] Compared with the prior art, the present invention has the following advantages:

[0163] This invention proposes a method for constructing sequencing libraries for short-chain ssDNA on a solid-phase carrier, which overcomes the shortcomings of existing technologies, is no longer limited by the fixed state of ssDNA, realizes the construction of libraries for special ssDNA, and the sequencing libraries constructed using the method of this invention have high sequencing accuracy. Attached Figure Description

[0164] Figure 1 To construct a library capillary electrophoresis quality control chart for adding A tails;

[0165] Figure 2 To construct a library capillary electrophoresis quality control chart for adding G tails;

[0166] Figure 3 To verify the electrophoretic image of the bands after DNA circularization using the method of Example 3;

[0167] Figure 4 This is an electrophoresis image used in Example 4 to verify rolling circle amplification after single-stranded circularization. Detailed Implementation

[0168] Exemplary embodiments will now be described in detail, examples of which are illustrated in the accompanying drawings. When the following description relates to the drawings, unless otherwise indicated, the same numbers in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this application as detailed in the appended claims.

[0169] The terminology used in this application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The singular forms “a,” “the,” and “the” used in this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the term “and / or” as used herein refers to and includes any or all possible combinations of one or more of the associated listed items.

[0170] The present application will be described in detail below through examples.

[0171] To better understand the above technical solutions, the following detailed descriptions will be provided in conjunction with specific implementation methods. Those skilled in the art should also understand that the reaction times and component additions involved in this application cannot be absolutely precise in actual production or experimentation, but are all within the allowable error range. For example, if the desired sample heating time is 30 minutes, the actual operation may be 30 minutes plus or minus 1 second; if the desired sample weight is 30g, the actual weight may be 30.001g or 29.998g.

[0172] The following are specific embodiments of the present invention. These embodiments are intended to further describe the present invention and are not intended to limit the present invention.

[0173] Unless otherwise specified, the reagents used in this application are generally commercially available. The specific sources of these reagents are listed below:

[0174] DNA polymerase: Novizan, product number P505-d1 Phanta Max Super-Fidelity DNA Polymerase reagent.

[0175] Magnetic beads: Novizan product number N411-03-AA VAHTSDNA Clean Beads.

[0176] DNA Library Construction Kit: Novizan ND607-02 VAHTS Universal DNA Library PrepKit for Illumina V3.

[0177] Terminal deoxynucleotidyl transferase: Thermo Fisher Scientific catalog number EP0162 terminal deoxynucleotidyl transferase.

[0178] End repair kit: BGI (Catalog No. 1000006985) MGIEasy Universal DNA Library Preparation Reagent Kit.

[0179] DNA Circulation Kit: Hieff (Catalog No. 13341ES16) Fast-Pace DNACyclization Kit for The Fast-Pace DNA circularization kit is a sequencing platform.

[0180] Circular DNA amplification polymerase: Novizan (Catalog No. N106-01) Phi29 MAX DNA Polymerase.

[0181] Circulated DNA amplified random hexamers: Bio-Rayborg (Catalog No. RFT106) Random hexamers.

[0182] Endonuclease: NEB (catalog number M0302S) T7 Endonuclease I

[0183] Oxford Nanopore Technologies (ONT) Library Preparation Kit: SQK-LSK109 Kit.

[0184] T4 Polynucleotide Kinase: Novizan (Catalog No. N102) T4 Polynucleotide Kinase.

[0185] Cycloylase: HaiGene (Catalog No. D2602A) ssDNA / RNA cyclization ligase.

[0186] Exonuclease: HaiGene (Catalog No. C5014) Exonuclease I (E. coli).

[0187] Those skilled in the art should understand that the above-mentioned reagents are provided for reference only and do not limit the scope of protection of this application. Unless otherwise specified, the above-mentioned reagents are not the only option.

[0188] Example 1: Adding an A tail to build a database

[0189] S1. Obtain the sample chip

[0190] A short ssDNA sequence is synthesized according to requirements. The 5' end of the ssDNA sequence is ligated to a fixation vector, and its length is 20-100 nt. In this embodiment, the sequence of this short ssDNA segment is as follows:

[0191] 5'-TTTTTTTTTT-GGCTAGAGACTCCTACGCGACTTGAGAAAGGATGATG-3'.

[0192] In some embodiments, the immobilization carrier is a gene chip.

[0193] S2, add polynucleotide A tail

[0194] Using the short-chain ssDNA obtained in step S1 as a template, a certain amount of polynucleotide tail of a certain length is added to the 3' end of the short-chain ssDNA using a TdT enzyme. The TdT enzyme can be selected from mouse-derived muTdT enzyme, and the tailing speed is controlled at 30-40 nt / min, with a tail length of 40-100 nt.

[0195] The specific steps for this process are as follows: Cut the sample chip to be used for library construction into a 4×4mm square and place it at the bottom of a 1.5mL centrifuge tube. Add the reaction solution prepared according to the components shown in Table 1, ensuring that the reaction solution evenly covers the chip and is free of air bubbles.

[0196] Table 1

[0197] Components Volume (μl) 5X terminal deoxynucleotidyl transferase reaction buffer 20μl Terminal deoxynucleotidyl transferase 5μl Enzyme-free water 73μl dATP (10 uM) 2μl

[0198] The muTdT enzyme reaction temperature is 30℃, the reaction time is 8-10 min, and it is finally inactivated at 98℃.

[0199] S3, PCR amplification reaction and purification

[0200] In this step, the product obtained in step S2 is used as a template, and a PCR amplification reaction is performed using a primer mixture that is complementary to the DNA polymerase and the template to enrich the target fragment.

