Gene library construction method, kit and use thereof

Abstract

A gene library construction method, the method comprising: providing a first gene fragment; providing a template gene fragment; combining the first gene fragment with the template gene fragment to obtain a target gene sequence having a sequence length greater than that of the first gene fragment; and performing PCR amplification on the target gene sequence to obtain a gene library.

Description

Gene library construction methods, reagent kits and their applications Technical Field

[0001] This disclosure relates to the field of biomedical technology, and in particular to a gene library construction method, a reagent kit, and their applications. Background Technology

[0002] Next-Generation Sequencing (NGS) technology enables massively parallel sequencing of hundreds of thousands to millions of DNA molecules simultaneously, providing a wealth of genetic information. Compared to previous generations of sequencing, NGS significantly reduces sequencing costs and time while maintaining high data volume and accuracy. Therefore, it is widely used in genomics, transcriptomics, epigenomics, and other analytical fields, as well as in cancer screening, pathological typing, pathogen identification, and other application scenarios. Summary of the Invention

[0003] On the one hand, a method for constructing a gene library is provided, the method comprising: providing a first gene fragment; providing a template gene fragment; combining the first gene fragment with the template gene fragment; obtaining a target gene sequence with a sequence length greater than that of the first gene fragment; and performing PCR amplification on the target gene sequence to obtain the gene library.

[0004] In some embodiments, the first gene fragment includes: a first sequence and a second sequence complementary to the first sequence; the template gene fragment includes: a third sequence and a fourth sequence at least partially complementary to the third sequence; the binding of the first gene fragment to the template gene fragment includes: binding at least one of the first sequence and the second sequence to a partially complementary portion of at least one of the third sequence and the fourth sequence, and / or, linking at least one of the first sequence and the second sequence to at least one of the third sequence and the fourth sequence.

[0005] In some embodiments, the third sequence includes: a first random sequence and a second random sequence, and a first extended sequence connecting the first random sequence and the second random sequence; obtaining a target gene sequence with a sequence length greater than the first gene fragment includes: one of the first sequence and the second sequence being complementary to either the first random sequence or the second random sequence; extending the sequence of the first gene fragment complementary to either the first random sequence or the second random sequence to obtain the target gene sequence.

[0006] In some embodiments, the number of bases in the first random sequence ranges from 15 to 25; and / or, the number of bases in the second random sequence ranges from 15 to 25.

[0007] In some embodiments, the number of bases in the first extended sequence ranges from 100 to 220.

[0008] In some embodiments, the third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 1 to SEQ ID No. 4.

[0009] In some embodiments, the third sequence and the fourth sequence are complementary; obtaining a target gene sequence with a sequence length greater than the first gene fragment includes: ligating the template gene fragment and the first gene fragment under the action of transposase to obtain the target gene sequence.

[0010] In some embodiments, the transposase includes at least one of Tn5 transposase, Leap-In transposase, and PiggyBac transposase.

[0011] In some embodiments, the number of bases in the third sequence ranges from 150 to 180.

[0012] In some embodiments, the third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 5 to SEQ ID No. 8.

[0013] In some embodiments, the third sequence includes: a third random sequence and a sticky t-base terminus attached to one end of the third random sequence, and the fourth sequence is complementary to the third random sequence; obtaining a target gene sequence with a sequence length greater than the first gene fragment includes: attaching an a-base sticky terminus to one end of the first sequence; connecting the first gene fragment and the template gene fragment through complementary binding of the t-base sticky terminus and the a-base sticky terminus to obtain a first ligation fragment; circularizing the first ligation fragment under the action of a circularization primer; and obtaining the target gene sequence through a rolling circle amplification reaction under the action of a rolling circle amplification primer.

[0014] In some embodiments, the number of bases in the third random sequence ranges from 2 to 8.

[0015] In some embodiments, the third sequence includes: tnnnnnnn; n is selected from any one of adenine, guanine, cytosine, and thymine.

[0016] In some embodiments, the third sequence includes: a second extended sequence and a sticky t-base terminus connected to one end of the second extended sequence; obtaining a target gene sequence with a sequence length greater than the first gene fragment includes: connecting one end of the first sequence to an sticky α-base terminus; connecting the first gene fragment and the template gene fragment through complementary binding of the sticky t-base terminus and the sticky α-base terminus; and obtaining the target gene sequence after ligase repair.

[0017] In some embodiments, the number of bases in the second extended sequence ranges from 150 to 180.

[0018] In some embodiments, the third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 9 to SEQ ID No. 12.

[0019] In some embodiments, the third sequence includes: a third extended sequence and a fourth random sequence linked to one end of the third extended sequence, wherein the fourth sequence is complementary to the third extended sequence; obtaining a target gene sequence with a sequence length greater than the first gene fragment includes: either the first sequence or the second sequence being complementary to the fourth random sequence; the sequence of the first gene fragment complementary to the fourth random sequence being ligated to the fourth sequence by a ligase to obtain the target gene sequence.

[0020] In some embodiments, the number of bases in the third extended sequence ranges from 125 to 185; and / or, the number of bases in the fourth random sequence ranges from 15 to 25.

[0021] In some embodiments, the third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 13 to SEQ ID No. 16.

[0022] In some embodiments, the first gene fragment has less than or equal to 50 bases.

[0023] On the other hand, a kit is provided, comprising: a template gene fragment; the template gene fragment comprising: a third sequence and a fourth sequence at least partially complementary to the third sequence; wherein the third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No. 1 to SEQ ID No. 16.

[0024] On the other hand, an application of the kit described in any of the above embodiments in the construction of cfDNA libraries is provided. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in this disclosure, the accompanying drawings used in some embodiments of this disclosure will be briefly described below. Obviously, the drawings described below are only drawings of some embodiments of this disclosure, and those skilled in the art can obtain other drawings based on these drawings. In addition, the drawings described below can be regarded as schematic diagrams and are not intended to limit the actual size of the product, the actual process of the method, etc. involved in the embodiments of this disclosure.

[0026] Figure 1 is a flowchart of a gene library construction method according to some embodiments of the present disclosure;

[0027] Figure 2 is a structural diagram of each step in the gene library construction method according to some embodiments of the present disclosure;

[0028] Figure 3 is a diagram showing the length distribution of cfDNA fragments in the gene library provided in Comparative Example 1 of this disclosure;

[0029] Figure 4 is a diagram showing the length distribution of cfDNA fragments in the gene library provided according to Example 1 of this disclosure;

[0030] Figure 5 is another structural diagram of the gene library construction method according to some embodiments of the present disclosure, corresponding to each step;

[0031] Figure 6 is a diagram showing the length distribution of cfDNA fragments in the gene library provided according to Example 2 of this disclosure;

[0032] Figure 7 is another structural diagram of the gene library construction method according to some embodiments of the present disclosure, corresponding to each step;

[0033] Figure 8 is a diagram showing the length distribution of cfDNA fragments in the gene library provided according to Example 3 of this disclosure;

[0034] Figure 9 is another structural diagram of the gene library construction method according to some embodiments of the present disclosure, corresponding to each step;

[0035] Figure 10 is a diagram showing the length distribution of cfDNA fragments in the gene library provided according to Example 4 of this disclosure;

[0036] Figure 11 is another structural diagram of the gene library construction method according to some embodiments of the present disclosure, corresponding to each step;

[0037] Figure 12 is a diagram showing the length distribution of cfDNA fragments in the gene library provided according to Example 5 of this disclosure. Detailed Implementation

[0038] The technical solutions in some embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments provided in this disclosure are within the scope of protection of this disclosure.

