Joint for single-stranded DNA library establishment, kit and application thereof

Abstract

The invention provides a linker for single-stranded DNA library establishment, a kit and application thereof. The connector is composed of a first double-chain connector and a second double-chain connector. The first double-chain joint is a double-chain joint which has complementary base pairs and contains a single-end protruding tail end, the 5'end of the first chain is subjected to phosphorylation modification and is used for being connected with the 3 'end of the single-chain DNA, and the 3' end of the second chain is provided with a protruding tail end formed by a random base sequence; the second double-chain joint is a double-chain joint with complementary base pairs and a single-end protruding tail end, the 3'end of the first chain is used for being connected with the 5 'end of the single-chain DNA, and the 5' end of the second chain is provided with a protruding tail end formed by a random base sequence. The two double-chain connectors are connected to the two ends of the single-chain DNA in the same reaction, PCR amplification is directly conducted after connection, the library building process is simplified, sample damage caused by multiple times of purification is avoided, and the method is suitable for low-initial-amount or low-quality sample library building.

Description

A linker for single-stranded DNA library construction, a kit and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of high-throughput sequencing technology, in particular to a linker for single-stranded DNA library construction, a kit and application thereof. BACKGROUND

[0002] High-throughput sequencing technology has developed rapidly and is widely used, and has analyzed many gene functions of humans, animals, plants and microorganisms, and has provided an effective tool for studying biological genetics, development and disease occurrence and development at the whole genome level.

[0003] Extracellular free DNA is a DNA fragment released into the body fluid in the process of apoptosis, necrosis, metabolism, etc., and carries genetic variation information that can reflect the disease, aging, etc. in the body. The body fluids such as serum, plasma, urine, saliva, amniotic fluid, etc. contain free DNA, and due to the convenience of sampling, free DNA sequencing analysis has the advantages of non-invasive / minimally invasive, and is widely used in many scientific researches and clinical detections such as cancer screening, prenatal diagnosis, etc.

[0004] DNA methylation is an important epigenetic mechanism, which regulates gene expression, cell differentiation, gene imprinting and other key biological processes without changing the DNA sequence, and further affects individual growth and development, aging, disease occurrence and development and other life activities. Due to the high stability and strong tissue specificity of DNA methylation, DNA methylation detection has become an important technology for studying gene expression regulation and disease pathogenesis, and in particular, free DNA methylation detection is gradually becoming a commonly used screening technology for liquid biopsy markers.

[0005] At present, the commonly used DNA methylation detection technology based on high-throughput sequencing includes whole genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS). The DNA methylation detection based on high-throughput sequencing mainly identifies whether the cytosine is methylated by converting the unmethylated cytosine into uracil while the methylated cytosine remains unchanged. The commonly used bisulfite treatment for conversion can cause damage to DNA and reduce the quality of DNA. Although the enzyme-based conversion method released in recent years has certain alleviation to the damage to DNA, due to the low content of free DNA in the sample, the methylation detection of free DNA still faces the demand of low initial amount. On the other hand, the double-linker commonly used in enzyme conversion method needs to be methylated to all cytosine bases contained in the linker, resulting in high cost of library construction, which restricts its application in scientific research and medical detection.

[0006] Library construction is a necessary step for high-throughput sequencing. The commonly used library construction process includes end repair, A base addition and double linker ligation. For trace and low-quality samples, the use of conventional double-stranded library construction has low library conversion efficiency and low sequencing data quality. For example, free DNA contains a large number of single-stranded gaps. If the double-stranded library construction method is used, denaturation occurs during the PCR amplification step after the linker ligation, which is easy to break at the gap and thus reduce the yield. The same problem also exists in DNA methylation research. The double linker ligation product obtained by the method of connecting first and then converting is subjected to bisulfite treatment, and a large number of DNA chains are broken and degraded, resulting in a significant decrease in effective templates for downstream amplification reaction, which cannot meet the library construction requirements of low starting amount of free DNA. On the other hand, the cytosine in the double linker needs to be methylated, which leads to high cost of DNA methylation detection, and it is difficult to be widely used in large sample scientific research and clinical detection.