[0201] The specific process steps are as follows: wash away the residual reaction solution in the product obtained in step S2, then wash twice with enzyme-free water, and then add the reaction solution formed by the components shown in Table 2, ensuring that the reaction solution covers the product and there are no air bubbles.

[0202] Table 2

[0203]

[0204] Place the above components in a PCR instrument and perform PCR amplification under the following conditions:

[0205] Pre-denaturation stage: reaction temperature is 95℃, reaction time is 3 min;

[0206] Denaturation stage: reaction temperature 95℃, reaction time 15s;

[0207] Annealing stage: reaction temperature is 50℃, reaction time is 15s;

[0208] Extension stage: The reaction temperature is 72℃, and the reaction time is 10s;

[0209] Final extension stage: reaction temperature 72℃, reaction time 5 min;

[0210] Storage stage: The reaction temperature is 4℃.

[0211] The PCR amplification reaction was performed in 30 cycles from denaturation to extension, with the pre-denaturation and final extension phases each performed once. After the PCR amplification reaction was completed, the product was recovered, and the DNA fragments were purified to obtain the dsDNA sample for library construction.

[0212] The obtained library-constructed dsDNA sample was further purified, and the following steps were performed:

[0213] Recover 100 μl of the dsDNA sample used for library preparation, then add 180 μl of magnetic beads (1.8×), mix thoroughly by pipetting, centrifuge at low speed to remove droplets from the EP tube wall, and ensure the magnetic beads are evenly dispersed in the solution. React at room temperature for 10 min.

[0214] Insert the EP tube into the magnetic rack. After the magnetic beads are attracted to the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution, being careful not to pick up the magnetic beads. After the surface of the magnetic beads is dry and without cracks, add 30 μl of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 min.

[0215] Insert the EP tube into the magnetic holder, wait for the magnetic beads to adhere to the tube wall, then recover the solution and use Qubit4.0 to detect the nucleic acid concentration.

[0216] S4, end repair and A addition

[0217] Using the purified product obtained in step S3 as a template, the DNA fragments on the template were subjected to end repair enzyme reaction and tailing reaction using the two enzyme mixture in the DNA library construction kit, so as to complete the 5' end protrusion of the DNA fragment and add an adenine deoxyribonucleotide tail (A tail) to the 3' end.

[0218] In the specific process steps, the volume x to be added is calculated based on the concentration, and 20 ng is taken to prepare the reaction solution shown in Table 3 below:

[0219] Table 3

[0220] Components Volume (μl) The product obtained in step S3 x End prep Mix 4 15 MQ To 65

[0221] Placed in a PCR instrument, the phosphorylation reaction of the terminal repair enzyme was carried out at a temperature of 20℃ for 15 min; the reaction of adding the A tail was carried out at a temperature of 65℃ for 15 min.

[0222] S5, Connecting connector

[0223] In this step, the product obtained in step S4 is used as a template, and a ligase is used to react and connect the A tail added to the 3' end in step S4 with the T protruding from the 5' end of the adapter sequence to form an "adapter-fragment-adapter" structure.

[0224] In this step, specifically in the process steps, the reaction solution formed by the components shown in Table 4 is prepared and mixed by blowing.

[0225] Table 4

[0226]

[0227] Place the sample in a PCR instrument. The optimal reaction temperature for this step is 20℃, and the reaction time is 15 minutes. After the adapter ligation step, two purification processes are required. The two purification processes are as follows:

[0228] For the first time, 100 μl of sample was recovered, and then 60 μl of magnetic beads (0.6×) were added. After mixing by blowing, the liquid droplets on the EP tube wall were removed by low-speed centrifugation to make the magnetic beads evenly dispersed in the solution. The reaction was carried out at room temperature for 10 min.

[0229] Insert the EP tube into the magnetic rack. After the magnetic beads are adsorbed onto the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution, being careful not to pick up the magnetic beads. Once the surface of the magnetic beads is dry and without cracks, add 22 μl of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 minutes.

[0230] Insert the EP tube into the magnetic frame, and after the magnetic beads are attracted to the tube wall, take 20 μl of solution.

[0231] The second time, 30 μl of magnetic beads (1.5×) were added to 20 μl of the recovered solution. After mixing by blowing and blowing, the liquid droplets on the tube wall were removed by low-speed centrifugation to make the magnetic beads evenly dispersed in the solution. The reaction was carried out at room temperature for 10 min.

[0232] Insert the EP tube into the magnetic rack. After the magnetic beads are adsorbed onto the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution, being careful not to pick up the magnetic beads. Once the surface of the magnetic beads is dry and without cracks, add 22 μl of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 minutes.

[0233] Insert the EP tube into the magnetic frame. After the magnetic beads are attracted to the tube wall, take 20 μl of the solution for later use.

[0234] S6, PCR enrichment and purification

[0235] In this step, the purified product obtained in step S5 is used as a template, and the fragment with adapter is amplified by PCR using enzyme mixture and universal primers to enrich the target fragment and obtain a DNA next-generation sequencing library.

[0236] In this specific process step, the components shown in Table 5 below are added to the PCR tube for PCR amplification.

[0237] Table 5

[0238] Components volume The purified product obtained in step S5 20μl PCR Mix (PCR Primer Mix 3 for Illumina) 5μl Amplification Mix (VAHTS HiFi Amplification Mix) 25μl

[0239] Place it in a PCR instrument and proceed under the following conditions:

[0240] Pre-denaturation stage: reaction temperature is 95℃, reaction time is 3 min;

[0241] Denaturation stage: reaction temperature 98℃, reaction time 20s;

[0242] Annealing stage: reaction temperature is 60℃, reaction time is 15s;

[0243] Extension stage: The reaction temperature is 72℃, and the reaction time is 30s;

[0244] Final extension stage: reaction temperature 72℃, reaction time 5 min;

[0245] Storage stage: The reaction temperature is 4℃.

