Method for separating nucleic acid sample

Abstract

The invention provides a method for separating a nucleic acid sample, and belongs to the technical field of biology. According to the method provided by the invention, the specific buffer solution is used for cleaning after the magnetic beads are used for separating the target fragments, so that the proportion of small-molecule non-target fragments can be remarkably reduced, and the effective proportion of the target fragments is increased.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and specifically relates to a method for isolating nucleic acid samples. Background Art

[0002] The process of preparing a next-generation sequencing (NGS) library is to transform a target DNA or RNA sample into a structure that can be sequenced on a sequencing platform. Taking the preparation of an NGS library by a conventional mechanical fragmentation method as an example, its steps include: (1) Sample preparation: First, high-quality DNA samples, which are called Insert DNA, need to be collected and prepared; (2) End repair: The Insert DNA is mechanically fragmented, and end repair enzymes are used to modify the fragment ends into a fixed structure; (3) Adapter ligation: The Insert DNA is ligated to a special DNA sequence called an adapter, which allows the fragments to be amplified and sequenced on the sequencing platform; (4) Library amplification: The library ligated with adapters is enriched by PCR to form a library ready for sequencing.

[0003] Among them, in the adapter ligation stage, excessive adapters will form adapter dimers. The structure of the dimer is similar to that of a normal library structure and is very short in length and easy to amplify. Therefore, it will be highly enriched in the library amplification stage, affecting the sequencing of the library ready for sequencing. Specifically, its negative impacts are as follows: (1) Adapter dimers can be highly enriched on the sequencing chip, but do not generate valid data, wasting sequencing costs; (2) Adapter dimers will occupy the chip capacity, affecting the sequencing depth of the data volume; (3) Adapter dimers are short repeat sequences, affecting the laser balance of the sequencing instrument, and thus reducing the quality of the data obtained from the sequencer.

[0004] In the application of targeted sequencing tNGS (target NGS), the first stage is to target and amplify specific fragments from the genome with hundreds to tens of thousands of primer pairs. Further, the products are amplified by Index primers for sequencing to form a complete amplicon library for sequencing. In the first stage of the amplicon library construction process of tNGS, gene-specific Panel primers are required for targeted amplification. Due to the large variety and quantity of primers, at the initial stage of targeted amplification, a large number of primers will form primer dimers through complementary pairing sequences at their 3' ends. The DNA polymerase in the amplification system can also capture such paired products as replication initiation sites for extension amplification. In subsequent multiple amplification cycles, primer dimers are enriched together with specific target products, resulting in a certain proportion of primer dimers in the amplification products and the final library. These primer dimers can also be sequenced, but the generated data is invalid data, which greatly wastes the chips and also has the same negative impact as adapter dimers.

[0005] In the prior art, the research on adapter dimers mainly focuses on the design and usage methods of special adapters. For example, US20170009227A1 discloses a method for reducing adapter dimers. By first ligating Insert DNA with the 3'-end adapter, then adding hairpin DNA to block the redundant 3'-end adapter, and then adding the 5'-end adapter for ligation, the contact between the two ends of the adapter to form dimers is avoided. CN105917002A designs an adapter containing a neck-loop structure and digestible bases. When adapter dimers exist after ligation, a digestive enzyme is used to remove the adapter dimers. WO2022256560A1 discloses the use of free endonucleases for cleavage or inhibition of the generation of adapter dimers during library construction, including T7 Endonuclease I, T4 Endonuclease VII, and Chaetomium thermophilum GEN 1, etc. However, the above methods still have deficiencies as a whole. For example, they require redesigning experimental methods and adapter production processes, have poor compatibility and high usage costs, or use special forms of adapters, which reduce the ligation efficiency compared with Y-shaped adapters. Summary of the Invention

[0006] The present application aims to provide a method for separating nucleic acid samples. By adding a step of washing with a washing solution when using magnetic beads to separate nucleic acid target fragments, the proportion of small molecule non-target fragments can be significantly reduced, and the effective proportion of target fragments can be increased. Further, the present application also provides a method for separating a DNA library. By treating with T7 endonuclease I before amplifying the ligation product and combining with a magnetic bead washing buffer, the effect of removing adapter dimers in the DNA library can be further enhanced.

[0007] A first aspect of the present application provides a method for separating nucleic acid samples, which includes: obtaining a nucleic acid sample containing a target fragment and a non-target fragment, wherein the molecular length of the target fragment is greater than that of the non-target fragment; adding a magnetic bead solution to adsorb the nucleic acid sample; applying an external magnetic field to separate the magnetic beads from the liquid, and removing the supernatant; adding a washing solution, mixing well, and incubating, wherein the washing solution contains 100-1000 mM NaCl and 20-200 mg / ml polyethylene glycol (PEG); applying an external magnetic field to separate the magnetic beads from the liquid, and removing the supernatant; eluting the target fragment on the magnetic beads.

[0008] In some embodiments, the magnetic bead solution contains magnetic beads and a binding buffer.

[0009] In some embodiments, the concentration of the PEG is 20-200 mg / ml, preferably 20-150 mg / ml, including 20 mg / ml, 30 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, and 150 mg / ml within the range.

[0010] In some embodiments, the PEG is polyethylene glycol of any molecular weight known to those of ordinary skill in the art, such as PEG200, PEG300, PEG400, PEG600, PEG1000, PEG2000, PEG4000, PEG5000, PEG6000, and PEG8000, preferably PEG8000.

[0011] In some embodiments, the concentration of the NaCl is 100-1000 mM, preferably 200-800 mM, including 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 320 mM, 325 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 375 mM, 380 mM, 390 mM, 400 mM, 420 mM, 430 mM, 440 mM, 450 mM, 460 mM, 470 mM, 475 mM, 480 mM, 500 mM, 520 mM, 540 mM, 550 mM, 570 mM, 590 mM, 600 mM, 620 mM, 650 mM, 680 mM, 700 mM, 720 mM, 750 mM, 780 mM, and 800 mM within the range.

[0012] In some embodiments, the washing solution further comprises a buffering component.

[0013] In some embodiments, the buffering component is one or more of, for example, Tris, Triton, HEPES, PIPES, and PBS, preferably Tris, more preferably 1-20 mM Tris, including 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, and 20 mM within this range.

[0014] In some embodiments, the washing solution further includes a chelating agent, which is, for example, EDTA, preferably 0.1-10 mM EDTA, including 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM within this range.