[0007] DNA single-stranded library construction can greatly improve the utilization rate of DNA molecules in the original sample and improve the complexity of the library. However, the commonly used single-stranded library construction technology needs to connect two end adapters in two steps, and after connecting the first adapter, it needs to use the existing DNA single-stranded template to synthesize the complementary double-stranded extension, which increases the probability of introducing cumulative errors. In recent years, the reported adapter ligation method without double-stranded synthesis needs two-step adapter ligation and purification, which increases the reaction time and reduces the library yield, thereby limiting its application in low starting amount and low quality samples.

[0008] Therefore, the library construction technology needs to be improved for trace and low-quality DNA samples, and an effective library construction method suitable for low starting amount samples such as body fluid free DNA and low quality samples such as DNA after methylation transformation needs to be developed.

[0009] SUMMARY

[0010] The purpose of the present application is to provide a new adapter for single-stranded DNA library construction, a kit and its application.

[0011] In order to achieve the above-mentioned purpose, the following technical solutions are adopted in the present application:

[0012] The first aspect of the present application discloses an adapter for single-stranded DNA library construction, which is composed of a first double-stranded adapter and a second double-stranded adapter. The first double-stranded adapter is a double-stranded adapter with complementary base pairs and containing a single-end overhanging end. The 5' end of the first strand has a phosphorylation modification for connecting the 3' end of single-stranded DNA, and the 3' end of the second strand has an overhanging end composed of a random base sequence. The second double-stranded adapter is a double-stranded adapter with complementary base pairs and containing a single-end overhanging end. The 3' end of the first strand is used to connect the 5' end of single-stranded DNA, and the 5' end of the second strand has an overhanging end composed of a random base sequence.

[0013] It should be noted that the linker for single-stranded DNA library construction in the present application can realize the connection of the 5' end and the 3' end of the single-stranded DNA in one reaction, that is, the first double-stranded linker can be connected to the 3' end of the single-stranded DNA, and the second double-stranded linker can be connected to the 5' end of the single-stranded DNA in the same reaction, thereby simplifying the reaction steps of the double-end linker connection. In an implementation mode of the present application, after the single-stranded DNA is connected by the linker of the present application, PCR amplification can be directly performed without purification, thereby realizing the condition that PCR reaction can be met without purification; the library construction process is simplified, and sample loss caused by multiple purifications is also avoided. Therefore, the library construction method based on the linker of the present application can construct a library for a low initial amount or low quality sample, and meet the use demand of high-throughput sequencing, especially can be applied to the library construction and high-throughput sequencing of free DNA, and the methylation high-throughput sequencing of free DNA; in an implementation mode of the present application, the initial input amount of the free DNA high-throughput sequencing can be as low as 250 pg, and the initial input amount of the free DNA methylation high-throughput sequencing can be as low as 2 ng.

[0014] It can be understood that the linker of the present application or the library construction method based on the linker of the present application can not only be applied to the high-throughput sequencing of free DNA or the methylation detection of free DNA, but also can be applied to the high-throughput sequencing or methylation detection of other low initial amount or low quality samples.

[0015] In an implementation mode of the present application, among the total of 8 end points of the four single-stranded oligonucleotides of the first double-stranded linker and the second double-stranded linker, the nucleotides of the remaining 6 end points except the two end points connected to the single-stranded DNA are modified by blocking, so as to prevent the connection of the 5' end phosphate group and the 3' end hydroxyl group between the linkers to cause self-connection between the linkers.

[0016] In an implementation mode of the present application, the blocking modification is C6 spacer modification or NH2-C6 modification at the 5' end, and C6 spacer modification, NH2-C6 modification or dideoxy modification at the 3' end.

[0017] It should be noted that the purpose of the blocking modification is to prevent the non-specific connection of the other ends of the first double-stranded linker and the second double-stranded linker, so as to improve the purity of the library main product. The C6 spacer modification, NH2-C6 modification, C6 spacer modification, NH2-C6 modification or dideoxy modification are only the specific modification modes adopted in an implementation mode of the present application, and other modifications that prevent the connection of the 5' end phosphate group and the 3' end hydroxyl group are not excluded, which are not limited here.