[0039] Unless the context otherwise requires, throughout the specification and claims, the term "comprising" is interpreted as open-ended and encompassing, meaning "including, but not limited to." In the description of the specification, terms such as "one embodiment," "some embodiments," "exemplary embodiment," "example," or "some examples" are intended to indicate that a particular feature, structure, material, or characteristic associated with that embodiment or example is included in at least one embodiment or example of this disclosure. The illustrative representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics mentioned may be included in any suitable manner in any one or more embodiments or examples.

[0040] Hereinafter, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of embodiments of this disclosure, unless otherwise stated, "a plurality of" means two or more.

[0041] "At least one of A, B and C" has the same meaning as "at least one of A, B or C", both including the following combinations of A, B and C: only A, only B, only C, combinations of A and B, combinations of A and C, combinations of B and C, and combinations of A, B and C.

[0042] "A and / or B" includes the following three combinations: A only, B only, and a combination of A and B.

[0043] As used herein, “about,” “approximately,” or “approximately” includes the stated value and the average value within an acceptable range of deviation from the given value, wherein the acceptable range of deviation is determined by a person skilled in the art taking into account the measurement under discussion and the error associated with the measurement of the given quantity (i.e., the limitations of the measurement system).

[0044] As used herein, “parallel,” “perpendicular,” and “equal” include the described situation and situations that are similar to the described situation, within an acceptable range of deviation, which is determined by those skilled in the art taking into account the measurement under discussion and the error associated with the measurement of a particular quantity (i.e., the limitations of the measurement system). For example, “parallel” includes absolute parallelism and approximate parallelism, where an acceptable range of deviation for approximate parallelism may be, for example, within 5°; “perpendicular” includes absolute perpendicularity and approximate perpendicularity, where an acceptable range of deviation for approximate perpendicularity may also be, for example, within 5°; “equal” includes absolute equality and approximate equality, where an acceptable range of deviation for approximate equality may be, for example, a difference between the two equals being less than or equal to 5% of either one.

[0045] In this application, DNA (deoxyribonucleic acid) is a long-chain polymer composed of four deoxynucleotides: adenine deoxynucleotide (dATP), thymine deoxynucleotide (dTTP), cytosine deoxynucleotide (dCTP), and guanine deoxynucleotide (dGTP). Deoxynucleotides are composed of phosphate, deoxyribose, and bases; among which, there are four main bases: α (adenine), γ (guanine), γ (cytosine), and t (thymine).

[0046] High-throughput sequencing (NGS, next-generation sequencing), also known as massively parallel sequencing or second-generation sequencing, can sequence multiple target regions of a single sample or multiple samples simultaneously. Its applications in clinical settings, including pharmacogenomics, genetic disease research and screening, tumor mutation gene detection, and clinical microbiology testing, are gaining increasing attention. Next-generation sequencing technology is currently the most widely used sequencing technology, offering advantages such as high sequencing depth, high throughput, high accuracy, and good sensitivity.

[0047] PCR (Polymerase Chain Reaction) is a process that uses a DNA template, with the participation of DNA polymerase and nucleotide substrates, to amplify the DNA to a sufficient quantity for structural and functional analysis. The principle of PCR is to amplify a DNA fragment located between two known sequences, similar to the replication process of natural DNA. Using the DNA molecule to be amplified as a template, and a pair of oligonucleotide fragments complementary to the 5' and 3' ends of the template as primers, DNA polymerase extends along the template strand according to a semi-conservative replication mechanism until new DNA is synthesized. Repeating this process amplifies the target DNA fragment.

[0048] UMI (Unique Molecular Identifier) ​​is used to distinguish the cells of origin.

[0049] High-throughput sequencing technology enables massively parallel sequencing of hundreds of thousands to millions of DNA molecules simultaneously, providing a wealth of genetic information. Compared to previous generations of sequencing, NGS significantly reduces sequencing costs and time while maintaining high data volume and accuracy. As a result, it is widely used in genomics, transcriptomics, epigenomics, and other analytical fields, as well as in various applications such as cancer screening, pathological typing, and pathogen identification.

[0050] Before performing NGS sequencing, the DNA sample to be tested usually needs to be fragmented and adapters with specific sequences ligated to both ends to meet the sequence reading requirements of the NGS platform. The main steps include: DNA fragmentation, end repair, α-tailing (also known as adding α-base sticky ends), adapter ligation, and library purification.

[0051] Cell-free DNA (cfDNA) in blood plasma is generally derived from DNA released after the rupture of damaged or senescent cells, such as tumor cells. It is cleaved into 160-170 bp fragments by nucleases and enters the bloodstream, with a half-life of approximately 5-150 minutes. Because cfDNA carries genetic characteristics such as mutations, copy number variations, and methylation status from the maternal cell, and can exhibit early-stage molecular features of disease, it is an ideal molecular marker for many diseases, especially tumors, serving as a supplement to imaging and tissue biopsies. However, the short length and low concentration of cfDNA in blood place high demands on the sensitivity and reliability of extraction and detection analysis techniques.

[0052] Most cfDNA fragments are concentrated in the 160bp to 170bp region, but some cfDNA fragments are longer than this range. The longer fragments can be extracted by further magnetic bead sorting and purification, and then shorter fragments can be obtained by ultrasonic disruption.

[0053] The size unit of a DNA fragment is the base pair, commonly bp (base pair), kbp (kilobase pair), and Mbp (megabase pair). The example shown here, where the DNA fragment size unit is bp (base pair), refers to a DNA fragment containing 160 to 170 base pairs.

[0054] However, even shorter cfDNA fragments, such as those in the tens of bp length, can be obtained through magnetic bead sorting and enrichment. However, during subsequent adapter ligation and primer PCR amplification, the PCR efficiency is very low due to the short length of the cfDNA fragments. The amplification is mainly due to the primer dimers themselves. Therefore, the library yield of short fragments is low, and information from these fragments is easily lost during subsequent sequencing, leading to errors in diagnostic analysis.

[0055] Based on this, as shown in Figure 1, an embodiment of this disclosure provides a gene library construction method, which includes: R1 to R4.

[0056] R1 provides the first gene fragment.

[0057] For example, the first gene fragment includes a small fragment of DNA, the first gene fragment having fewer than or equal to 50 bases.