[0015] In some embodiments, the washing solution further contains a surfactant, which is one or more of, for example, anionic surfactants, cationic surfactants, and nonionic surfactants.

[0016] In some embodiments, the surfactant is a nonionic surfactant, such as one or more of Tween20, Tween80, NP40, APEO, and AEO, preferably Tween20, more preferably 0.01%-5% v / v Tween20, including 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, and 5% within this range.

[0017] In some embodiments, the target fragment is a DNA library fragment, and the non-target fragment is an adapter dimer and / or a primer dimer.

[0018] In some embodiments, the target fragment is a DNA amplicon, and the non-target fragment is a primer dimer.

[0019] In some embodiments, the amount of the magnetic bead solution is in excess relative to the target fragment, or the magnetic bead solution can adsorb all of the target fragments, or the amount of the magnetic bead solution is sufficient to adsorb all of the target fragments.

[0020] In some embodiments, those of ordinary skill in the art can select the amount of the magnetic bead solution sufficient to adsorb all target fragments according to the size of the target fragments. For example, when the target fragment to be recovered is 350 bp - 500 bp and the non-target fragment is 50 - 100 bp, 0.8X - 1X magnetic bead solution is used; when the target fragment to be recovered is 350 bp - 500 bp and the non-target fragment is 100 - 200 bp, 0.6X - 0.8X magnetic bead solution is used; when the target fragment to be recovered is more than 1000 bp and the non-target fragment is 50 - 500 bp, 0.5X - 0.6X magnetic bead solution is used.

[0021] In some embodiments, 1X magnetic bead solution means that the volume ratio of the magnetic bead solution to the nucleic acid sample is 1.

[0022] In some embodiments, the amount of the magnetic beads is selected based on the amount theoretically capable of distinguishing target fragments and non-target fragments. Actually, the magnetic beads adsorb both the target fragments and the non-target fragments, and the proportion of the adsorbed non-target fragments is, for example, 1 - 40%, including 1%, 2%, 5%, 6%, 8%, 10%, 15%, 16%, 18%, 20%, 21%, 22%, 24%, 25%, 28%, 29%, 30%, 32%, 35%, 38% and 40% within the range.

[0023] In some embodiments, the washing solution washes away all non-target fragments and does not affect the binding of the target fragments and the magnetic beads, or the washing solution washes away all non-target fragments and washes away less than 40% of the target fragments, such as less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%.

[0024] In some embodiments, the size of the non-target fragment is 50 - 500 bp, preferably 50 - 300 bp, including 50 bp, 60 bp, 80 bp, 100 bp, 150 bp, 200 bp, 250 bp and 300 bp within the range.

[0025] In some embodiments, the size of the target fragment is 350 - 5000 bp, such as 350 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1200 bp, 1300 bp, 1400 bp, 1500 bp, 1600 bp, 1800 bp, 1900 bp, 2000 bp, 2200 bp, 2500 bp, 2600 bp, 2800 bp, 3000 bp, 3500 bp, 3800 bp, 4000 bp, 4500 bp and 5000 bp.

[0026] In some embodiments, the target fragment is larger than the non-target fragment, and the difference in molecular length between the target fragment and the non-target fragment is 50 - 2500 bp, including 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp, 1100 bp, 1200 bp, 1300 bp, 1400 bp, 1500 bp, 1600 bp, 1700 bp, 1800 bp, 1900 bp, 2000 bp, 2100 bp, 2200 bp, 2300 bp, 2400 bp, and 2500 bp within this range.

[0027] In some embodiments, the elution includes adding an eluent, which is, for example, an alcohol solution, such as ethanol or isopropanol, for example, 70% - 95% ethanol or isopropanol.

[0028] The second aspect of the present application provides a method for separating a nucleic acid sample, the method comprising: obtaining a nucleic acid sample containing DNA library fragments and adapter dimers, wherein the molecular length of the DNA library fragments is greater than that of the adapter dimers; adding T7 endonuclease I and incubating; adding a magnetic bead solution to adsorb the nucleic acid sample; applying an external magnetic field to separate the magnetic beads from the liquid and removing the supernatant; adding a washing solution to wash the magnetic beads adsorbing the nucleic acid sample, the washing solution containing 100 - 1000 mM NaCl and 20 - 200 mg / ml PEG; applying an external magnetic field to separate the magnetic beads from the liquid and removing the supernatant; and eluting the DNA library fragments on the magnetic beads.

[0029] In some embodiments, the magnetic bead solution contains magnetic beads and a binding buffer.

[0030] In some embodiments, the PEG is polyethylene glycol of any molecular weight known to those of ordinary skill in the art, such as PEG200, PEG300, PEG400, PEG600, PEG1000, PEG2000, PEG4000, PEG5000, PEG6000, and PEG8000, preferably PEG8000.

[0031] In some embodiments, the concentration of the PEG is 20 - 200 mg / ml, preferably 20 - 150 mg / ml, including 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 320 mM, 325 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 375 mM, 380 mM, 390 mM, 400 mM, 420 mM, 430 mM, 440 mM, 450 mM, 460 mM, 470 mM, 475 mM, 480 mM, 500 mM, 520 mM, 540 mM, 550 mM, 570 mM, 590 mM, 600 mM, 620 mM, 650 mM, 680 mM, 700 mM, 720 mM, 750 mM, 780 mM and 800 mM within the range.

[0032] In some embodiments, the concentration of the NaCl is 100 - 1000 mM, preferably 200 - 800 mM, including 300 mM, 320 mM, 325 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 375 mM, 380 mM, 390 mM, 400 mM, 420 mM, 430 mM, 440 mM, 450 mM, 460 mM, 470 mM, 475 mM, 480 mM, 500 mM, 510 mM, 520 mM, 530 mM, 540 mM, 550 mM, 560 mM, 570 mM, 580 mM, 590 mM and 600 mM within the range.

[0033] In some embodiments, the cleaning solution further comprises a buffering component.

[0034] In some embodiments, the buffering component is one or more of, for example, Tris, Triton, HEPES, PIPES and PBS, preferably Tris, more preferably 1 - 20 mM Tris, including 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM and 20 mM within the range.

[0035] In some embodiments, the cleaning solution further comprises a chelating agent, which is, for example, EDTA, preferably 0.1-10 mM of EDTA, including 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM within this range.