[0018] In an implementation form of the application, the complementary base pairs in the first and second double-linkers have a length of 18-58 bp.

[0019] In an implementation form of the application, the complementary base pairs in the first and second double-linkers have a length of 25-30 bp.

[0020] It should be noted that the complementary base pairs in the first and second double-linkers of the application can be designed according to the library structure requirements, for example, the primer binding sites of the sequencing library can be designed into the complementary base pairs, or other complementary base pairs can be designed according to the requirements of the sequencing platform.

[0021] In an implementation form of the application, the overhanging end composed of random base sequences in the first and second double-linkers has a length of 2-12 bp.

[0022] In an implementation form of the application, the overhanging end composed of random base sequences in the first and second double-linkers has a length of 6-10 bp.

[0023] It should be noted that the overhanging end composed of random base sequences in the application is randomly complementary to the single-stranded DNA fragment, so as to facilitate the connection of the first and second double-linkers to the 3' end and 5' end of the single-stranded DNA, respectively. Therefore, the length of the overhanging end is generally 2-12 bp, which can meet the use requirements, and the overhanging end with a length of 6-10 bp is preferred. The random base sequence can be any combination of A, T, C and G.

[0024] The second aspect of the application discloses a kit for single-stranded DNA library construction, which contains the linker for single-stranded DNA library construction of the application.

[0025] In an implementation form of the application, the kit of the application further contains a primer pair, which contains two single-stranded primers corresponding to the first and second double-linkers respectively for PCR amplification.

[0026] It should be noted that the two single-stranded primers corresponding to the first and second double-linkers respectively for PCR amplification means that one single-stranded primer can bind to the partial sequence or partial complementary sequence of the first double-linker, and the other single-stranded primer can bind to the partial complementary sequence or partial sequence of the second double-linker, so as to realize PCR amplification of the fragments in the combined region.

[0027] In an implementation form of the application, at least one of the two single-stranded primers in the primer pair contains a tag sequence.

[0028] It should be noted that the role of the barcode sequence is to distinguish different samples or DNA sequences, and therefore, the barcode sequence can be designed in one of the two single-stranded primers according to requirements, or the barcode sequence can be designed in the two single-stranded primers respectively.

[0029] In an implementation manner of the present application, the 5' end of one of the two single-stranded primers in the primer pair has a phosphorylation modification.

[0030] It should be noted that the phosphorylation modification designed at the 5' end is to facilitate the circularization of the single strand amplified by the primer in the subsequent circularization process, so as to obtain a single-stranded circular library.

[0031] In an implementation manner of the present application, the kit of the present application further contains a splint oligo for circularizing one of the single-stranded DNA in the PCR amplification product of the primer pair.

[0032] It should be noted that the specific design of the splint oligo can refer to the existing single-stranded circular library construction technology.

[0033] The third aspect of the present application discloses the application of the adapter for single-stranded DNA library construction of the present application or the kit of the present application in library construction of a low initial amount or a low quality sample, high-throughput sequencing of a low initial amount or a low quality sample, methylation library construction or methylation high-throughput sequencing.

[0034] It should be noted that the library construction method based on the adapter of the present application can construct a library for a low initial amount or a low quality sample, and meet the use requirements of high-throughput sequencing; therefore, the adapter for single-stranded DNA library construction of the present application or the kit of the present application can be used for library construction of a low initial amount or a low quality sample, and of course, can be used for high-throughput sequencing of a low initial amount or a low quality sample. Similarly, the adapter for single-stranded DNA library construction of the present application or the kit of the present application can also be used for methylation library construction or methylation high-throughput sequencing.

[0035] The fourth aspect of the present application discloses a library construction method, comprising the following steps:

[0036] In one reaction system, a ligase is used to connect the adapter for single-stranded DNA library construction of the present application to the two ends of the single-stranded DNA of the sample to be tested respectively;

[0037] The primer pair in the kit of the present application is used to perform PCR amplification on the adapter connection product of the single-stranded DNA of the sample to be tested, so as to obtain a sequencing library.