[0246] The PCR process consists of 7 cycles from denaturation to extension, with the pre-denaturation and final extension phases each performed once. After PCR amplification, the products are recovered, and the DNA fragments are purified to obtain the target library. Purification is performed as follows:

[0247] Recover 50 μl of sample, add 60 μl of magnetic beads (1.2×), mix by blowing and stirring, then centrifuge at low speed to remove droplets from the tube wall, so that the magnetic beads are evenly dispersed in the solution, and react at room temperature for 10 min.

[0248] Insert the EP tube into the magnetic rack. After the magnetic beads are attracted to the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution, being careful not to pick up the magnetic beads. After the surface of the magnetic beads is dry and without cracks, add 30 μl of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 min.

[0249] Insert the EP tube into the magnetic frame. After the magnetic beads are attracted to the tube wall, recover the solution, measure the concentration, and perform capillary electrophoresis for quality control.

[0250] Example 2: Adding the "G" suffix to build the database

[0251] S1. Obtain the sample chip

[0252] A short ssDNA sequence is synthesized according to requirements. The 5' end of the ssDNA sequence is ligated to a fixation vector, and its length is 20-100 nt. In this embodiment, the sequence of this short ssDNA segment is as follows:

[0253] 5'-TTTTTTTTTT-GGCTAGAGACTCCTACGCGACTTGAGAAAGGATGATG-3'.

[0254] In some embodiments, the immobilization carrier is a gene chip.

[0255] S2, G-tail of polynucleotide

[0256] Using the short-chain ssDNA obtained in step S1 as a template, a certain amount of polynucleotide tail of a certain length is added to the 3' end of the short-chain ssDNA using a TdT enzyme. The TdT enzyme can be selected from mouse-derived muTdT enzyme, and the tailing speed is controlled at 30-40 nt / min, with a tail length of 40-100 nt.

[0257] During the experiment, the applicant found that the synthesis speed of the G-tail was easier to control than that of the A-tail. Therefore, in Example 1, the added A-tail can be directly used for complementary amplification on the vector to achieve one amplification. In this example, since the length of the added G-tail is easier to control, a poly-C+ fixed sequence is designed to complement the added G-tail for amplification. The fixed sequence is used as the down-index for the second amplification, achieving two amplifications and making the library construction process easier to operate.

[0258] The specific steps for this process are as follows: Cut the sample chip to be used for library construction into a 4×4mm square and place it at the bottom of a 1.5mL centrifuge tube. Add the reaction solution prepared according to the components shown in Table 6 below, ensuring that the reaction solution evenly covers the chip and is free of air bubbles.

[0259] Table 6

[0260] Components Volume (μl) 5X terminal deoxynucleotidyl transferase reaction buffer 20μl Terminal deoxynucleotidyl transferase 5μl Enzyme-free water 73μl dATP (10 uM) 2μl

[0261] The muTdT enzyme reaction temperature is 30℃, the reaction time is 8-10 min, and it is finally inactivated at 98℃.

[0262] S3, PCR amplification reaction and purification

[0263] In this step, the product obtained in step S2 is used as a template to perform PCR amplification using phanta enzyme. The first-stage primer is designed with a sequence complementary to and extended to the G tail, and the second-stage primer mixture is designed with a primer complementary to the extended sequence to enrich the target fragment.

[0264] The specific process steps are as follows: remove the residual reaction solution from the product obtained in step S2, wash the tablets twice with enzyme-free water, and then add the reaction solution prepared according to the components shown in Table 7, ensuring that the liquid covers the product and there are no air bubbles.

[0265] Table 7

[0266] Components Volume (μl) Enzyme-free water 42 2×buffer 50 dNTP 2 Second-stage primer mixture 2 First-stage primers 2

[0267] Place the above components in a PCR instrument and proceed under the following conditions:

[0268] Pre-denaturation stage: reaction temperature is 98℃, reaction time is 10 min;

[0269] First denaturation stage: reaction temperature 98℃, reaction time 30s;

[0270] First annealing stage: reaction temperature is 60℃, reaction time is 15s;

[0271] First extension stage: reaction temperature is 72℃, reaction time is 20s;

[0272] Second denaturation stage: reaction temperature is 98℃, reaction time is 30s;

[0273] Second annealing stage: reaction temperature is 45℃, reaction time is 15s;

[0274] Second extension stage: reaction temperature is 72℃, reaction time is 20s;

[0275] Final extension stage: reaction temperature 72℃, reaction time 5 min;

[0276] Storage stage: The reaction temperature is 4℃.

[0277] The PCR process consists of 10 cycles from the first denaturation stage to the first extension stage (stage 1 PCR); 24 cycles from the second denaturation stage to the second extension stage (stage 2 PCR); and one cycle each for the pre-denaturation stage and the final extension stage. After PCR amplification, the product is recovered, and the DNA fragment is purified to obtain the dsDNA sample for library construction.

[0278] The obtained library-constructed dsDNA sample was further purified, and the following steps were performed:

[0279] Recover 100 μl of sample, then add 180 μl of Novizan magnetic beads (1.8×), mix by blowing and stirring, then centrifuge at low speed to remove droplets from the tube wall, so that the magnetic beads are evenly dispersed in the solution, and react at room temperature for 10 min.