[0036] In some embodiments, the cleaning solution further contains a surfactant, which is one or more of, for example, an anionic surfactant, a cationic surfactant, and a non-ionic surfactant.

[0037] In some embodiments, the surfactant is a non-ionic surfactant, such as one or more of Tween20, Tween80, NP40, APEO, and AEO, preferably Tween20, more preferably 0.01%-5% by volume of Tween20, including 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, and 5% within this range.

[0038] In some embodiments, the amount of the magnetic bead solution is in excess relative to the target fragment, or the magnetic bead solution can adsorb all the DNA library fragments, or the amount of the magnetic bead solution is sufficient to adsorb all the target fragments.

[0039] In some embodiments, those of ordinary skill in the art can select the amount of the magnetic bead solution sufficient to adsorb all the DNA library fragments according to the size of the DNA library fragments. For example, when the recovered DNA library fragments are 350 bp-500 bp and the adapter dimers are 50-100 bp, 0.8X-1X magnetic bead solution is used; when the recovered DNA library fragments are 350 bp-500 bp and the adapter dimers are 100-200 bp, 0.6X-0.8X magnetic bead solution is used; when the recovered DNA library fragments are more than 1000 bp and the adapter dimers are 50-500 bp, 0.5X-0.6X magnetic bead solution is used.

[0040] In some embodiments, the amount of magnetic beads is selected such that it can theoretically distinguish DNA library fragments from adapter dimers. In fact, the magnetic beads adsorb both the DNA library fragments and the adapter dimers simultaneously, and the proportion of the adsorbed adapter dimers is, for example, 1-40%, including 1%, 2%, 5%, 6%, 8%, 10%, 15%, 16%, 18%, 20%, 21%, 22%, 24%, 25%, 28%, 29%, 30%, 32%, 35%, 38%, and 40% within this range.

[0041] In some embodiments, the washing solution washes away all non-target fragments without affecting the binding of the target fragments to the magnetic beads, or the washing solution washes away all non-target fragments and washes away less than 40% of the target fragments, such as less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, and less than 5%.

[0042] In some embodiments, the DNA library fragments are library fragments suitable for next-generation sequencing (NGS), and the size of the DNA library fragments is, for example, 350-500 bp.

[0043] In some embodiments, the DNA library fragments are library fragments suitable for third-generation sequencing (TGS, such as Pacbio platform and ONT platform), and the size of the DNA library fragments is, for example, greater than 1000 bp, such as 2000 bp, 3000 bp, 5000 bp, 10 kbp, 20 kbp or more.

[0044] In some embodiments, the size of the adapter dimer is 50-500 bp, preferably 50-300 bp, including 50 bp, 60 bp, 80 bp, 100 bp, 150 bp, 200 bp, 250 bp, and 300 bp within this range.

[0045] In some embodiments, the working concentration of T7 endonuclease I is 0.1-1 U / μl, including 0.1 U / μl, 0.2 U / μl, 0.3 U / μl, 0.4 U / μl, 0.5 U / μl, 0.6 U / μl, 0.7 U / μl, 0.8 U / μl, 0.9 U / μl, and 1 U / μl within this range.

[0046] In some embodiments, the incubation temperature of the T7 endonuclease I is 30 - 42 °C, including 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, and 42 °C within this range; the incubation time of the T7 endonuclease I is 5 - 20 minutes, including 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, and 20 minutes within this range; including, but not limited to, any combination of the aforementioned incubation temperatures and incubation times.

[0047] In some embodiments, the T7 endonuclease I can be added alone to the nucleic acid sample for reaction, or added to the nucleic acid sample together with the amplification reagent and reacted before the amplification step.

[0048] In some embodiments, the elution includes adding an eluent, which is, for example, an alcohol solution, such as ethanol or isopropanol, for example, 70% - 95% ethanol or isopropanol.

[0049] The third aspect of the present application provides a library construction method, which includes obtaining a nucleic acid template and constructing a DNA library. The library contains target fragments and non-target fragments. The target fragments are DNA library fragments, and the non-target fragments are adapter dimers and / or primer dimers; using the method described in the first aspect to recover the DNA library.

[0050] In some embodiments, the mass of the nucleic acid template is at least 100 pg, at least 500 pg, at least 1 ng, at least 5 ng, at least 10 ng, at least 50 ng, at least 100 ng, at least 200 ng, at least 300 ng, at least 400 ng, or at least 500 ng.

[0051] In some embodiments, the method for constructing the library is any library construction method known to those of ordinary skill in the art, such as library construction by transposase method, amplicon library construction, TA ligation adapter library construction, blunt-end ligation adapter library construction, library construction by Nanopore technology, and library construction by PacBio technology.

[0052] In some embodiments, the method further includes amplifying the DNA library.

[0053] In some embodiments, the method further includes using the method described in the first aspect to recover the amplified library.

[0054] The fourth aspect of the present application provides a library construction method, which includes obtaining a nucleic acid template and constructing a DNA library. The library contains target fragments and non-target fragments. The target fragments are DNA library fragments, and the non-target fragments are adapter dimers; the DNA library is recovered using the method described in the second aspect.

[0055] In some embodiments, the quality of the nucleic acid template is at least 100 pg, at least 500 pg, at least 1 ng, at least 5 ng, at least 10 ng, at least 50 ng, at least 100 ng, at least 200 ng, at least 300 ng, at least 400 ng, or at least 500 ng.

[0056] In some embodiments, the method for constructing the library is TA ligation adapter library construction and blunt-end ligation adapter library construction.

[0057] In some embodiments, the method further includes amplifying the DNA library.

[0058] In some embodiments, the method further includes recovering the amplified library using the method described in the second aspect.

[0059] The fifth aspect of the present application provides a kit for implementing the method described in the first aspect or the second aspect.

[0060] In some embodiments, the kit contains a magnetic bead solution and a washing solution, and the washing solution contains 100 - 1000 mM NaCl and 20 - 200 mg / ml PEG.

[0061] In some embodiments, the PEG is polyethylene glycol with any molecular weight known to those of ordinary skill in the art, such as PEG200, PEG300, PEG400, PEG600, PEG1000, PEG2000, PEG4000, PEG5000, PEG6000, and PEG8000, preferably PEG8000.

[0062] In some embodiments, the concentration of the PEG is 20 - 200 mg / ml, preferably 20 - 150 mg / ml, including 20 mg / ml, 30 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, and 150 mg / ml within this range.