[0038] In an implementation of the present application, the library construction method further comprises using the bridging oligonucleotide in the kit to circularize one single strand in the PCR amplification product, to obtain a single-stranded circular library.

[0039] It should be noted that the library construction method of the present application is particularly suitable for library construction of low starting amount or low quality samples, such as free DNA or other low quality DNA. The sample of the library construction method of the present application is a tissue, cell or body fluid sample, and the body fluid sample can be plasma, serum, urine, cerebrospinal fluid, saliva or other samples containing free DNA. The DNA of the present application is genomic DNA or free DNA extracted from the sample.

[0040] It should be further noted that the library construction method of the present application simplifies the reaction steps, realizes the double-end linker ligation of the single-stranded DNA insert in the same reaction, and then uses a primer pair containing a tag sequence to amplify and add a tag to the linker ligation product, so that the signal can be effectively amplified in a small number of cycles, solving the problem of high requirement for the amount and quality of the starting DNA in the traditional library construction method.

[0041] In an implementation of the present application, in the reaction system for linker ligation, the ratio of the molar concentration of the linker to the molar concentration of the single-stranded DNA is 100-1000.

[0042] Preferably, in the reaction system for linker ligation, a macromolecular crowding agent is further added.

[0043] Preferably, the macromolecular crowding agent is polyethylene glycol.

[0044] It should be noted that in the reaction system for linker ligation, the present application uses high-concentration linkers and adds macromolecular crowding agents, mainly considering the characteristics of low amount and small fragments of free DNA and methylated DNA, and using macromolecular crowding agents and high-concentration linkers to improve the efficiency and quality of linker ligation.

[0045] The fifth aspect of the present application discloses a method for constructing a methylation library, comprising methylating a DNA sample to be tested to obtain single-stranded DNA of the methylated DNA sample to be tested; using a ligase to connect the linker for single-stranded DNA library construction of the present application to both ends of the single-stranded DNA after methylation in a reaction system; using a primer pair in the kit of the present application to perform PCR amplification on the linker ligation product, to obtain a methylation library.

[0046] In an implementation of the present application, the methylation library construction method of the present application further comprises using the bridging oligonucleotide in the kit to circularize one single strand in the PCR amplification product, to obtain a single-stranded circular library.

[0047] It should be noted that the methylation library construction method of the present application can effectively connect the single-stranded DNA after transformation using a non-methylated linker, so as to obtain a high complexity library at a low cost and at a low initial amount. That is, the first double-stranded linker and the second double-stranded linker of the present application can be directly connected to the methylated single-stranded DNA without methylating treatment.

[0048] In an implementation manner of the present application, the ratio of the molar concentration of the linker to the molar concentration of the single-stranded DNA in the reaction system of the linker connection of the methylation library construction is 100-1000.

[0049] Preferably, the reaction system of the linker connection of the methylation library construction is further added with a macromolecular crowding agent.

[0050] Preferably, the macromolecular crowding agent is polyethylene glycol.

[0051] It should be noted that the present application uses a high concentration of linker and macromolecular crowding agent in the reaction system of the linker connection, which is mainly considered the characteristics of small amount and small fragments of the DNA after methylation transformation. The macromolecular crowding agent and the high concentration of linker are used to improve the efficiency and quality of the linker connection.

[0052] The present application has the following beneficial effects:

[0053] The linker for single-stranded DNA library construction of the present application can connect the first double-stranded linker and the second double-stranded linker to both ends of the single-stranded DNA in the same reaction, and after connection, there is no need for purification, and PCR amplification can be directly performed, which simplifies the library construction process and also avoids sample damage caused by multiple purifications. Therefore, the linker and the library construction method of the present application can construct a library for a low initial amount or low quality sample, and meet the use demand of high-throughput sequencing, and are especially suitable for library construction and high-throughput sequencing of free DNA, and high-throughput sequencing of methylation of free DNA. Moreover, the linker of the present application can effectively connect the methylated single-stranded DNA without methylating treatment, which reduces the cost of methylation detection. BRIEF DESCRIPTION OF DRAWINGS