[0280] Insert the EP tube into the magnetic rack. After the magnetic beads are attracted to the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution, being careful not to pick up the magnetic beads. After the surface of the magnetic beads is dry and without cracks, add 30 μl of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 min.

[0281] Insert the EP tube into the magnetic holder, wait for the magnetic beads to adhere to the tube wall, then recover the solution and use Qubit4.0 to detect the nucleic acid concentration.

[0282] S4, end repair and A addition

[0283] Using the purified product obtained in step S3 as a template, the DNA fragments on the template were subjected to end repair enzyme reaction and tailing reaction using the two enzyme mixture in the DNA library construction kit, so as to complete the 5' end protrusion of the DNA fragment and add an adenine deoxyribonucleotide tail (A tail) to the 3' end.

[0284] In the specific process steps, the volume to be added is calculated based on the concentration, and 20 ng is taken to prepare the reaction solution shown in Table 8 below:

[0285] Table 8

[0286] Components Volume (μl) The product obtained in step S3 x End prep Mix 4 15 MQ To 65

[0287] Placed in a PCR instrument, the end-repair phosphorylase reaction was carried out at a temperature of 20℃ for 15 min; the A-tailing reaction was carried out at a temperature of 65℃ for 15 min.

[0288] The subsequent steps are the same as in Example 1.

[0289] Experimental Example 1

[0290] This experimental example investigated the sequencing accuracy of the next-generation sequencing library of short-strand ssDNA constructed in this invention on the Illumina platform.

[0291] Three different sample chips were taken, and the next-generation sequencing Illumina platform libraries with short-stranded ssDNA were constructed according to the method of Example 1 of the present invention, namely A-tailed sample 1, A-tailed sample 2 and A-tailed sample 3.

[0292] Three more batches of different sample chips were taken, and short-stranded ssDNA libraries for next-generation sequencing on the Illumina platform were constructed according to the method in Example 2 of this invention. These were G-tailed sample 1, G-tailed sample 2, and G-tailed sample 3, respectively. The six samples were sequenced using the MiSeq next-generation sequencing platform. The quality control chart is shown below. Figure 1 and Figure 2 .

[0293] The results are shown in Table 9:

[0294] Table 9

[0295]

[0296]

[0297] As can be seen from the results in Table 9, the accuracy and Q30 of each sample in Examples 1 and 2 of this application are basically within a reasonable range, and the sequencing effect is basically at the level of ordinary sequences. It is understandable that the sequencing library with short-stranded ssDNA fixed in the vector constructed using the method of this invention can be applied to sequencing experiments, such as those on the Illumina next-generation sequencing platform, and can obtain relatively accurate experimental results, achieving efficient and accurate sequencing of special sequences.

[0298] Example 3: Double-chain cyclication scheme

[0299] S1. Obtain the sample chip

[0300] A short ssDNA sequence is synthesized according to requirements. The 5' end of the ssDNA sequence is ligated to a fixation vector, and its length is 20-100 nt. In this embodiment, the sequence of this short ssDNA segment is as follows:

[0301] 5'-TTTTTTTTTT-CCGATCTCTGAGGATGCGCTGAACT-3'.

[0302] S2, add polynucleotide A tail

[0303] Using the short-chain ssDNA obtained in step S1 as a template, a certain amount of polynucleotide tail of a certain length is added to the 3' end of the short-chain ssDNA using a TdT enzyme. The TdT enzyme can be selected from mouse-derived muTdT enzyme, and the tailing rate is controlled at 30-40 nt / min, with a tail length of 8-20 nt.

[0304] The specific steps for this process are as follows: Cut the sample chip to be used for library construction into a 4×4mm square and place it at the bottom of a 1.5mL centrifuge tube. Add the reaction solution prepared according to the components shown in Table 10, ensuring that the reaction solution evenly covers the chip and is free of air bubbles.

[0305] Table 10

[0306] Components Volume (μl) 5× terminal deoxynucleotidyl transferase reaction buffer 20μl Terminal deoxynucleotidyl transferase 5μl dATP (10 uM) 2μl Enzyme-free water Make up to 100μl sample x

[0307] The muTdT enzyme reaction temperature was 30℃, the reaction time was 8-10 min, and it was finally inactivated at 98℃. The supernatant was collected after centrifugation at 12000 rpm for 2 min.

[0308] S3. PCR amplification of circular single-stranded DNA S301 and short-stranded ssDNA.

[0309] In this step, the product obtained in step S2 is used as a template, and a PCR amplification reaction is performed using a mixture of Novozymes reagent P505-d1 and the first primer complementary to the template to enrich the target fragment.

[0310] The specific process steps are as follows: wash the residual reaction solution in the product obtained in step S2, then wash it twice with enzyme-free water, and then add the reaction solution formed by the components shown in Table 11, ensuring that the reaction solution covers the product and there are no air bubbles.

[0311] Table 11

[0312]

[0313] Place the sample in a PCR instrument and perform the PCR amplification reaction under the following conditions:

[0314] Pre-denaturation stage: reaction temperature is 95℃, reaction time is 3 min;

[0315] Denaturation stage: reaction temperature 95℃, reaction time 15s;

[0316] Annealing stage: reaction temperature is 50℃, reaction time is 15s;

[0317] Extension stage: The reaction temperature is 72℃, and the reaction time is 10s;

[0318] Final extension stage: reaction temperature 72℃, reaction time 5 min;

[0319] Storage stage: The reaction temperature is 4℃.

[0320] The PCR amplification reaction consisted of 30 cycles from denaturation to extension, with one cycle each for the pre-denaturation and final extension stages. After the reaction was complete, the product was recovered, and the DNA fragments were purified to obtain the dsDNA sample for library construction.