[0058] For example, the number of bases in the first gene fragment ranges from 20 to 30. For instance, the number of bases in the first gene fragment may be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, etc., and there is no limit here.

[0059] For example, the first gene fragment includes a small fragment of cfDNA.

[0060] R2 provides template gene fragments.

[0061] For example, the template gene fragment is a gene fragment with a known sequence.

[0062] R3. Combine the first gene fragment with the template gene fragment to obtain the target gene sequence with a sequence length greater than that of the first gene fragment.

[0063] For example, the first gene fragment includes a first sequence and a second sequence complementary to the first sequence, and the template gene fragment includes a third sequence and a fourth sequence at least partially complementary to the third sequence. The step of binding the first gene fragment to the template gene fragment includes: binding at least one of the first and second sequences to a partially complementary portion of at least one of the third and fourth sequences, and / or linking at least one of the first and second sequences to at least one of the third and fourth sequences.

[0064] In other words, by ligating the sequence of the first gene fragment to the template gene fragment, the template gene fragment elongates the first gene fragment. Furthermore, since the template gene fragment is a known sequence, it also helps to locate the first gene fragment. By combining the first gene fragment with the template gene fragment, a target gene sequence with a length greater than the first gene fragment is obtained, which can effectively solve the problem of low PCR efficiency caused by the short fragment length of small cfDNA fragments.

[0065] For details on the implementation of combining the first gene fragment with the template gene fragment, please refer to the following content; it will not be described in detail here.

[0066] R4. The target gene sequence is amplified by PCR to obtain a gene library.

[0067] Through steps R1 to R4, a gene library containing short DNA fragment gene information was obtained. Due to the binding of the first gene fragment with the template gene fragment, a target gene sequence with a sequence length longer than the first gene fragment was obtained, thereby increasing the yield of the first gene fragment in the gene library. This effectively prevents the loss of sequencing information from the short first gene fragment, improving the accuracy and reliability of sequencing data. Furthermore, the extended sequence does not interfere with the sequence information of the first gene fragment itself and is easily identifiable, allowing it to be excluded in subsequent analysis. Therefore, the gene library construction method provided in the embodiments of this disclosure helps improve the accuracy and reliability of sequencing analysis results.

[0068] The following is an example of the combination of a first gene fragment and a template gene fragment.

[0069] In some embodiments, as shown in FIG2, the third sequence includes: a first random sequence and a second random sequence, and a first extended sequence connecting the first random sequence and the second random sequence.

[0070] The random sequence refers to a sequence that includes at least one random base n, where the random base n is selected from any one of a (adenine), g (guanine), c (cytosine), and t (thymine).

[0071] For example, the number of bases in the first random sequence ranges from 15 to 25. For instance, the number of bases in the first random sequence can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, etc., and there is no limit here.

[0072] For example, the number of bases in the second random sequence ranges from 15 to 25. For instance, the number of bases in the second random sequence can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, etc., and there is no limit here.

[0073] For example, the number of bases in the first extension sequence ranges from 100 to 220. For instance, the number of bases in the first extension sequence can be 100, 110, 120, 135, 140, 150, 166, 175, 185, 190, 210, or 220, etc., and there is no limitation here.

[0074] By including a first random sequence and a second random sequence, and a first extension sequence connecting the first random sequence and the second random sequence, the number of bases in the third sequence can range from 150 to 250, and the gene fragment of this sequence length meets the needs of gene library construction.

[0075] In some examples, as shown in Figure 2, the steps to obtain the target gene sequence with a sequence length greater than that of the first gene fragment include: R31 and R32.

[0076] R31, one of the first sequence and the second sequence is combined complementaryly with either the first random sequence or the second random sequence.

[0077] For example, the first gene fragment and the template gene fragment are denatured and annealed to achieve complementary binding between one of the first sequence and the second sequence and either the first random sequence or the second random sequence.

[0078] R32. Extend the sequence of the first gene fragment that is complementary to either the first random sequence or the second random sequence to obtain the target gene sequence.

[0079] For example, the first sequence of the first gene fragment is complementary to the second random sequence, and then the first sequence is extended using the third sequence as a template to form two complementary gene chains, thus obtaining the target gene sequence.

[0080] Steps R31 and R32 achieve complementary binding of at least one of the first and second sequences with portions of at least one of the third and fourth sequences, resulting in the target gene sequence after sequence extension. This method can be called template gene fragment amplification. The target gene sequence has a base count ranging from 150 to 250, a length suitable for gene library construction, and the reaction steps are simple and easy to implement.

[0081] In some embodiments, the third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 1 to SEQ ID No. 4.

[0082] Wherein, n is selected from any one of a (adenine), g (guanine), c (cytosine), and t (thymine). In the above example, both the first random sequence and the second random sequence comprise 15 random bases, and both the first random sequence and the second random sequence are represented as nnnnnnnnnnnnnnn. The sequence between the first random sequence and the second random sequence is the first extended sequence. The embodiments of this disclosure are not limited thereto.

[0083] Example 1 is provided based on the gene library construction method including the template gene fragment amplification method described above.

[0084] Example 1

[0085] This example provides a gene library construction method that includes template gene fragment amplification processing of small cfDNA fragments. Small cfDNA fragments can be understood as cfDNA fragments with a length range of less than 50 bp, for example, cfDNA fragments with a length range of 20 bp to 30 bp.

[0086] This method uses denatured and unwound single-stranded cfDNA as a primer, binding to a template gene fragment of approximately 200 bp in length to amplify the fragment. Only the two ends of the template gene fragment, where cfDNA binds, are random sequences used to "capture" the single-stranded cfDNA; the first extension sequence in the middle region is a known, defined sequence. Once the cfDNA binds to the template, it is amplified by polymerase and an amplification buffer system. The resulting product sequence contains the cfDNA sequence, effectively extending the original small cfDNA fragment, which can then be used for subsequent library construction and amplification. During sequencing analysis, the cfDNA sequences at both ends can be located by identifying the first extension sequence in the middle.

[0087] For example, as shown in Figure 2, the steps for constructing a gene library using the template gene fragment amplification method are as follows.

[0088] I. Sorting of small fragment cfDNA

[0089] (1) Vortex the magnetic bead suspension to mix well. Add about 10 ng to 50 ng of cfDNA extraction product to enzyme-free water to make up to 50 μL.

[0090] (2) Add 25 μL (0.5 x) of resuspended magnetic beads to the product, mix by blowing more than 10 times, incubate at room temperature for 5 min, briefly centrifuge, and place on a magnetic rack until the solution is clear (about 5 min).

[0091] The amount of magnetic beads added in this step is 25 μL (0.5 x), at which point the magnetic beads capture long fragments of cfDNA.

[0092] (3) Transfer the supernatant (approximately 75 μL) to a new 0.2 mL enzyme-free PCR tube. Do not disturb the magnetic beads. Add 65 μL (1.8 x) magnetic beads, mix by pipetting more than 10 times, incubate at room temperature for 5 min, centrifuge briefly, and place on a magnetic rack until the solution is clear. At this point, the cfDNA fragments adsorbed by the magnetic beads can be used for ordinary library construction.