[0063] In some embodiments, the concentration of the NaCl is 100 - 1000 mM, preferably 200 - 800 mM, including 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 320 mM, 325 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 375 mM, 380 mM, 390 mM, 400 mM, 420 mM, 430 mM, 440 mM, 450 mM, 460 mM, 470 mM, 475 mM, 480 mM, 500 mM, 520 mM, 540 mM, 550 mM, 570 mM, 590 mM, 600 mM, 620 mM, 650 mM, 680 mM, 700 mM, 720 mM, 750 mM, 780 mM, and 800 mM within the range.

[0064] In some embodiments, the cleaning solution further comprises a buffering component.

[0065] In some embodiments, the buffering component is one or more of, for example, Tris, Triton, HEPES, PIPES, and PBS, preferably Tris, more preferably Tris at 1 - 20 mM, including 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, and 20 mM within the range.

[0066] In some embodiments, the cleaning solution further includes a chelating agent, and the chelating agent is, for example, EDTA, preferably EDTA at 0.1 - 10 mM, including 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM within the range.

[0067] In some embodiments, the cleaning solution further contains a surfactant, and the surfactant is one or more of, for example, anionic surfactants, cationic surfactants, and non - ionic surfactants.

[0068] In some embodiments, the surfactant is a non-ionic surfactant, such as one or more of Tween20, Tween80, NP40, APEO, and AEO, preferably Tween20, and more preferably Tween20 with a volume fraction of 0.01%-5%, including 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, and 5% within the range.

[0069] In some embodiments, the kit further comprises T7 endonuclease I.

[0070] The sixth aspect of the present application provides a kit for implementing the method described in the third or fourth aspect.

[0071] In some embodiments, the kit comprises a magnetic bead solution and a washing solution, and the washing solution comprises 100-1000 mM NaCl and 20-200 mg / ml PEG.

[0072] In some embodiments, the PEG is polyethylene glycol with any molecular weight known to those of ordinary skill in the art, such as PEG200, PEG300, PEG400, PEG600, PEG1000, PEG2000, PEG4000, PEG5000, PEG6000, and PEG8000, preferably PEG8000.

[0073] In some embodiments, the concentration of the PEG is 20-200 mg / ml, preferably 20-150 mg / ml, including 20 mg / ml, 30 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, and 150 mg / ml within the range.

[0074] In some embodiments, the concentration of the NaCl is 100 - 1000 mM, preferably 200 - 800 mM, including 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, 290 mM, 300 mM, 320 mM, 325 mM, 330 mM, 340 mM, 350 mM, 360 mM, 370 mM, 375 mM, 380 mM, 390 mM, 400 mM, 420 mM, 430 mM, 440 mM, 450 mM, 460 mM, 470 mM, 475 mM, 480 mM, 500 mM, 520 mM, 540 mM, 550 mM, 570 mM, 590 mM, 600 mM, 620 mM, 650 mM, 680 mM, 700 mM, 720 mM, 750 mM, 780 mM, and 800 mM within the range.

[0075] In some embodiments, the cleaning solution further contains a buffering component.

[0076] In some embodiments, the buffering component is one or more of, for example, Tris, Triton, HEPES, PIPES, and PBS, preferably Tris, and more preferably 1 - 20 mM Tris, including 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, and 20 mM within the range.

[0077] In some embodiments, the cleaning solution further includes a chelating agent, and the chelating agent is, for example, EDTA, preferably 0.1 - 10 mM EDTA, including 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, and 10 mM within the range.

[0078] In some embodiments, the cleaning solution further contains a surfactant, and the surfactant is one or more of, for example, an anionic surfactant, a cationic surfactant, and a non - ionic surfactant.

[0079] In some embodiments, the surfactant is a non-ionic surfactant, such as one or more of Tween20, Tween80, NP40, APEO, and AEO, preferably Tween20, more preferably Tween20 with a volume fraction of 0.01% - 5%, including 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, and 5% within the range.

[0080] In some embodiments, the kit further comprises T7 endonuclease I.

[0081] In some embodiments, the kit further includes all or part of the reagents required for library construction, such as fragmentation reagents, end-filling reagents, library amplification reagents, and library adapters, and the library adapter is, for example, the Y-shaped adapter of the Illumina platform. Description of the Drawings

[0082] Figure 1 : Schematic diagram of the principle of cleaning adapter dimers with Clean Buffer;

[0083] Figure 2-1 : Removal effect of different proportions of 60bp small fragments in the 380bp main library using Control Group 1 and Control Group 2;

[0084] Figure 2-2 : Removal effect of different proportions of 60bp small fragments in the 380bp main library using Control Group 3 and Experimental Group 1;

[0085] Figure 2-3 : Removal effect of different proportions of 60bp small fragments in the 380bp main library using Experimental Group 2 and Experimental Group 3;

[0086] Figure 3-1 : Removal effect of different proportions of 100bp small fragments in the 380bp main library using Control Group 1 and Control Group 2;

[0087] Figure 3-2 : Removal effect of different proportions of 100bp small fragments in the 380bp main library using Control Group 3 and Experimental Group 1;

[0088] Figure 3-3 : Removal effect of different proportions of 100bp small fragments in the 380bp main library using Experimental Group 2 and Experimental Group 3;

[0089] Figure 4-1: Removal effects of different proportions of 150bp small fragments in the 380bp main library using Control Group 1 and Control Group 2;

[0090] Figure 4-2 : Removal effects of different proportions of 150bp small fragments in the 380bp main library using Control Group 3 and Experimental Group 1;

[0091] Figure 4-3 : Removal effects of different proportions of 150bp small fragments in the 380bp main library using Experimental Group 2 and Experimental Group 3;

[0092] Figure 5-1 : Removal effects of T7 endonuclease I digestion on adapter dimers in the ADL(1 / 5) and ADL(1 / 10) library models;

[0093] Figure 5-2 : Removal effects of T7 endonuclease I digestion on the ADL(1 / 20) and ADL(1 / 50) library models;

[0094] Figure 6 : Library output obtained by digesting adapter dimers contained in the 380bp main library with T7 endonuclease I;

[0095] Figure 7 : Proportion of adapter dimers obtained by digesting adapter dimers contained in the 380bp main library with T7 endonuclease I;

[0096] Figure 8-1 : Removal effects of T7 endonuclease I in combination with Clean Buffer a-c on adapter dimers in the ADL(1 / 5) and ADL(1 / 10) library models;