[0054] Fig. 1 is a schematic diagram of the library construction process of methylation detection in the embodiment of the present application;

[0055] Fig. 2 is the analysis result of the library fragments constructed in the embodiment of the present application;

[0056] Fig. 3 is the analysis result of the library fragments constructed in another embodiment of the present application;

[0057] Fig. 4 is the correlation analysis result of the methylation patterns of repeated samples in the embodiment of the present application. DETAILED DESCRIPTION

[0058] For low initial amount samples (such as free DNA) or low quality samples (such as DNA after methylation conversion), there is still a lack of effective library construction methods.

[0059] To this end, the present application creatively develops a single-stranded DNA library construction based on a linker, and a low-cost, high-yield, and high-quality library construction method based on the linker of the present application. Based on the linker and library construction method of the present application, the initial input amount of free DNA high-throughput sequencing can be as low as 250 pg, and the initial input amount of free DNA methylation high-throughput sequencing can be as low as 2 ng.

[0060] The library construction method based on the linker of the present application simplifies the reaction steps, realizes the double-end linker connection of the single-stranded DNA insert fragment in the same reaction, and then uses the primer pair containing the tag sequence to amplify and add the tag to the linker connection product, so as to effectively amplify the signal in a small number of cycles, solving the problem of high requirement for initial DNA input amount and DNA quality in the traditional library construction method. When applied to methylation detection, the linker without methylation treatment can effectively connect the converted single-stranded DNA, so as to obtain a library with high complexity at a low cost at a low initial amount.

[0061] The invention points of the technical solutions provided by the present application include linker sequence design, linker end point closure design, linker random sequence base number design, amplification primer pair sequence design, reaction system optimization design, etc. First, the present application provides a design method of a group of linkers and primers. The linker of the present application includes a double-linker containing a single-end random overhang sequence, and the primer of the present application is a primer containing a barcode sequence corresponding to the linker. Based on the linker and primer of the present application, the present application provides a library construction method for low initial amount and low quality samples. Based on the linker and primer of the present application, the present application provides a methylation library construction method. Based on the linker and primer of the present application, the present application provides a set of library construction kits. The methylation library construction process of the present application is shown in FIG. 1, which includes methylation conversion of double-stranded DNA samples, denaturation to form single-stranded DNA, connection of the linker of the present application to both ends of the single-stranded DNA, and then PCR amplification to obtain a library.

[0062] The linker and primer design method of the present application is used for constructing a sequencing library based on a single-stranded DNA molecule. The linker is a double-linker containing a single-end random overhang sequence, and the primer is a primer pair corresponding to the linker. According to the requirements, a splint oligo for single-stranded circularization can also be included. The double-linker includes a first double-linker and a second double-linker. The primer pair includes two single-stranded primers, one or both of which contains a barcode sequence.

[0063] The sequence of the adaptor is designed according to the structure of the library to be constructed. The double-stranded portion of the adaptor is obtained by cutting a proper number of complementary base pairs from both ends of the inserted fragment. The first double-stranded adaptor is a double-stranded adaptor with a single-end overhang, which has a 5' end of the first strand to be ligated to the 3' end of the single-stranded DNA fragment, and a 3' end of the second strand with a random base sequence overhang. The second double-stranded adaptor is a double-stranded adaptor with a single-end overhang, which has a 3' end of the first strand to be ligated to the 5' end of the single-stranded DNA fragment, and a 5' end of the second strand with a random base sequence overhang. Further, among the 8 end points of the 4 oligonucleotide single strands of the first and second double-stranded adaptors, the nucleotides of 6 end points except for the 2 end points of the nucleotides of the single-stranded DNA fragment are blocked to prevent the ligation of the 5' phosphate group and the 3' hydroxyl group between the adaptors, thereby preventing self-ligation between the adaptors. Further, the blocking modification is a C6 spacer modification or a NH2-C6 modification at the 5' end, a C6 spacer modification, a NH2-C6 modification or a dideoxy modification at the 3' end, or other modifications that can prevent the ligation of the 5' phosphate group and the 3' hydroxyl group, thereby improving the purity of the library product. Further, the length of the complementary base pairs of the first and second double-stranded adaptors is between 18-58 base pairs, preferably 25-30 base pairs. Further, the length of the random base sequence overhang of the second strand of the first and second double-stranded adaptors is between 2-12 bases, preferably 6-10 bases. The random base sequence can be any combination of A, T, C and G.