[0093] The amount of magnetic beads added in this step is 65 μL (1.8 x). At this time, the cfDNA captured by the magnetic beads has a length range of 160 bp to 170 bp. The cfDNA fragments adsorbed by the magnetic beads in steps (2) and (3) can be used for ordinary library construction. That is, since the length of this part of cfDNA meets the requirements for gene library construction, it can be directly used for cfDNA gene library construction, with high library yield, and the obtained library includes cfDNA information, which can be used for disease diagnosis and analysis.

[0094] (4) Collect the clear solution (approximately 140 μL) into a new 0.2 mL enzyme-free PCR tube. Do not disturb the magnetic beads. Add 20 μL (2.2 x) magnetic beads, mix by pipetting more than 10 times, incubate at room temperature for 5 min, centrifuge briefly, place on a magnetic rack until the solution is clear, carefully discard the supernatant, and do not disturb the magnetic beads.

[0095] In this step, the amount of magnetic beads added is 20 μL (2.2x), at which point the cfDNA captured by the magnetic beads is a small fragment of cfDNA.

[0096] (5) Keep the PCR tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for 30 seconds, and carefully remove the supernatant; keep the magnetic beads on the magnetic rack open to dry (it takes about 2 min to 3 min), do not dry them too much, for example, the judgment standard is dark brown and shiny but no visible liquid.

[0097] This step involves cleaning the magnetic beads with 80% ethanol to remove adsorbed protein impurities.

[0098] (6) Remove the magnetic rack, add 21 μL of enzyme-free water to wash, mix by pipetting more than 10 times, centrifuge briefly, and incubate at room temperature for 2 min; place on the magnetic rack until the solution is clear (about 3 min), transfer 20 μL of supernatant to a new enzyme-free PCR tube, and you can obtain a small fragment of cfDNA for subsequent library construction.

[0099] II. Template gene fragment amplification

[0100] (1) Design a template gene fragment, wherein the regions at both ends of the template gene fragment that bind to cfDNA are random sequences (including: a first random sequence and a second random sequence) used to “capture” single strands of cfDNA, and the first extension sequence in the middle region is a known sequence that does not pair complementaryly with a human genome sequence. For example, the third sequence of the template gene fragment is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of SEQ ID No. 1.

[0101] Wherein, n is selected from any one of a (adenine), g (guanine), c (cytosine), and t (thymine), and the template gene fragment is 200 bp in length.

[0102] (2) Formation of amplification product solution

[0103] Prepare the following reagents.

[0104] After mixing, proceed with the reaction according to the following procedure.

[0105] The amplified products were purified using 0.5× to 1.8× magnetic beads, following the same steps as the small fragment cfDNA sorting (1) to (3) in step one above. After quantification by Qubit nucleic acid, a cfDNA library was constructed.

[0106] III. Construction of cfDNA gene libraries

[0107] For example, a commercial DNA library construction kit was used to construct a library of cfDNA.

[0108] (1) Dilute 10 ng of the product from step 2 with 10 mM Tris-HCl at pH 8.0 to 35 μL in a 0.2 mL PCR tube or well plate, prepare the reaction solution on ice, vortex to mix, and immediately proceed to the next step on ice.

[0109] (2) Incubate in a thermal cycler with the cover set to 65°C; after the reaction is complete, quickly transfer to ice and proceed to the next step immediately.

[0110] Among them, end repair with an α tail is to introduce an α-base sticky end; ∞ indicates that there is no time limit.

[0111] (3) Connector connection: Keep the reaction solution on ice, add 5 μL of universal connector to each reaction volume, then add 10 μL of the linking reaction mixture, vortex to mix, and centrifuge briefly to a final volume of 75 μL.

[0112] (4) Incubate in a thermal cycler with the cover set to 50°C; proceed to the next step immediately after the reaction is complete.

[0113] (5) Post-ligation purification: Add 60 μL of magnetic beads to each 75 μL reaction system to resuspend the uniform magnetic beads at room temperature, for a final volume of 135 μL; ensure uniform mixing and centrifuge quickly.

[0114] Incubate at room temperature for 5 min, place on a magnetic rack until the solution is clear, and carefully discard the supernatant; keep the centrifuge tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for ≥30 sec, and carefully discard the ethanol; repeat the ethanol wash once, carefully removing all remaining ethanol without disturbing the magnetic beads; air dry at room temperature, avoiding excessive drying; remove the centrifuge tube from the magnetic rack; resuspend the magnetic beads in 22 μL of 10 mM Tris-HCl solution at pH 8.0, and incubate at room temperature for 2 min; place on a magnetic rack until the solution is clear, transfer 20 μL of elution buffer to a new tube, and immediately proceed to the next stage of library amplification.

[0115] (6) Library amplification and UDI (Unique Device Identification) ligation: Prepare the PCR amplification mixture as follows, mix well, centrifuge quickly, and immediately proceed with subsequent amplification.

[0116] (7) Amplify in a thermal cycler with the cover plate set to 105°C; proceed to the next step immediately after the reaction is complete.

[0117] (8) Library purification: Add 70 μL of the magnetic beads resuspended and mixed at room temperature to the amplified sample, ensuring that the mixture is uniform, and centrifuge quickly; incubate at room temperature for 5 min, place on a magnetic rack until the solution is clear, and carefully discard the supernatant; keep the centrifuge tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for ≥30 sec, and carefully discard the ethanol; repeat the ethanol washing once, carefully and completely removing the remaining ethanol without disturbing the magnetic beads; air dry at room temperature, avoiding excessive drying; remove the centrifuge tube from the magnetic rack; resuspend the magnetic beads in 32 μL of PCR water, incubate at room temperature for 2 min; place on a magnetic rack until the solution is clear, and transfer 30 μL of elution buffer to a new tube.

[0118] To facilitate data analysis, Comparative Example 1 is provided. Compared with Example 1, the gene library of cfDNA in Comparative Example 1 did not undergo template gene fragment amplification during construction, while the remaining steps were the same as in Example 1.

[0119] Figure 3 shows the cfDNA fragment length distribution in the gene library provided in Comparative Example 1; Figure 4 shows the cfDNA fragment length distribution in the gene library provided in Example 1. The horizontal axis represents the fragment length in bp; the vertical axis represents the normalized fluorescence intensity; "minimum" and "maximum" are reference settings for fragment length determination. As can be seen from Figure 3, the cfDNA fragment length distribution in the gene library without template gene fragment amplification is in the range of 160bp to 220bp, with a peak at 190bp and a small gap width; and the normalized fluorescence intensity is low, meaning the peak in the gene library is very small and cannot be used for gene sequencing.