[0097] Figure 8-2 : Removal effects of T7 endonuclease I in combination with Clean Buffer a-c on adapter dimers in the ADL(1 / 20) and ADL(1 / 50) library models;

[0098] Figure 9 : Library output obtained by combining T7 endonuclease I with Clean Buffer a-c;

[0099] Figure 10 : Proportion of adapter dimers obtained by combining T7 endonuclease I with Clean Buffer a-c;

[0100] Figure 11 : Removal effects of T7 endonuclease I in combination with Clean Buffer b on adapter dimers at different DNA input amounts;

[0101] Figure 12-1 : Removal effect of adapter dimers in ADL(1 / 5) and ADL(1 / 10) library models by AMPure XP Beads combined with Clean Buffer b;

[0102] Figure 12-2 : Removal effect of adapter dimers in ADL(1 / 20) and ADL(1 / 50) library models by AMPure XP Beads combined with Clean Buffer b;

[0103] Figure 13-1 : Removal effect of adapter dimers in ADL(1 / 5) and ADL(1 / 10) library models by Clean Buffer d;

[0104] Figure 13-2 : Removal effect of adapter dimers in ADL(1 / 20) and ADL(1 / 50) library models by Clean Buffer d;

[0105] Figure 14 : Removal effect of adapter dimers by Clean Buffer e;

[0106] Figure 15 : Electrophoresis diagram of the removal effect of different Clean Buffers on small fragments of 250 - 500bp in 3000bp long fragments.

[0107] Specific implementation mode (Example)

[0108] The technical solution of the present application will be further described below in conjunction with the accompanying drawings and through specific implementation modes. However, the following examples are only simple examples of the present application and do not represent or limit the scope of the protection of the rights of the present application. The scope of protection of the present application shall be subject to the claims.

[0109] In the following examples, unless otherwise specified, the reagents and consumables used are purchased from conventional reagent manufacturers in the art; unless otherwise specified, the experimental methods and technical means used are conventional methods and means in the art.

[0110] Example 1

[0111] Removal of specific small fragments by Clean Buffer.

[0112] First, 1 μg of fragmented salmon DNA was used to construct a library with Vazyme#ND607 to obtain a PCR-free library (Standard Library, the main peak of the library was at 380 bp). The library construction process was carried out according to the Vazyme#ND607 instruction manual, which specifically included three steps: end repair, adapter ligation, and library amplification. At the same time, using the 293T genome as a template, specific fragment products with sizes of 60 bp, 100 bp, and 150 bp were obtained by PCR amplification respectively to simulate dimers of different sizes, such as primer dimers. The amplification primers for preparing specific small fragments are shown in Table 1 below:

[0113] Table 1

[0114]

[0115]

[0116] Specific small fragments of different sizes were mixed with the standard library in different proportions to obtain a library model containing simulated dimers of different proportions and different sizes. The removal effect of Clean Buffer on small fragments of simulated dimers was tested in the aforementioned model. The naming rule for the constructed library test model was as follows: the high dimer template containing 60 bp simulated dimer was named high 60, the medium dimer template containing 60 bp simulated dimer was named medium 60, the low dimer template containing 60 bp simulated dimer was named low 60, and so on. The specific preparation method is shown in Table 2 below:

[0117] Table 2

[0118] Design template Simulated dimer mass Simulated library mass Ratio Volume after mixing High dimer template 600 ng (60 bp / 100 bp / 150 bp) 300 ng 2:1 20 μl Medium dimer template 450 ng (60 bp / 100 bp / 150 bp) 450 ng 1:1 20 μl Low dimer template 300 ng (60 bp / 100 bp / 150 bp) 600 ng 1:2 20 μl

[0119] The 9 test models prepared above were respectively recovered using magnetic beads in different proportions. The magnetic bead recovery reagent used in this example was Vazyme#N411. Referring to the purification conditions in the instruction manual, for samples with a 380 bp target fragment, 0.6× - 1× magnetic beads (the use of magnetic beads refers to the volume of the recovered sample, where 1× represents using magnetic beads equal to 1 times the sample system) were used for recovery, and further elution was carried out with Clean Buffer on the basis of 1× magnetic beads. The CleanBuffer used in the experiment included a, b, and c, and the specific main components were as follows:

[0120] The components of Clean Buffer a were 3 mM Tris, 0.5 mM EDTA, 0.05% Tween20, 500 mM NaCl, and 100 mg / ml PEG8000;

[0121] The components of Clean Buffer b are 3 mM Tris, 0.5 mM EDTA, 0.05% Tween20, 440 mM NaCl, and 90 mg / ml PEG8000;

[0122] The components of Clean Buffer c are 3 mM Tris, 0.5 mM EDTA, 0.05% Tween20, 375 mM NaCl, and 75 mg / ml PEG8000.

[0123] The control group and the experimental groups are set as shown in Table 3 (the specific volume depends on the ratio of magnetic beads used in different experimental groups in the experimental protocol; for example, for 1× magnetic beads, the volume of magnetic beads used is 1 times the volume of the sample to be used and recovered).

[0124] Table 3

[0125]

[0126]

[0127] The magnetic bead recovery process can refer to the instruction manual of Vazyme#N411. Specifically, for the use of Clean Buffer, after the magnetic beads bind to DNA and before eluting with 75 - 80% ethanol, for each sample, 50 μl of Clean Buffer is used to resuspend the magnetic beads bound with DNA. After gently pipetting to mix evenly, it is placed back on the magnetic stand for adsorption and precipitation, and the subsequent operation steps are completed. The specific details are as follows:

[0128] 1.1: Take out the Vazyme#N411 magnetic bead solution and Clean Buffer a - c from 2 - 8°C 30 minutes in advance, and let it stand to equilibrate the temperature to room temperature.

[0129] 1.2: Invert or vortex - oscillate to fully mix the magnetic bead solution. According to the settings in Table 3, respectively pipette the N411 magnetic bead solution into the DNA samples, and gently pipette 10 times with a pipette to mix evenly.

[0130] 1.3: Incubate at room temperature for 5 minutes to allow the DNA to bind to the magnetic beads.

[0131] 1.4: Place the sample on the magnetic stand. After the solution becomes clear (about 5 minutes), carefully remove the supernatant.

[0132] 1.5: Remove the PCR tubes of the experimental groups from the magnetic stand. For the control groups 1 - 3, no treatment is performed. For the experimental groups 1 - 3, add 50 μl of Clean Buffer a - c respectively, and gently pipette 10 times with a pipette to mix evenly. Incubate at room temperature for 3 minutes.