[0064] The primer pair of the present application comprises two single-stranded primers for PCR amplification corresponding to the sequence of the adaptor. For example, the first primer in the primer pair has a complementary sequence to the first strand of the first double-stranded adaptor. The second primer in the primer pair has a complementary sequence to the second strand of the second double-stranded adaptor. Further, the primer pair has a complementary sequence of more than 15 bp between the corresponding adaptor oligonucleotide strands. The 3' end of the two oligonucleotide single strands of the primer pair has a common sequence of no more than 1 nucleotide to prevent the primer from amplifying non-target regions. One or both primers in the primer pair contain a barcode for distinguishing different samples or DNA sequences. Further, the 5' end of the second primer in the primer pair is phosphorylated. The bridge oligonucleotide can bind to both ends of the single strand containing the barcode forward sequence in the PCR product, thereby facilitating the circularization reaction. According to the above idea, Table 1 and Table 2 provide examples of adaptor and primer design suitable for MGI platform and Illumina platform, respectively, wherein N represents a random base and the X region is a barcode.

[0065] Table 1 Adaptor and primer oligonucleotide sequences suitable for MGI platform

[0066] Table 2 Sequences of adapter and primer oligonucleotides for Illumina platform

[0067] In Table 1 and Table 2, A11 refers to the first strand of the first double adapter, A12 refers to the second strand of the first double adapter, A21 refers to the first strand of the second double adapter, A22 refers to the second strand of the second double adapter, P1 and P2 refer to the first primer and the second primer of the primer pair, i.e. two single-stranded primers, and S1 refers to the bridging oligo (Splint oligo).

[0068] Based on the adapters of the present application, the present application develops a high-throughput sequencing library construction method suitable for low starting amount and low quality DNA samples. The library construction is performed using the adapters and primer pair of the present application, including the following steps:

[0069] Step 1) In a reaction system, the first double adapter and the second double adapter of the present application are respectively connected to the two ends of the single-stranded DNA fragments using a ligase.

[0070] The volume of the ligation reaction system of the present application is generally 15-50 μL; preferably, the volume of the ligation reaction system for free DNA or other low starting amount or low quality DNA library construction is generally 20 μL or less, such as 15 μL, 10 μL, etc., and a macromolecular crowding agent such as polyethylene glycol (PEG) is added to improve the efficiency of the ligation of the DNA insert and the adapter. The working concentration of the adapter is 0.5-2.5 μM. Before the ligation reaction, the DNA is denatured into a single-stranded state. Optionally, before the DNA is denatured, the DNA is treated with a polynucleotide kinase to phosphorylate the nucleotides at the 5' end of the DNA and dephosphorylate the phosphorylated nucleotides at the 3' end, so as to facilitate the smooth occurrence of the ligation reaction. The ligase is T4 DNA ligase, or other DNA ligase. The polynucleotide kinase is T4 polynucleotide kinase. Preferably, after the ligation reaction, the ligation product can be purified, or can be directly used for PCR amplification.

[0071] Step 2) The ligation product obtained in step 1) is subjected to PCR amplification using a primer pair under the action of a DNA polymerase, and the amplification product is purified, thereby obtaining a DNA library. The working concentration of the primer pair is 0.75-6 μM, and the DNA polymerase can be various DNA polymerases. The PCR amplification is generally performed for 8-13 cycles.

[0072] The samples used in this application are tissue, cell, or body fluid samples. Body fluid samples include plasma, serum, urine, cerebrospinal fluid, saliva, and other samples containing cell-free DNA. The DNA is genomic DNA or cell-free DNA extracted from the sample.