[0120] As shown in Figure 4, the cfDNA fragment length distribution range in the gene library obtained in Example 1 is 180bp to 700bp, with a peak at 289bp and a maximum normalized fluorescence intensity of 2100. The normalized fluorescence intensity of cfDNA in the gene library obtained in Example 1 is much greater than that of cfDNA in the gene library obtained in Comparative Example 1. That is, the peak height and suture width of the gene library obtained in Example 1 are normal and meet the requirements of gene sequencing.

[0121] Therefore, the template gene fragment amplification process for small cfDNA fragments in Example 1 increases the yield of cfDNA in the gene library, effectively prevents the loss of sequencing information of small cfDNA fragments, improves the accuracy and reliability of sequencing data, and the template gene fragments do not interfere with the sequence information of the cfDNA itself, are easy to identify, and can be excluded in subsequent analysis. The gene library construction method provided by the embodiments of this disclosure helps to improve the accuracy and reliability of sequencing analysis results, and the reaction steps are simple and easy to implement.

[0122] The following provides another example of the combination of a first gene fragment and a template gene fragment.

[0123] In some embodiments, as shown in Figure 5, the third and fourth sequences of the template gene fragment are complementary.

[0124] For example, the number of bases in the third sequence ranges from 150 to 180. For instance, the number of bases in the third sequence may be 150, 160, 170, or 180, etc., and there is no limit here.

[0125] In some examples, obtaining a target gene sequence with a sequence length greater than that of the first gene fragment involves ligating the template gene fragment and the first gene fragment using transposase to obtain the target gene sequence.

[0126] This method, known as the transposase method, connects a first gene fragment with a sequence length ranging from 20bp to 30bp and a template gene fragment with a sequence length ranging from 150bp to 180bp using a transposase to obtain a target gene sequence with a sequence length of approximately 200bp. This gene fragment of this length meets the requirements for gene library construction, and the reaction steps are simple and easy to implement.

[0127] For example, transposases include at least one of Tn5 transposase, Leap-In transposase, and PiggyBac transposase.

[0128] The recognition sequences for the Tn5 transposase include SEQ ID No. 17 and SEQ ID No. 18. SEQ ID No. 17: ctgactcttatacacaagt; SEQ ID No. 18: ctgtctcttatacacatct.

[0129] The recognition sequences of Leap-In transposases include ttaa and ttat.

[0130] The recognition sequence of the PiggyBac transposase includes: ttaa.

[0131] For example, the third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 5 to SEQ ID No. 8.

[0132] Example 2 is provided based on the gene library construction method including the transposase method described above.

[0133] Example 2

[0134] This example provides a method for constructing a gene library that includes transposase treatment of small fragments of cfDNA.

[0135] This method utilizes the insertion sequence characteristics of transposases. A mixture of several transposases that recognize different sites is applied to a small cfDNA fragment to insert a template gene fragment with known sequence content into the cfDNA fragment, forming a new long sequence. Then, an adapter is added for sequencing. The cfDNA sequence can be located using the template gene fragment.

[0136] For example, as shown in Figure 5, the steps for constructing a gene library using the transposase method are as follows.

[0137] I. Sorting of small fragment cfDNA

[0138] For an introduction to the sorting of small fragment cfDNA, please refer to the content of Example 1, which will not be repeated here.

[0139] II. Transposable Enzymatic Method

[0140] 1. Template gene fragment design

[0141] The template gene fragment can be any known sequence fragment that does not pair complementaryly with the target, and the third sequence of the template gene fragment is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of SEQ ID No. 5.

[0142] The length of the third sequence of the template gene fragment is 160 bp.

[0143] 2. Insertion reaction and purification

[0144] Prepare the following reaction on ice, mix by pipetting, briefly centrifuge, and then immediately place it in a PCR instrument and incubate at 55°C for 5 min with the cover plate temperature set to 70°C; place it on ice immediately after the reaction is complete.

[0145] The reaction product was added to 50 μL of purification magnetic beads, and subsequent product purification and elution were performed according to the same magnetic bead purification steps as in Example 1.

[0146] III. The reaction products were processed in the following steps as described in Example 1 for constructing the gene library of cfDNA.

[0147] Figure 6 shows the length distribution of cfDNA fragments in the gene library provided in Example 2. As can be seen from Figure 6, the length distribution of cfDNA fragments in the gene library obtained in Example 2 ranges from 250bp to 600bp, with a peak at 340bp, and the normalized fluorescence intensity reaches a maximum of 1200. That is, the peak height and suture width of the gene library obtained in Example 2 are normal, meeting the requirements for gene sequencing.

[0148] The following provides another example of the combination of a first gene fragment and a template gene fragment.

[0149] In some embodiments, as shown in FIG7, the third sequence of the template gene fragment includes: a third random sequence and a sticky t-base terminus connected to one end of the third random sequence, and the fourth sequence is complementary to the third random sequence.

[0150] For example, the number of bases in the third random sequence ranges from 2 to 8. For instance, the number of bases in the third random sequence can be 2, 3, 4, 5, 6, or 8, etc., and there is no limitation here.

[0151] The third sequence includes: tnnnnnnn; n is selected from any one of adenine, g (guanine), c (cytosine), and t (thymine). In this case, the template gene fragment can be called a unique molecular tag (UMI).

[0152] For example, as shown in Figure 7, the target gene sequences with a sequence length greater than that of the first gene fragment include: S31 to S34.

[0153] S31. Connect one end of the first sequence to the sticky end of the α base.

[0154] S32. The first gene fragment and the template gene fragment are connected by complementary binding of the sticky t base and the sticky a base to obtain the first linker fragment.

[0155] S33. Under the action of the circularization primer, the first linker fragment is circularized and linked.

[0156] S34. Under the action of rolling circle amplification primers, the target gene sequence is obtained through rolling circle amplification reaction.

[0157] Prior to the rolling circle amplification reaction, the process includes a step of denaturing the first cyclized linker fragment, so that the first cyclized linker fragment is called a single-stranded ring.

[0158] The target gene sequence was obtained by designing UMI and following steps S31 to S34 above. This method can be called rolling circle amplification. The length of the target gene sequence obtained by rolling circle amplification can be set according to the sequencing depth of the sequence. The number of rolling circles can be, for example, 10 to 100. The more rolling circles, the higher the sequencing depth, and the higher the sequencing depth, the higher the sequencing accuracy.

[0159] Example 3 is provided based on the gene library construction method including rolling circle amplification.

[0160] Example 3

[0161] This example provides a method for constructing a gene library that includes processing small fragments of cfDNA using rolling circle amplification.

[0162] Based on the basic principle of rolling circle amplification (RBA), a small cfDNA fragment is first ligated with a unique molecular tag of a known sequence, and then circular DNA is synthesized under the action of a catalytic enzyme. Subsequently, using a primer complementary to the UMI, a complementary long fragment is synthesized by polymerase catalysis, rotating around the circular DNA template. Each fragment can contain several UMI and cfDNA short sequences, which can be used for subsequent library construction. The cfDNA sequence can be located by adjacent UMI sequences during analysis.

[0163] For example, as shown in Figure 7, the steps for constructing a gene library using the rolling circle amplification method are as follows.