[0133] 1.6: Place the experimental group samples on a magnetic stand. After the experimental group solution becomes clear (about 3 min), carefully remove the supernatant (Note: Steps 1.5 and 1.6 are not performed on the control group).

[0134] 1.7: Keep the samples on the magnetic stand all the time. Add 200 μl of freshly prepared 80% ethanol to wash the magnetic beads, incubate at room temperature for 30 sec, and carefully remove the supernatant.

[0135] 1.8: Repeat Step 1.7 once for a total of two washes.

[0136] 1.9: Keep the samples on the magnetic stand all the time and dry the magnetic beads with the lid open at room temperature for about 5 - 10 min.

[0137] 1.10: Remove the samples from the magnetic stand, add an appropriate amount of nuclease - free water, vortex or pipette to mix well, and let stand at room temperature for 2 min. Let stand on the magnetic stand for 5 min until the solution becomes clear, then carefully aspirate the supernatant into a new nuclease - free centrifuge tube. The purified product can be stored at - 20°C for a long time.

[0138] In the library quality control step, the Vazyme#EQ121 is used to detect the product concentration, and the measurement steps are carried out according to the Vazyme#EQ121 instruction manual. At the same time, the Houze Bio Qsep instrument is used to detect the distribution range of the library as Figure 2-1 、 Figure 2-2 、 Figure 2-3 、 Figure 3-1 、 Figure 3-2 、 Figure 3-3 、 Figure 4-2 、 Figure 4-2 and Figure 4-3 shown.

[0139] The calculation method of the adapter dimer ratio is as follows: The adapter dimer length is between 130 bp - 170 bp, and the main library is distributed between 200 bp - 1000 bp. The number of molecules in each interval is calculated by the instrument. The number of molecules in the dimer interval / the number of molecules in the main library interval = the dimer ratio. The statistical results are shown in Tables 4 - 6.

[0140] Table 4 DNA recovery amounts under different conditions in a 380 - bp library containing 60 - bp small fragments

[0141] Control group 1 Control group 2 Control group 3 Experimental group 1 Experimental group 2 Experimental group 3 60 - High dimer 436 370 178 402 368 264 60 - Medium dimer 444 372 172 398 348 246 60 - Low dimer 412 366 170 396 376 248

[0142] Table 5 DNA recovery amounts under different conditions in a 380 - bp library containing 100 - bp small fragments

[0143] Control group 1 Control group 2 Control group 3 Experimental group 1 Experimental group 2 Experimental group 3 100 - High dimer 448 398 120 428 366 244 100 - Medium dimer 428 378 170 446 368 256 100 - Low dimer 468 370 174 438 352 248

[0144] Table 6 shows the DNA recovery amounts in the 380bp library with 150bp small fragments under different conditions

[0145] CleanBuffer Control group 1 Control group 2 Control group 3 Experimental group 1 Experimental group 2 Experimental group 3 150 - High dimer 424 360 178 408 348 222 150 - Medium dimer 428 376 176 436 376 230 150 - Low dimer 448 396 174 410 364 270

[0146] Result analysis:

[0147] As Figure 2-1 、 Figure 2-2 、 Figure 2-3 、 Figure 3-1 、 Figure 3-2 、 Figure 3-3 、 Figure 4-1 、 Figure 4-2 and Figure 4-3 shown, under the dimer models with different fragment sizes and ratios, in Control Group 1 - Control Group 3, using less magnetic beads for recovery can reduce the ratio of dimers, but still cannot completely remove dimers, and will change the peak shape of the main library. Especially when the molecular size of the target library is relatively close to that of the non - target dimer fragments, reducing only the amount of magnetic beads to remove non - target products will also result in a large loss of target products.

[0148] Compared with Control Group 1, in Experimental Groups 1, 2, and 3 using three kinds of Clean Buffer a, b, and c, the ratio of dimers can be significantly reduced. Among them, Experimental Group 2 using Clean Buffer b has the best effect, which can completely remove dimers without affecting the peak shape of the main library.

[0149] Example 2

[0150] First, establish an adapter dimer model. Use 1μg fragmented salmon DNA and perform library construction with Vazyme#ND607 to obtain a PCR - free library (Standard Library). The library construction process follows the Vazyme#ND607 instruction manual, specifically including two steps: end repair and adapter ligation.

[0151] Meanwhile, without using a template, only use Vazyme#N801 adapter for ligation. Specifically, follow the library construction process in the Vazyme#ND607 instruction manual, including two steps: end repair and adapter ligation. After ligation, use 2× magnetic beads (Vazyme#N411) for purification to obtain a PCR - free adapter dimer (Adapter Dimmer).

[0152] The PCR-free adapter dimers and the PCR-free libraries were mixed at mass ratios of 1 / 1, 1 / 2, 1 / 5, 1 / 10, 1 / 20, and 1 / 50, respectively. The mixed products were named ADL (Adapter Dimer Library). Among them, ADL(1 / 1), ADL(1 / 2), and ADL(1 / 5) are PCR-free libraries with high dimer content, and ADL(1 / 10), ADL(1 / 20), and ADL(1 / 50) are PCR-free libraries with low dimer content. Two methods were used to remove adapter dimers: digestion with T7 endonuclease I and digestion with Clean Buffer (prepared in the same way as in Example 1). The specific experimental settings are shown in Table 7 below.

[0153] Table 7

[0154]

[0155] 1. Digestion treatment of adapter dimers

[0156] T7 endonuclease I (Vazyme#EN303, T7 Endonuclease I) was used as the enzyme for digesting adapter dimers. T7 endonuclease I with a concentration of 25 U / μl was prepared and named T7 EndoA. Similarly, T7 EndoB with a concentration of 40 U / μl and T7 EndoC with a concentration of 50 U / μl were prepared. The library amplification systems for different concentrations of T7 endonuclease I experimental groups were prepared as shown in Table 8 below, and adapter dimer digestion was carried out. Among them, the working concentrations of T7 EndoA, T7 EndoB, and T7 EndoC in the system were 0.5 U / μl, 0.8 U / μl, and 1 U / μl, respectively.