[0073] Optionally, the PCR product obtained in step 2) is circularized using a single-stranded circularization bridging oligonucleotide (Splint oligo) with the aid of a ligase to prepare a single-stranded circularized library, wherein the single-stranded circularization bridging oligonucleotide can simultaneously bind to both ends of the single strand containing the barcode sequence in the PCR product. Preferably, uncirculated DNA is removed using a digestive enzyme after circularization.

[0074] Based on the adapters of this application, this application develops a method for constructing a DNA methylation library. Using the adapters and primer pairs of this application, the methylation library is constructed, including the following steps:

[0075] Step 1) Methylate the DNA.

[0076] The methylation conversion method described in this application involves treating DNA with reagents that differentially modify methylated and unmethylated DNA. The conversion can be achieved using methods such as bisulfite, ammonium bisulfite, or enzymatic conversion, which specifically convert unmethylated cytosine, causing the unmethylated cytosine in the DNA to deaminate and convert into uracil, which can be recognized as thymine by DNA polymerase during amplification, while the methylated cytosine remains unchanged. Alternatively, the conversion can be achieved using methods such as TAPS, which specifically convert methylated cytosine, causing the methylated cytosine in the DNA to convert into dihydrouracil, which can be recognized as thymine by DNA polymerase during amplification, while the unmethylated cytosine remains unchanged.

[0077] Step 2) Ligate paired-end adapters to the DNA treated in Step 1) in its single-stranded state. The adapters used for methylation library construction, namely the first and second paired-end adapters of this application, do not require methylation modification of cytosine in their nucleotide sequences. Optionally, before denaturing the DNA to single strands, a polynucleotide kinase can be used to phosphorylate the 5' end of the DNA and dephosphorylate the phosphorylated nucleotides at the 3' end, promoting successful ligation. The ligase is T4 DNA ligase or other DNA ligases. The polynucleotide kinase is T4 polynucleotide kinase.

[0078] Step 3) The ligation product obtained in Step 2) is amplified by PCR amplification primer pair under the action of DNA polymerase to construct a PCR library containing barcode. The DNA polymerase is a uracil-compatible DNA polymerase. The amplification primer pair is two amplification primers corresponding to the adapters, i.e. the primer pair in the kit of the present application. Similarly, the PCR amplification is generally performed for 8-13 cycles.

[0079] Step 4) The PCR amplification product obtained in Step 3) is purified using magnetic beads to remove excess primers and reagent components that may affect subsequent reactions.

[0080] Optionally, the PCR product obtained in Step 4) is subjected to circularization treatment to prepare a single-stranded circular library under the action of a ligase using a single-stranded circular bridging oligonucleotide (Splint oligo) which can bind to both ends of the single strand containing the barcode sequence in the PCR product. Preferably, a digestion enzyme is used to remove the non-circularized DNA after circularization.

[0081] For convenience of use, the adapters and other components of the present application can be assembled into a kit, i.e. a single-stranded DNA library kit of the present application, which contains the adapters for single-stranded DNA library construction of the present application. Further, the kit of the present application also contains a primer pair, i.e. two single-stranded primers for PCR amplification corresponding to the first double-stranded adapter and the second double-stranded adapter, respectively. Further, according to the needs, the kit of the present application can also contain a bridging oligonucleotide (Splint oligo) for circularizing one of the single strands of the PCR amplification product of the primer pair.

[0082] Optionally, the kit of the present application can contain other reagents used in the library construction process in addition to the adapters, primer pair and bridging oligonucleotide, such as ligase, ligation reaction buffer, DNA polymerase and matching reaction buffer, etc. Of course, these reagents can also be purchased separately. Preferably, the DNA polymerase contained in the kit for constructing a methylation library is a uracil-compatible DNA polymerase. Optionally, the methylation library construction kit can also contain a methylation conversion reagent, such as a reagent or reagent set that can differentially modify methylated or non-methylated cytosine, a reagent or reagent set for specific conversion of non-methylated cytosine by bisulfite, sodium bisulfite or enzymatic conversion method, or a reagent or reagent set for specific conversion of methylated cytosine by TAPS conversion method. The present application mainly relates to library construction for high-throughput sequencing, which can be adapted to DNA sequencing library construction without conversion, or to various methylation conversion library construction steps.