[0164] I. Sorting of small fragment cfDNA

[0165] For an introduction to the sorting of small fragment cfDNA, please refer to the content of Example 1, which will not be repeated here.

[0166] II. Rolling Circle Amplification Method

[0167] 1. Template gene fragment design

[0168] Select a nucleotide chain of 2bp to 8bp that is completely random but whose sequence is known, with a sticky t-base extension at one end, for ligation to a small cfDNA fragment; the possible sequence is tnnnnnnn, where n is selected from a (adenine), g (guanine), c (cytosine), and t (thymine).

[0169] 2. Ligation of template gene fragment with small cfDNA fragment

[0170] (1) Dilute 10 ng of small cfDNA fragments with 10 mM Tris-HCl at pH 8.0 to 35 μL in a 0.2 mL PCR tube or well plate, prepare the following reaction solution on ice, vortex to mix, and immediately proceed to the next step on ice.

[0171] (2) Incubate in a thermal cycler with the cover set to 65°C; after the reaction is complete, quickly transfer to ice and proceed to the next step immediately.

[0172] (3) Template gene fragment ligation: Keep the reaction solution on ice, add 5 μL of UMI to each reaction volume, then add 10 μL of ligation reaction mix, vortex to mix, centrifuge briefly, and the final volume is 75 μL.

[0173] (4) Incubate in a thermal cycler with the cover set to 50°C; proceed to the next step immediately after the reaction is complete.

[0174] (5) Post-ligation purification: Add 60 μL of magnetic beads to each 75 μL reaction system to resuspend the uniform magnetic beads at room temperature, for a final volume of 135 μL; ensure uniform mixing and centrifuge quickly.

[0175] Incubate at room temperature for 5 min, place on a magnetic rack until the solution is clear, and carefully discard the supernatant; keep the centrifuge tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for ≥30 sec, and carefully discard the ethanol; repeat the ethanol wash once, carefully removing all remaining ethanol without disturbing the magnetic beads; air dry at room temperature, avoiding excessive drying; remove the centrifuge tube from the magnetic rack; resuspend the magnetic beads in 22 μL of 10 mM Tris-HCl solution at pH 8.0, and incubate at room temperature for 2 min; place on a magnetic rack until the solution is clear, and transfer 20 μL of eluent to a new tube.

[0176] 3. Cyclization reaction

[0177] (1) Add enzyme-free water to the above product to 35 μL, prepare the following reaction system on ice, mix well, denature at 96 °C for 3 min on a PCR instrument, and then place on ice for 2 min.

[0178] (2) Prepare the reaction system on ice as follows, and mix thoroughly by inverting.

[0179] (3) Set the PCR instrument to perform the cyclization reaction as follows.

[0180] (4) The reaction products are prepared into the following system and reaction procedure for enzymatic digestion to remove uncyclized products.

[0181] Remove the uncirculated product to prevent it from interfering with gene library construction.

[0182] (5) After the reaction is complete, add 120 μL of purified magnetic beads and follow the same magnetic bead purification steps as in Example 1 to purify and elute the subsequent products.

[0183] 5. Rolling ring amplification reaction

[0184] (1) Denaturation: Take 5 μL of purified product and add 5 μL of denaturing solution, mix by blowing and stirring, and let stand at room temperature for 2 min.

[0185] For example, the denaturing solution comprises 4% formaldehyde. This step is used to form a cyclized single-chain product from the double-chain product of the cyclization reaction.

[0186] (2) Prepare the reaction solution according to the following system, mix it by blowing and stirring, and then perform the amplification and inactivation reaction under programmed temperature control.

[0187] The extinguishing reaction is used to terminate the amplification of the sequence.

[0188] Phi29 DNA polymerase is a thermophilic DNA polymerase cloned from Bacillus subtilis phage Phi29. It has 3' to 5' exonuclease reading capabilities and unique multiple substitution and continuous synthesis characteristics.

[0189] III. The reaction products were processed in the following steps as described in Example 1 for constructing the gene library of cfDNA.

[0190] Figure 8 shows the length distribution of cfDNA fragments in the gene library provided in Example 3. As can be seen from Figure 8, the length distribution of cfDNA fragments in the gene library obtained in Example 3 ranges from 200bp to 1000bp, with peak values ​​including 342bp and 502bp, and the normalized fluorescence intensity reaches a maximum of 3000. That is, the peak height and suture width of the gene library obtained in Example 3 are normal, meeting the requirements for gene sequencing.

[0191] The following provides another example of the combination of a first gene fragment and a template gene fragment.

[0192] In some embodiments, as shown in FIG9, the third sequence of the template gene fragment includes: a second extension sequence and a sticky t-base terminus connected to one end of the second extension sequence.

[0193] For example, the number of bases in the second extension sequence ranges from 150 to 180. For instance, the number of bases in the second extension sequence can be 150, 155, 160, 165, 170, 175, or 180, etc., and there is no limit here.

[0194] The number of bases in the second extension sequence ranges from 150 to 180, so that the number of bases in the target gene sequence obtained after the first gene fragment is linked with the template gene fragment is about 200. This sequence length meets the requirements for gene library construction.

[0195] For example, target gene sequences with a sequence length greater than that of the first gene fragment include T31 and T32.

[0196] T31. Connect one end of the first sequence to the sticky end of the α base.

[0197] T32. The first gene fragment and the template gene fragment are ligated by complementary binding of the sticky t bases and sticky a bases. After ligase repair, the target gene sequence is obtained.

[0198] The first gene fragment and the template gene fragment were joined through steps T31 and T32. This method can be called the double-stranded ligase method. The length of the target gene sequence obtained meets the requirements for gene library construction, and the reaction steps are simple and easy to implement.

[0199] In some embodiments, the third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 9 to SEQ ID No. 12.

[0200] Example 4 is provided based on the gene library construction method including the double-stranded ligase method described above.

[0201] Example 4

[0202] This example provides a method for constructing a gene library that includes enzymatic processing of small fragments of cfDNA using double-strand ligation.

[0203] This method involves end-completing and adding an α-tail to a small cfDNA fragment, which is then ligated to a template gene fragment (a known double-stranded DNA sequence) using double-stranded DNA ligase. The cfDNA fragment is then extended to obtain a target gene sequence of approximately 200 bp, which can then be used in subsequent gene library construction processes. The cfDNA sequence can be located using adjacent known DNA sequences.

[0204] For example, as shown in Figure 9, the steps for constructing a gene library using the double-stranded ligase method are as follows.

[0205] I. Sorting of small fragment cfDNA

[0206] For an introduction to the sorting of small fragment cfDNA, please refer to the content of Example 1, which will not be repeated here.

[0207] II. Double-stranded ligation enzyme method

[0208] 1. Template gene fragment design

[0209] Design a known double-stranded sequence as a template gene fragment, one end of which has an extended T-base sticky end for ligation with a small cfDNA fragment, and the third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of SEQ ID No. 9.