[0157] Library amplification was carried out according to the procedure in Table 9. Among them, 37°C for 10 min in Table 9 was the condition for T7 endonuclease I to function. The library amplification systems of the control group and the Clean Buffer experimental group were the same in other components except that they did not contain T7 endonuclease I. The corresponding amplification procedures removed the first step of 37°C for 10 min based on Table 9.

[0158] Table 8

[0159]

[0160] Table 9

[0161]

[0162] 2. Cleaning treatment of adapter dimers

[0163] The control group and the T7 endonuclease I experimental group were only recovered using 0.8× magnetic beads (Vazyme#N411). The CleanBuffer experimental group was first recovered using 0.8× magnetic beads (Vazyme#N411), and at the same time, Clean Buffer was prepared for further elution of adapter dimers. The operation steps of magnetic bead recovery and the preparation method were the same as those in Example 1. Further, library quality control and data analysis were the same as those in Example 1.

[0164] Result analysis:

[0165] As Figure 5-1 、 Figure 5-2 、 Figure 6 and Figure 7 shown: Only using T7 endonuclease I for testing can reduce the proportion of adapter dimers. It can significantly reduce adapter dimers under the usage condition of 0.8 U / μl, and has a relatively small impact on the target library.

[0166] As Figure 8-1 、 Figure 8-2 、 Figure 9 and Figure 10 shown, in the library containing adapter dimers, using Clean Buffer can also significantly reduce the proportion of adapter dimers, and the highest can reach more than 80%. By comprehensively comparing the removal effects of CleanBuffer a - c on adapter dimers, it can be seen that Clean Buffer c has the strongest removal effect, and Clean Buffer a has the least loss on the normal library. Considering the loss of the comprehensive library and the removal effect, Clean Buffer b has the best effect, which can not only significantly reduce adapter dimers but also retain the target library to the greatest extent, indicating that using CleanBuffer can achieve the same technical effect as T7 endonuclease I.

[0167] Example 3

[0168] Test the removal effect of the combination of T7 endonuclease I and Clean Buffer on adapter dimers.

[0169] To further test whether the combination of T7 endonuclease I and Clean Buffer can achieve better adapter dimer removal effect during the library construction process of real samples, the library construction test was carried out using the conventional DNA library construction kit Vazyme#ND607. 1 ng and 500 ng of 293T gDNA were respectively input and ligated with sequencing adapters (Vazyme#N801) for library construction. The specific library construction process was carried out according to the instructions of Vazyme#ND607 (the usage of the adapter was undiluted), including three steps: end repair, adapter ligation, and library amplification. Among them, the first magnetic bead recovery was carried out after the adapter ligation was completed, and the second magnetic bead recovery was carried out after the library amplification. The specific experimental scheme is shown in Table 10 below:

[0170] Table 10

[0171]

[0172] Among them, the magnetic bead recovery steps involved in the specific library construction process, as well as the preparation of Clean Buffer b and T7 Endo A, were the same as those in Examples 1-2; the treatment method of T7 Endo A was the same as that in Example 2. The subsequent library quality inspection steps were the same as those in Example 1, and the library output was counted as shown in Table 11.

[0173] Table 11

[0174]

[0175] As Figure 11 shown, it can be seen that when using real samples to ligate with undiluted adapters for library construction, higher adapter dimers will appear in the final library. In the same library construction system, the use of T7 EndoA and Clean Buffer b can completely remove the adapter dimers generated during the library construction process, and can also achieve the construction of libraries with a low input amount of 1 ng DNA template, improving the library construction efficiency. Further, the method provided in this application can also achieve a unified library construction process for DNA library construction experiments with different contents, thus realizing automated operation.

[0176] Example 4

[0177] Test the effect of the combination of Clean Buffer and AMPure XP Beads (Beckman#A63880) on the removal of adapter dimers.

[0178] The test model and experimental operation process are the same as those in Example 2. Specifically, the difference between this example and Example 2 is that in the cleaning treatment step of the adapter dimer, 0.8×AMpure XP Beads (Beckman #A63880) is used to replace Vazyme #N411 for recovery. The cleaning solution used in this example is Clean Buffer b (the components are the same as those in Example 1), and the library peak diagram obtained is as Figure 12-1 and Figure 12-2 shown.

[0179] As Figure 12-1 and Figure 12-2 shown, using Clean Buffer b in combination with AMpure XP Beads to recover DNA can also well remove small fragment adapter dimers, indicating that the method of this application can be applicable to magnetic beads of various brands.

[0180] Example 5

[0181] Test the removal effect of Clean Buffer with different core components on dimers.

[0182] The test model and experimental operation process are the same as those in Example 2. The specific components of Clean Buffer d used in this example are: Tris 3 mM, EDTA 0.5 mM, Tween 20 0.05%, NaCl 440 mM, PEG5000 90 mg / ml. Other experimental conditions are the same as those in Example 2, and the library peak diagram obtained is as Figure 13-1 and Figure 13-2 shown.

[0183] As Figure 13-1 and Figure 13-2 shown, Clean Buffer d formulated based on PEG5000 can also well remove adapter dimers with different proportions.

[0184] Example 6

[0185] Test the removal effect of the core components in Clean Buffer on dimers.

[0186] The test model in this example is the same as ADL(1 / 5) and ADL(1 / 20) in Example 2. The specific components of Clean Buffer e used in this example are: NaCl 440 mM, PEG8000 90 mg / ml.

[0187] The specific experimental operation process is the same as that in Example 2, and the library peak diagram obtained is as Figure 14 shown.

[0188] As Figure 14As shown, Clean Buffer e containing only the core components NaCl and PEG8000 can also remove dimers in both the high-dimer ADL(1 / 5) and low-dimer ADL(1 / 20) systems, indicating that the presence of NaCl and PEG8000 in Clean Buffer is sufficient to achieve the effect of removing small-fragment dimers.

[0189] Example 7

[0190] To further test the removal effect of Clean Buffer on small fragments in a large-fragment library, 10 ng of 293T gDNA was used as a template and amplified using Vazyme#NA301 to obtain amplification products of 237 bp, 392 bp, 528 bp, and 3703 bp, respectively. The primer sequences are shown in Table 12, the amplification system is shown in Table 13, the amplification programs for the 237 bp, 392 bp, and 528 bp amplification products are shown in Table 14, and the amplification program for the 3703 bp amplification product is shown in Table 15. After amplification, the amplification products of different fragment sizes were mixed in equal volumes to obtain a mixed simulated sample, which was purified using Vazyme#N001. Among them, 3703 bp is the target fragment, and the 237 bp, 392 bp, and 528 bp products are interfering non-target small fragments. The mixed simulated sample was further recovered, and 1x magnetic beads (Vazyme#N411) were used to match different ratios of Clean Buffer b (1×, with the same components as in Example 1).