[0321] Further, the obtained library-constructed dsDNA sample is purified, and the following steps are performed:

[0322] Recover 100 μl of the dsDNA sample used for library preparation, then add 180 μl of magnetic beads (1.8×), mix thoroughly by pipetting, centrifuge at low speed to remove droplets from the tube wall, and ensure the magnetic beads are evenly dispersed in the solution. React at room temperature for 10 min.

[0323] Insert the EP tube into the magnetic rack. After the magnetic beads are attracted to the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution, being careful not to pick up the magnetic beads. After the surface of the magnetic beads is dry and without cracks, add 30 μl of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 min.

[0324] Insert the EP tube into the magnetic holder, wait for the magnetic beads to adhere to the tube wall, then recover the solution and use Qubit4.0 to detect the nucleic acid concentration.

[0325] S302, End Repair Procedures

[0326] The following steps can be completed using reagents from the MGIEasy Universal DNA Library Preparation Reagent Kit. Details are as follows:

[0327] Using the purified product obtained in step S301 as a template, the DNA fragments on the template were subjected to end repair enzyme reaction and A-tailing reaction using a mixture of two enzymes from the MGIEAsy universal DNA library preparation kit, thereby achieving the completion of the 5' end protrusion of the DNA fragment and the addition of an A tail to the 3' end.

[0328] In the specific process steps, 50 ng of the purified product from step 301 is added, and the total volume is supplemented to 40 μl with MQ to prepare the reaction solution shown in Table 12 below:

[0329] Table 12

[0330] Components Volume (μl) ERAT Buffer 7.1 ERAT Enzyme Mix 2.9 Ultrapure water (MQ) containing 50 ng of purified product 40

[0331] Use a pipette to add 10 μl of the prepared end-repair reaction solution to a PCR tube, vortex 3 times for 3 seconds each time, briefly centrifuge to collect the reaction solution to the bottom of the tube, place it in a PCR instrument, and perform the following reaction:

[0332] The terminal repair enzyme reaction was carried out at a temperature of 37℃ for 30 min; the reaction with A-tailed enzyme was carried out at a temperature of 65℃ for 15 min.

[0333] S303, Connection steps

[0334] Using the product obtained in step S302 as a template, a ligase was used to connect the A added at the 3' end to the T protruding at the 5' end of the adapter sequence, forming an "adapter-fragment-adapter" structure.

[0335] In this step, specifically in the process steps, the reaction solution formed by the components shown in Table 13 below is prepared and mixed by blowing.

[0336] Table 13

[0337] Components Volume (μl) End-of-life repair products 50 Rapid Ligation Buffer 23.4 DNA ligase (rapid DNA ligase) 1.6 DNA adapter 1.9 Ultrapure water (MQ) 3.1

[0338] Slowly pipette 25 μl of the prepared adapter ligation reaction solution into the PCR tube from the previous step. Vortex 6 times for 3 seconds each time, and then briefly centrifuge to collect the reaction solution at the bottom of the tube. Place the tube in a PCR instrument. The optimal reaction temperature for this step is 20°C, and the reaction time is 15 min. After the adapter ligation step, purification is required. The purification procedure is as follows:

[0339] Recover 80 μl of sample, then add 40 μl of Novizan magnetic beads (0.5×), mix by blowing and stirring, then centrifuge at low speed to remove droplets from the tube wall, so that the magnetic beads are evenly dispersed in the solution, and react at room temperature for 10 min.

[0340] Insert the EP tube into the magnetic rack. After the magnetic beads are attracted to the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution, being careful not to pick up the magnetic beads. After the surface of the magnetic beads is dry and without cracks, add 22ul of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 minutes.

[0341] Insert the EP tube into the magnetic frame, and after the magnetic beads are attracted to the tube wall, take 20 μl of solution.

[0342] S304 and dsDNA PCR enrichment and purification steps

[0343] Using the purified product obtained in step S303 as a template, PCR amplification was performed to enrich the target fragment and obtain a DNA sequencing library.

[0344] In this specific process step, the components shown in Table 14 below are added to the PCR tube for PCR amplification.

[0345] Table 14

[0346]

[0347] Place in a PCR tube and proceed under the following conditions:

[0348] Pre-denaturation stage: reaction temperature is 95℃, reaction time is 3 min;

[0349] Denaturation stage: reaction temperature 98℃, reaction time 20s;

[0350] Annealing stage: reaction temperature is 60℃, reaction time is 15s;

[0351] Extension stage: The reaction temperature is 72℃, and the reaction time is 30s;

[0352] Final extension stage: reaction temperature 72℃, reaction time 10 min;

[0353] Storage stage: The reaction temperature is 4℃.

[0354] The PCR process consists of 7 cycles from denaturation to extension, with one cycle each for the pre-denaturation and final extension phases. After PCR amplification is complete, the products are recovered.

[0355] Next, the DNA fragments were purified, and the purification was performed as follows:

[0356] Recover 50 μl of sample, then add 75 μl of Novizan magnetic beads (1.2×), mix by blowing and stirring, then centrifuge at low speed to remove droplets from the tube wall, so that the magnetic beads are evenly dispersed in the solution, and react at room temperature for 10 min.

[0357] Insert the EP tube into the magnetic rack. After the magnetic beads are attracted to the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution, being careful not to pick up the magnetic beads. After the surface of the magnetic beads is dry and without cracks, add 22ul of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 minutes.

[0358] Insert the EP tube into the magnetic frame, and recover the solution after the magnetic beads are attracted to the tube wall.