[0210] The length of the third sequence is 150 bp.

[0211] 2. Template gene fragment ligation

[0212] (1) Dilute 10 ng of small cfDNA fragments with 10 mM Tris-HCl at pH 8.0 to 35 μL in a 0.2 mL PCR tube or well plate, prepare the following reaction solution on ice, vortex to mix, and immediately proceed to the next step on ice.

[0213] (2) Incubate in a thermal cycler with the cover set to 65°C; after the reaction is complete, quickly transfer to ice and proceed to the next step immediately.

[0214] (3) Double-stranded DNA ligation: Keep the reaction solution on ice. Add 5 μL of double-stranded DNA to each reaction volume, then add 10 μL of ligation reaction mixture, vortex to mix, and centrifuge briefly to a final volume of 75 μL.

[0215] (4) Incubate in a thermal cycler with the cover set to 50°C; proceed to the next step immediately after the reaction is complete.

[0216] (5) Post-ligation purification: Add 60 μL of magnetic beads to each 75 μL reaction system to resuspend the uniform magnetic beads at room temperature, for a final volume of 135 μL; ensure uniform mixing and centrifuge quickly.

[0217] Incubate at room temperature for 5 min, place on a magnetic rack until the solution is clear, and carefully discard the supernatant; keep the centrifuge tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for ≥30 sec, and carefully discard the ethanol; repeat the ethanol wash once, carefully removing all remaining ethanol without disturbing the magnetic beads; air dry at room temperature, avoiding excessive drying; remove the centrifuge tube from the magnetic rack; resuspend the magnetic beads in 22 μL of 10 mM Tris-HCl solution at pH 8.0, and incubate at room temperature for 2 min; place on a magnetic rack until the solution is clear, and transfer 20 μL of eluent to a new tube.

[0218] III. The reaction products were processed in the following steps as described in Example 1 for constructing the gene library of cfDNA.

[0219] Figure 10 shows the length distribution of cfDNA fragments in the gene library provided in Example 4. As can be seen from Figure 10, the length distribution of cfDNA fragments in the gene library obtained in Example 4 ranges from 170bp to 700bp, with a peak value of 291bp, and the normalized fluorescence intensity reaches a maximum of 2200. That is, the peak height and suture width of the gene library obtained in Example 4 are normal, meeting the requirements for gene sequencing.

[0220] The following provides another example of the combination of a first gene fragment and a template gene fragment.

[0221] In some embodiments, as shown in FIG11, the third sequence includes: a third extended sequence and a fourth random sequence connected to one end of the third extended sequence, the fourth sequence being complementary to the third extended sequence.

[0222] For example, the number of bases in the third extension sequence ranges from 125 to 185. For instance, the number of bases in the third extension sequence can be 125, 130, 140, 150, 160, 165, 170, 180, or 185, etc., and there is no limit here.

[0223] For example, the number of bases in the fourth random sequence ranges from 15 to 25. For instance, the number of bases in the fourth random sequence can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, etc., and there is no limit here.

[0224] The third sequence includes a third extended sequence and a fourth random sequence attached to one end of the third extended sequence, such that the number of bases in the third sequence is about 200. This sequence length of gene fragment meets the requirements for gene library construction.

[0225] For example, a target gene sequence with a sequence length greater than that of the first gene fragment is obtained, including: U31 and U32.

[0226] U31, either the first sequence or the second sequence, is combined complementaryly with the fourth random sequence.

[0227] For example, the first sequence is combined complementaryly with the fourth random sequence.

[0228] U32, the sequence of the first gene fragment that is complementary to the fourth random sequence is ligated with the fourth sequence by ligase to obtain the target gene sequence.

[0229] For example, the first sequence is ligated with the fourth sequence by a ligase to obtain the target gene sequence.

[0230] The first gene fragment was ligated to the template gene fragment through steps U31 and U32. This method can be called the single-strand ligase method. The number of bases in the target gene sequence obtained is about 200. The gene fragment of this length meets the needs of gene library construction, and the reaction steps are simple and easy to implement.

[0231] In some embodiments, the third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 13 to SEQ ID No. 16.

[0232] Wherein, n is selected from any one of a (adenine), g (guanine), c (cytosine), and t (thymine). In the above example, the fourth random sequence comprises 15 random bases, and the fourth random sequence is nnnnnnnnnnnnnnnn, but the embodiments of this disclosure are not limited thereto.

[0233] Example 5 is provided based on the gene library construction method including the single-stranded ligase method described above.

[0234] Example 5

[0235] This example provides a method for constructing a gene library that includes single-stranded ligation of small cfDNA fragments.

[0236] This method denatures and unwinds a small cfDNA fragment and a template gene fragment into single strands. The template gene fragment contains a random sequence as the sticky end, and the remaining part is the third extension sequence, which is a known sequence. Subsequently, the reaction system is annealed, and the cfDNA is "captured" by a specific complementary random sequence and binds to the template gene fragment. Finally, a single-stranded DNA ligase fills in the gap, thus achieving the elongation of the small cfDNA fragment. Its sequence can be located by adjacent known DNA sequences.

[0237] For example, as shown in Figure 11, the steps for constructing a gene library using the single-stranded ligase method are as follows.

[0238] I. Sorting of small fragment cfDNA

[0239] For an introduction to the sorting of small fragment cfDNA, please refer to the content of Example 1, which will not be repeated here.

[0240] II. Single-strand ligation enzyme method

[0241] 1. Template gene fragment design

[0242] The template gene fragment includes a third sequence and a fourth sequence. The third sequence includes a third extension sequence and a fourth random sequence attached to one end of the third extension sequence. The fourth sequence is complementary to the third extension sequence. The fourth random sequence binds to cfDNA and is a single-stranded, sticky end for "capturing" single strands of cfDNA. The third sequence of the template gene fragment is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of SEQ ID No. 13.

[0243] The length of the third sequence is 180 bp.

[0244] 2. Template gene fragment ligation

[0245] (1) Prepare the reaction solution according to the following reaction and carry out the denaturation annealing reaction according to the set program temperature.

[0246] (2) Template gene fragment ligation: Prepare the ligation reaction solution according to the following system, vortex to mix, briefly centrifuge, and carry out the ligation reaction according to the set temperature.

[0247] (3) Post-ligation purification: Add 60 μL of magnetic beads to each 75 μL reaction system to resuspend the uniform magnetic beads at room temperature, for a final volume of 135 μL; ensure uniform mixing and centrifuge quickly.

[0248] Incubate at room temperature for 5 min, place on a magnetic rack until the solution is clear, and carefully discard the supernatant; keep the centrifuge tube on the magnetic rack, add 200 μL of freshly prepared 80% ethanol, incubate at room temperature for ≥30 sec, and carefully discard the ethanol; repeat the ethanol wash once, carefully removing all remaining ethanol without disturbing the magnetic beads; air dry at room temperature, avoiding excessive drying; remove the centrifuge tube from the magnetic rack; resuspend the magnetic beads in 22 μL of 10 mM Tris-HCl solution at pH 8.0, and incubate at room temperature for 2 min; place on a magnetic rack until the solution is clear, and transfer 20 μL of eluent to a new tube.