[0191] Table 12

[0192]

[0193] Table 13

[0194]

[0195]

[0196] Table 14

[0197]

[0198] Table 15

[0199]

[0200] The purification operation of the recycled mixed simulation samples was the same as that in Example 1. Among them, Control Group 1 was set to purify the mixed simulation samples only using 1× magnetic beads, Control Group 2 was the unpurified mixed simulation samples, Control Group 3 was the unpurified 3703bp library target fragment, and Control Group 4 was the unpurified 237bp, 392bp, and 528bp non-target small fragments; Test Group 1 was the mixed simulation samples treated with 1× magnetic bead purification + 0.9× Clean Buffer b washing; Test Group 2 was the mixed simulation samples treated with 1× magnetic bead purification + 0.8× Clean Buffer washing.

[0201] The samples of the aforementioned Control Groups 1-4 and Test Groups 1-2 were subjected to gel electrophoresis, and the results were as Figure 15 shown. The non-target small fragment bands in Test Group 1 and Test Group 2 were cleared to varying degrees, and among them, the effect of Test Group 2 was the best, and the non-target small fragment band almost disappeared. This result indicates that Clean Buffer also has a good recovery effect on long fragments >1000bp, indicating that its application scenario is not limited to the fragment size of magnetic bead purification and has the potential to be extended to third-generation sequencing.

Claims

1. A method for separating nucleic acid samples, comprising: Obtain a nucleic acid sample, wherein the nucleic acid sample contains a target fragment and a non-target fragment, and the molecular length of the target fragment is greater than that of the non-target fragment; Add a magnetic bead solution to adsorb the nucleic acid sample; apply an external magnetic field to separate the magnetic beads from the liquid, and remove the supernatant; add a washing solution to wash the magnetic beads adsorbed with the nucleic acid sample, and the washing solution contains 100-1000 mM NaCl and 20-200 mg / ml polyethylene glycol (PEG); apply an external magnetic field to separate the magnetic beads from the liquid, and remove the supernatant; elute the target fragment on the magnetic beads.

2. A method for separating nucleic acid samples, comprising: Obtain a nucleic acid sample, wherein the nucleic acid sample contains DNA library fragments and adapter dimers, and the molecular length of the DNA library fragments is greater than that of the adapter dimers; Add T7 endonuclease I and incubate; Add a magnetic bead solution to adsorb the nucleic acid sample; apply an external magnetic field to separate the magnetic beads from the liquid, and remove the supernatant; add a washing solution to wash the magnetic beads adsorbed with the nucleic acid sample, and the washing solution contains 100-1000 mM NaCl and 20-200 mg / ml PEG; apply an external magnetic field to separate the magnetic beads from the liquid, and remove the supernatant; elute the DNA library fragments on the magnetic beads.

3. The method according to claim 1 or 2, wherein the concentration of the PEG is 20-150 mg / ml.

4. The method according to claim 1 or 2, wherein the concentration of the NaCl is 200-800 mM.

5. The method according to claim 1 or 2, wherein the washing solution further contains a buffering component, and the buffering component is one or more of Tris, Triton, HEPES, PIPES and PBS, preferably Tris, more preferably 1-20 mM Tris.

6. The method according to claim 1 or 2, wherein the washing solution further includes a chelating agent, and the chelating agent is EDTA, preferably 0.1-10 mM EDTA.

7. The method according to claim 1 or 2, wherein the washing solution further contains a surfactant, and the surfactant is one or more of an anionic surfactant, a cationic surfactant and a nonionic surfactant; preferably, the surfactant is a nonionic surfactant; preferably, the nonionic surfactant is one or more of Tween20, Tween80, NP40, APEO and AEO, preferably Tween20, more preferably 0.01%-5% by volume of Tween20.

8. The method according to claim 1 or 2, wherein the elution includes adding an elution solution, and the elution solution is an alcohol solution; preferably, the alcohol solution is ethanol or isopropanol, preferably 70%-95% ethanol or isopropanol.

9. The method according to claim 1, wherein the target fragment is a DNA library, the non-target fragment is an adapter dimer and / or a primer dimer, or, the target fragment is a DNA amplicon, and the non-target fragment is a primer dimer.

10. The method according to claim 1, wherein the size of the non-target fragment is 50-500 bp, and the size of the target fragment is 350-5000 bp; preferably, the size of the non-target fragment is 50-300 bp, and the size of the target fragment is 350-2000 bp.

11. The method according to claim 2, wherein the DNA library fragment is a library fragment suitable for next-generation sequencing (NGS), and the size of the DNA library fragment is 350-500 bp, or the DNA library fragment is a library fragment suitable for third-generation sequencing (TGS), and the size of the DNA library fragment is greater than 1000 bp.

12. The method according to claim 2, wherein the size of the adapter dimer is 50-300 bp.

13. The method according to claim 2, wherein the working concentration of T7 endonuclease I is 0.1-1 U / μl.

14. The method according to claim 2, wherein the incubation temperature of T7 endonuclease I is 30-42 °C, and the incubation time of T7 endonuclease I is 5-20 minutes.

15. The method according to claim 2, wherein T7 endonuclease I is added alone to the nucleic acid sample for reaction, or is added to the nucleic acid sample together with the amplification reagent and reacts before the amplification step.

16. A library construction method, the method comprising obtaining a nucleic acid template, constructing a DNA library, the library containing a target fragment and a non-target fragment, the target fragment being a DNA library fragment, and the non-target fragment being an adapter dimer and / or a primer dimer; recovering the DNA library using the method according to any one of claims 1, 3-10.

17. A library construction method, the method comprising obtaining a nucleic acid template, constructing a DNA library, the library containing a target fragment and a non-target fragment, the target fragment being a DNA library fragment, and the non-target fragment being an adapter dimer; recovering the DNA library using the method according to any one of claims 2-8, 11-15.

18. A kit for implementing the method according to any one of claims 1-15.

19. A kit for implementing the method according to claim 16 or 17.

20. The kit according to any one of claims 18-19, the kit comprising a magnetic bead solution and a washing solution, the washing solution containing 100-1000 mM NaCl and 20-200 mg / ml PEG.

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