[0359] S305, DNA circularization step

[0360] The following steps can be taken using Hieff Fast-Pace DNA Cyclization Kit for The reagents in the Fast-Pace DNA Circulation Kit for the sequencing platform are used to perform chain circularization.

[0361] In this step, the double strand is first converted into a single strand, and then the single strand is circularized.

[0362] The specific process steps are as follows: transfer the sample obtained in step S304 to a PCR tube, add MQ to make up to 34ul, and place the reagent on ice to prepare the reaction system shown in Table 15 below.

[0363] Table 15

[0364] name Volume (μl) Step S304 Sample 34 Bridging oligonucleotides (Splint oligo) 6

[0365] After mixing, denature the DNA at 98°C for 3 min in a PCR instrument, immediately place it on ice, incubate on ice for 2 min, and then centrifuge to denature the dsDNA into single strands for subsequent DNA cyclization reaction.

[0366] In the specific process steps, the reaction solution from the previous step is placed on ice to prepare the reaction system shown in Table 16 below.

[0367] Table 16

[0368] name Volume (μl) Previous reactants 40 Splint buffer (linking buffer) 15 DNA ligase 5

[0369] The enzyme was placed in a PCR instrument for chain cyclization reaction. The optimal reaction temperature of the enzyme was 37℃, and the reaction time was 15 min.

[0370] After cyclization, the non-cyclized fragments are digested by enzyme digestion. In the specific process steps, the sample is placed on ice to prepare the reaction system shown in Table 17 below.

[0371] Table 17

[0372] name Volume (μl) Previous reactants 60 DNA digestion buffer 8 DNA Digestion enzyme 2

[0373] The enzyme was placed in a PCR instrument for digestion. The optimal reaction temperature was 37℃, and the reaction time was 10 min. The reacted sample was then purified. The purification procedure is as follows:

[0374] Recover 70 μL of sample, then add 112 μL of Novizan magnetic beads (1.6×), mix by blowing and stirring, centrifuge at low speed to remove droplets from the tube wall, so that the magnetic beads are evenly dispersed in the solution, and react at room temperature for 10 min.

[0375] Insert the EP tube into the magnetic rack. After the magnetic beads are attracted to the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution, being careful not to pick up the magnetic beads. After the surface of the magnetic beads is dry and without cracks, add 22ul of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 minutes.

[0376] Insert the EP tube into the magnetic frame, and recover the solution after the magnetic beads are attracted to the tube wall.

[0377] In this step, DNA is circularized through complementarity between single-stranded DNA and bridging oligonucleotides (Splint oligos).

[0378] S306, circularized DNA amplification

[0379] This step uses phi29 DNA polymerase and random hexamer primers to perform rolling circle amplification, amplifying the circular single-stranded DNA strand obtained in step S305.

[0380] The specific process steps are as follows: transfer the sample from the previous step into a PCR tube as a replication template and prepare the system shown in Table 18 below.

[0381] Table 18

[0382]

[0383] Mix thoroughly by pipetting, centrifuge briefly to the bottom, incubate overnight at 30°C, and then add the reaction solution to 300 μl for later use.

[0384] After DNA circularization using a clip, the gel image used to verify the bands is shown below. Figure 3 As shown, where, Figure 3 The leftmost one is a marker, and the other four are samples after circulation.

[0385] S4, DNA strand fragmentation

[0386] The product obtained in step S306 is fragmented using T7 endonuclease.

[0387] The specific process steps are as follows: Transfer the product obtained in step S306 into a PCR tube, prepare the system shown in Table 19 below, and place it on ice for later use:

[0388] Table 19

[0389] name Volume (μl) T7 endonuclease reaction buffer (NEB buffer 2) 10 T7 endonuclease 5 S306 product 50 Ultrapure water (MQ) 40

[0390] Add 0.5× Novizan magnetic beads to the product, discard the supernatant, add the prepared reaction solution to the magnetic beads, mix well by pipetting, incubate the magnetic beads in a 37°C metal bath for 2 hours with constant shaking, separate the magnetic beads on a magnetic rack, collect the supernatant, and add the magnetic beads for a first purification. Purification is performed as follows:

[0391] 105 μl of sample was recovered, and 52.5 μl of Novizan magnetic beads (0.5×) were added. After mixing by blowing, the liquid droplets on the tube wall were removed by low-speed centrifugation to make the magnetic beads evenly dispersed in the solution. The reaction was carried out at room temperature for 10 min.

[0392] Insert the EP tube into the magnetic rack. After the magnetic beads are attracted to the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution, being careful not to pick up the magnetic beads. After the surface of the magnetic beads is dry and without cracks, add 32 μl of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 min.

[0393] Insert the EP tube into the magnetic frame, and recover the solution after the magnetic beads are attracted to the tube wall.

[0394] S5, Oxford Nanopore Technologies (ONT) library construction

[0395] Library construction was performed using the SQK-LSK109 kit.

[0396] Example 4: Single-chain cyclication scheme

[0397] S1. Obtain the sample chip

[0398] A short ssDNA sequence is synthesized according to requirements. The 5' end of the ssDNA sequence is ligated to a fixation vector, and its length is 20-100 nt. In this embodiment, the sequence of this short ssDNA segment is as follows:

[0399] 5'-TTTTTTTTTT-CCGATCTCTGAGGATGCGCTGAACT-3'.

[0400] S2, add polynucleotide A tail

[0401] Using the short-chain ssDNA obtained in step S1 as a template, a certain amount of polynucleotide tail of a certain length is added to the 3' end of the short-chain ssDNA using a TdT enzyme. The TdT enzyme can be selected from mouse-derived muTdT enzyme, and the tailing rate is controlled at 30-40 nt / min, with a tail length of 8-20 nt.