[0249] III. The reaction products were processed in the following steps as described in Example 1 for constructing the gene library of cfDNA.

[0250] Figure 12 shows the length distribution of cfDNA fragments in the gene library provided in Example 5. As can be seen from Figure 12, the length distribution of cfDNA fragments in the gene library obtained in Example 5 ranges from 180bp to 700bp, with a peak value of 294bp, and the normalized fluorescence intensity reaches a maximum of 2200. That is, the peak height and suture width of the gene library obtained in Example 5 are normal, meeting the requirements for gene sequencing.

[0251] Embodiments of this disclosure also provide a kit comprising: a template gene fragment; the template gene fragment comprising: a third sequence and a fourth sequence at least partially complementary to the third sequence; wherein the third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No. 1 to SEQ ID No. 16.

[0252] For information on SEQ ID No. 1 to SEQ ID No. 16, please refer to the above content and it will not be repeated here.

[0253] For example, the kit may also include at least one of the following: PCR premix, end-repair and α-base mixture, cyclization primers, cyclization reaction mixture, ligase, digestive enzyme, denaturing solution, rolling circle amplification primers, rolling circle amplification mixture, MnCl2, polymerase, universal adapter, ligation reaction solution, magnetic beads, UDI premix, PCR amplification mixture, and transposase.

[0254] This kit can be used to construct cfDNA libraries with 50 or fewer bases.

[0255] This kit can be used to construct small fragment cfDNA libraries. Therefore, the kit provided in the embodiments of this disclosure has all the beneficial effects of constructing gene libraries provided in any of the above embodiments, and will not be elaborated here.

[0256] The above description is merely a specific embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. A method for constructing a gene library, comprising: Provide the first gene fragment; Provide template gene fragments; The first gene fragment is combined with the template gene fragment; Obtain the target gene sequence with a sequence length greater than the first gene fragment; The target gene sequence was amplified by PCR to obtain the gene library.

2. The genetic library construction method according to claim 1, wherein, The first gene fragment includes: a first sequence and a second sequence complementary to the first sequence; the template gene fragment includes: a third sequence and a fourth sequence at least partially complementary to the third sequence; The step of combining the first gene fragment with the template gene fragment includes: Partial complementary combination of at least one of the first sequence and the second sequence with at least one of the third sequence and the fourth sequence, and / or connection of at least one of the first sequence and the second sequence with at least one of the third sequence and the fourth sequence.

3. The method of constructing a genetic library according to claim 2, wherein, The third sequence includes: a first random sequence and a second random sequence, and a first extended sequence connecting the first random sequence and the second random sequence; The obtained target gene sequence with a sequence length greater than the first gene fragment includes: One of the first sequence and the second sequence is combined complementaryly with either the first random sequence or the second random sequence; The target gene sequence is obtained by extending the sequence of the first gene fragment that is complementary to either the first random sequence or the second random sequence.

4. The method of constructing a genetic library according to claim 3, wherein, The number of bases in the first random sequence ranges from 15 to 25; and / or, the number of bases in the second random sequence ranges from 15 to 25.

5. The method of constructing a genetic library according to claim 3 or 4, wherein, The number of bases in the first extended sequence ranges from 100 to 220.

6. The method of constructing a gene library according to any one of claims 3 to 5, wherein, The third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 1 to SEQ ID No.

4.

7. The method of constructing a genetic library according to claim 2, wherein, The third sequence and the fourth sequence are complementary; The obtained target gene sequence with a sequence length greater than the first gene fragment includes: The template gene fragment and the first gene fragment are ligated under the action of transposase to obtain the target gene sequence.

8. The method of constructing a genetic library according to claim 7, wherein, The transposase includes at least one of Tn5 transposase, Leap-In transposase, and PiggyBac transposase.

9. The method of constructing a genetic library according to claim 7 or 8, wherein, The number of bases in the third sequence ranges from 150 to 180.

10. The method of constructing a genetic library according to any one of claims 7 to 9, wherein, The third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 5 to SEQ ID No.

8.

11. The method of constructing a genetic library according to claim 2, wherein, The third sequence includes: a third random sequence and a sticky t-base terminus connected to one end of the third random sequence; the fourth sequence is complementary to the third random sequence. The obtained target gene sequence with a sequence length greater than the first gene fragment includes: Connect one end of the first sequence to the sticky α-base end; The first linker fragment is obtained by linking the first gene fragment and the template gene fragment through complementary binding of the sticky t bases and sticky a bases. The first linker fragment is circularized and linked by the action of the circularization primer; The target gene sequence was obtained through rolling circle amplification reaction using rolling circle primers.

12. The method of constructing a genetic library according to claim 11, wherein, The number of bases in the third random sequence ranges from 2 to 8.

13. The method of constructing a genetic library according to claim 11 or 12, wherein, The third sequence includes: tnnnnnnn; n is selected from any one of adenine, guanine, cytosine, and thymine.

14. The method of constructing a genetic library according to claim 2, wherein, The third sequence includes: a second extended sequence and a sticky t-base terminus connected to one end of the second extended sequence; The obtained target gene sequence with a sequence length greater than the first gene fragment includes: Connect one end of the first sequence to the sticky α-base end; The first gene fragment and the template gene fragment are joined by complementary binding of the sticky t-base and sticky a-base, and the target gene sequence is obtained after ligase repair.

15. The genetic library construction method according to claim 14, wherein, The number of bases in the second extended sequence ranges from 150 to 180.

16. The method of constructing a genetic library according to claim 14 or 15, wherein, The third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 9 to SEQ ID No.

12.

17. The method of constructing a genetic library according to claim 2, wherein, The third sequence includes: a third extended sequence and a fourth random sequence connected to one end of the third extended sequence, wherein the fourth sequence is complementary to the third extended sequence; The obtained target gene sequence with a sequence length greater than the first gene fragment includes: Either the first sequence or the second sequence is combined complementaryly with the fourth random sequence; The sequence of the first gene fragment that is complementary to the fourth random sequence is ligated to the fourth sequence by a ligase to obtain the target gene sequence.

18. The genetic library construction method according to claim 17, wherein, The third extended sequence has a base number ranging from 125 to 185; and / or, the fourth random sequence has a base number ranging from 15 to 25.

19. The method of constructing a genetic library according to claim 17 or 18, wherein, The third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 13 to SEQ ID No.

16.

20. The genetic library construction method according to any one of claims 1 to 19, wherein, The first gene fragment has less than or equal to 50 bases.

21. A kit comprising: Template gene fragment; The template gene fragment includes: a third sequence and a fourth sequence that is at least partially complementary to the third sequence; The third sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70%, or 80% of any one of SEQ ID No. 1 to SEQ ID No.

16.

22. The application of the kit as described in claim 21 in the construction of cfDNA libraries.

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