[0402] The specific steps for this process are as follows: Cut the sample chip to be used for library construction into a 4×4mm square and place it at the bottom of a 1.5mL centrifuge tube. Add the reaction solution prepared according to the components shown in Table 20, ensuring that the reaction solution evenly covers the chip and is free of air bubbles.

[0403] Table 20

[0404] Components Volume (μl) 5× terminal deoxynucleotidyl transferase reaction buffer 20μl Terminal deoxynucleotidyl transferase 5μl dATP (10 uM) 2μl Enzyme-free water Make up to 100μl sample x

[0405] The muTdT enzyme reaction temperature was 30℃, the reaction time was 8-10 min, and it was finally inactivated at 98℃. The supernatant was collected after centrifugation at 12000 rpm for 2 min.

[0406] S3, Obtaining circularized single-stranded DNA S301, 5' end phosphorylation step

[0407] Using the product obtained in step S2 as a template, phosphorylation was performed using T4 polynucleotide kinase (T4 PNK).

[0408] The specific process steps are as follows: Measure the concentration and calculate the input amount of 50 ng, and prepare the system shown in Table 21 below.

[0409] Table 21

[0410] Components Volume (μl) S2 step product DNA (50 ng) x 10× Polynucleotide kinase buffer 2 0.1mM ATP 1 T4 polynucleotide kinase (T4 PNK) 1 Ultrapure water (MQ) To 20ul

[0411] Place it in a PCR instrument. The optimal reaction temperature for this step is 37℃ for 30 minutes, and then react at 75℃ for 10 minutes to inactivate it.

[0412] S302, ssDNA cyclization step

[0413] The 5' end of the purified product obtained in step S301 is linked to the 3' end to circularize the single-stranded DNA.

[0414] In the specific process steps, the reaction system shown in Table 22 is prepared on ice:

[0415] Table 22

[0416]

[0417]

[0418] The sample was placed in a PCR instrument for single-strand cyclization. The optimal temperature for single-strand cyclization was 60°C for 1 hour. Inactivation was performed at 80°C for 10 minutes.

[0419] After ssDNA is circularized, the non-circularized ssDNA is digested using exonucleases. The specific process steps are as follows: Prepare the reaction system shown in Table 23 below on ice:

[0420] Table 23

[0421] The following reaction was performed on a PCR instrument. The optimal temperature for the enzyme reaction was 37℃, and the reaction time was 30 min. For inactivation, the reaction was performed at 80℃ for 20 min. After purification of the circular DNA, a pure sample was obtained. Purification was carried out according to the following steps:

[0422] Recover 21 μL of sample, then add 33.6 μL of Novizan magnetic beads (1.6×), mix by blowing and stirring, then centrifuge at low speed to remove droplets from the tube wall, so that the magnetic beads are evenly dispersed in the solution, and react at room temperature for 10 min.

[0423] Insert the EP tube into the magnetic rack. After the magnetic beads are attracted to the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution, being careful not to pick up the magnetic beads. After the surface of the magnetic beads is dry and without cracks, add 22ul of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 minutes.

[0424] Insert the EP tube into the magnetic frame, and recover the solution after the magnetic beads are attracted to the tube wall.

[0425] S303, circularized DNA amplification

[0426] This step uses phi29 DNA polymerase and random hexamer primers to perform rolling circle amplification, amplifying the circular single-stranded DNA strand obtained in step S302.

[0427] The specific process steps are as follows: transfer the sample from the previous step into a PCR tube as a replication template and prepare the system shown in Table 24 below.

[0428] Table 24

[0430]

[0431]

[0432] Mix thoroughly by pipetting, centrifuge briefly to the bottom, incubate overnight at 30°C, and then add the reaction solution to 300 μl for later use.

[0433] After DNA circularization using a clip, the gel image used to verify the bands is shown below. Figure 4 As shown, where, Figure 4 The leftmost one is a marker, and the other four are samples after circulation.

[0434] S4, DNA strand fragmentation

[0435] The product obtained in step S303 is fragmented using T7 endonuclease.

[0436] The specific process steps are as follows: Transfer the product obtained in step S303 into a PCR tube, prepare the system shown in Table 25 below, and place it on ice for later use:

[0437] Table 25

[0438] name Volume (μl) T7 endonuclease reaction buffer (NEB buffer 2) 10 T7 endonuclease 5 S303 product 50 MQ 40

[0439] Add 0.5× Novizan magnetic beads to the product, discard the supernatant, add the prepared reaction solution to the magnetic beads, mix well by pipetting, incubate the magnetic beads in a 37°C metal bath for 2 hours with constant shaking, separate the magnetic beads on a magnetic rack, collect the supernatant, and add the magnetic beads for a first purification. Purification is performed as follows:

[0440] 105 μl of sample was recovered, and 52.5 μl of Novizan magnetic beads (0.5×) were added. After mixing by blowing, the liquid droplets on the tube wall were removed by low-speed centrifugation to make the magnetic beads evenly dispersed in the solution. The reaction was carried out at room temperature for 10 min.

[0441] Insert the EP tube into the magnetic rack. After the magnetic beads are attracted to the tube wall, remove the solution. Rinse the tube wall with 80% alcohol and remove the solution, being careful not to pick up the magnetic beads. After the surface of the magnetic beads is dry and without cracks, add 32 μl of enzyme-free water and mix the magnetic beads by blowing. React at room temperature for 5 min.

[0442] Insert the EP tube into the magnetic frame, and recover the solution after the magnetic beads are attracted to the tube wall.

[0443] S5, Oxford Nanopore Technologies (ONT) library construction

[0444] Library construction was performed using the SQK-LSK109 kit.

[0445] Experimental Example 2

[0446] This experimental example examines the sequencing accuracy of the short ssDNA library constructed for the third-generation sequencing ONT platform according to this invention.

[0447] Two batches of different sample chips were taken, and short-stranded ssDNA libraries for the third-generation sequencing ONT platform were constructed according to the method of Example 1 of this invention, namely Sample 1-1 and Sample 1-2; two more batches of different sample chips were taken, and short-stranded ssDNA libraries for the third-generation sequencing ONT platform were constructed according to the method of Example 2 of this invention, namely Sample 2-1 and Sample 2-2. The four samples were sequenced using the third-generation sequencing ONT platform, and the results are shown in Table 26:

[0448] Table 26

[0449]

[0450] Combination Figures 3-4 , Figure 3 Electrophoresis images of samples 1-1 (RCA-1, RCA-2) and 1-2 (RCA-3, RCA-4) after DNA circularization were used to verify the results. The results showed that the circularized DNA sequences in each sample were all above 100 bp, which met the experimental requirements. Figure 4 The images are electrophoresis results of rolling circle amplification after single-stranded circularization of samples 2-1 and 2-2. They show that the sequence length of each sample after rolling circle amplification is greater than 1500bp, and the gel images are clean and uniform, meeting the requirements for library construction.

[0451] The results above show that the sequencing accuracy using the ONT platform after library construction using the method of this invention has reached over 96%, even higher than the 90% accuracy of the ONT chip R9.4 results. Therefore, it is understandable that the sequencing library with short-stranded ssDNA fixed in the vector constructed using the method of this invention can not only be applied to sequencing experiments on platforms such as third-generation sequencing, but also achieves detection results that are even superior to the accuracy of existing third-generation sequencing technologies for normal sequences.

[0452] In summary, the method for constructing sequencing libraries, sequencing methods, and applications of short-chain ssDNA provided in this invention solves the technical problems of low sequencing accuracy in direct library construction of short ssDNA in existing technologies by extending short-chain ssDNA and making it circular, and realizes the library construction of special ssDNA, which has good commercial prospects.

[0453] It should be noted that, for those skilled in the art, the technical features in the above embodiments can be freely combined, and the resulting technical solutions also belong to the embodiments disclosed in this application.

[0454] Furthermore, without departing from the principles of this application, several improvements and modifications may be made to this application, and these improvements and modifications also fall within the protection scope of the claims of this application.

Claims

1. A method for constructing a sequencing library of short-chain ssDNA, characterized in that: The 5' end of the short-chain ssDNA is ligated to the immobilization vector, and the 3' end is exposed. The sequencing library construction method includes the following steps: Vector ligation steps: The 5' end of the first ssDNA fragment is ligated to the immobilization vector; Adding a polynucleotide tail: Using the first ssDNA fragment as a template, an enzymatic reaction is performed using terminal deoxynucleotidyl transferase to add a polynucleotide tail to the 3' end of the first ssDNA fragment to obtain tailed ssDNA. PCR amplification steps for tailed ssDNA: Using tailed ssDNA as a template, design the first primer to perform PCR amplification reaction, and purify the amplification product. Sequencing library construction steps: Use the purified amplification product as a template to construct a sequencing library.

2. The sequencing library construction method according to claim 1, characterized in that: The short-chain ssDNA has a length of 20-100 nt.

3. The sequencing library construction method according to claim 1, characterized in that: The sequencing library construction steps include: End repair step: Using the purified amplification product as a template, 5' end repair and 3' end tailing reaction are performed to obtain the repair product; Adapter ligation step: Using the repair product as a template, a ligase is used to react and obtain a template with adapters, which is then purified. PCR enrichment and purification steps for dsDNA: Using the purified product obtained from the adapter ligation step as a template, PCR enrichment and purification are performed to obtain a sequencing library.

4. The sequencing library construction method according to claim 1, characterized in that: The PCR enrichment and purification steps for the dsDNA include: Steps to obtain circularized single-stranded DNA: The tailed short-stranded ssDNA is treated and ligated into a circular form, followed by purification of the circular DNA product, and amplification is performed using the purified product as a template; DNA strand fragmentation step: The product of the step of obtaining circular single-stranded DNA is fragmented using endonuclease and a sequencing library is constructed.

5. The sequencing library construction method according to claim 1, characterized in that: In the step of adding a polynucleotide tail, the polynucleotide is polyadenine deoxyribonucleotide or polyguanine deoxyribonucleotide.

6. The sequencing library construction method according to claim 1, characterized in that: In the polynucleotide tailing step, the tailing reaction rate is controlled at 30-40 nt / min.

7. The sequencing library construction method according to claim 4, characterized in that: The DNA circularization step includes: The dsDNA was denatured into a single strand, and the ssDNA was linked into a circular DNA loop using clips.

8. The sequencing library construction method according to claim 4, characterized in that: The DNA circularization step includes: 5' phosphorylation step: Phosphorylate the 5' end of the tailed short ssDNA; ssDNA circularization step: The phosphorylated ssDNA is circularized so that its 5' end is connected to its 3' end.

9. A sequencing method, the sequencing method comprising the method for constructing a short-strand ssDNA sequencing library as described in any one of claims 1-8.

10. The application of the method for constructing a short-chain ssDNA sequencing library as described in any one of claims 1-8 in the synthesis of short-chain ssDNA.

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