[0083] Compared with the existing adapters and library construction methods, the adapter and library construction method for single-stranded DNA library construction has the advantages of wide application range, simple operation steps, automation, low cost, etc.

[0084] Firstly, the library construction method provided by the present application has low DNA starting amount requirement, which is suitable for genomic DNA extracted from tissues or cells, and can also be suitable for low starting amount, low quality or severely degraded DNA, such as free DNA, circulating tumor DNA, DNA after methylation transformation, paraffin-embedded DNA, ancient microbial DNA, etc. When the free DNA used for direct library construction is as low as 250 pg or the free DNA used for library construction after methylation transformation is as low as 2 ng, the library obtained by using the adapter and library construction method of the present application can still be sufficient for downstream detection and analysis.

[0085] Secondly, the adapter and library construction method of the present application is convenient to obtain materials and easy to operate, which can be used for research or packaged into a kit. The adapter and library construction method of the present application uses commonly used reagents such as DNA ligase and DNA polymerase in the laboratory, and devices such as PCR instrument, and can realize library construction for high-throughput sequencing within 3 hours through simple operations such as ligation, amplification and purification and rapid reactions. The library construction method of the present application also has the advantage of automation, which can be suitable for high-throughput detection of large samples.

[0086] Thirdly, the library construction method of the present application can add adapters to both ends of the single-stranded DNA insert fragment through one-step ligation reaction, which reduces the ligation steps and purification steps, simplifies the operation and saves time, and also saves reagent consumption. At the same time, the reduction of the purification step directly reduces the loss of target product in the purification process, which can effectively improve the library yield.

[0087] Fourthly, the adapter of the present application does not need to be methylated when used for constructing a library for methylation detection, which can effectively reduce the library construction cost of methylation detection.

[0088] The present application will be further described in detail through specific examples. The following examples are only for further description of the present application and should not be understood as limiting the present application.

[0089] If not specifically indicated, the technical means used in the examples is the conventional means familiar to those skilled in the art.

[0090] Example 1

[0091] In this example, the free DNA extracted from human serum is constructed into a library. The adapter and primer sequences designed in this example are shown in Table 3, and the underlined sequences in the primer sequences are tag sequences.

[0092] Table 3 Oligonucleotide sequences of free DNA adapter and primer

[0093] The steps of library construction are as follows:

[0094] 1) DNA pretreatment

[0095] Take 1 ng of free DNA solution extracted from serum and place it on ice for 2 minutes, react at 95°C for 5 minutes, and then immediately place it on ice for 5 minutes. The purpose of this step is to denature the DNA into a single-stranded state.

[0096] 2) Ligation reaction

[0097] Prepare the reaction system as follows: 12 μL of single-stranded DNA denatured in the previous reaction, 1 μL of the first double-stranded linker (20 μM), 1 μL of the second double-stranded linker (20 μM), mix thoroughly; then continue to add, 2 μL of 10×T4 ligase buffer, 2 μL of 50% PEG 4000, 0.6 μL of nuclease-free water, 1.4 μL of T4 DNA ligase (600 U / μL), a total of 20 μL.

[0098] After thorough mixing, place the above reaction system in a PCR instrument at 20°C for 60 minutes, and after the reaction is completed, purify and recover the product with 1-fold volume of magnetic beads, and dissolve the purified product in 23 μL of nuclease-free water.

[0099] It should be noted that in the case of small amounts of DNA, magnetic bead purification can also be omitted, and the ligation product can be directly subjected to subsequent PCR amplification.

[0100] 3) PCR amplification

[0101] In the purified DNA ligation product, prepare the PCR reaction system as follows: 22 μL of DNA ligation product, 1.5 μL of the first primer (50 μM), 1.5 μL of the second primer (50 μM), 25 μL of 2×DNA polymerase mixture, a total of 50 μL.

[0102] After thorough mixing, perform the amplification reaction in a PCR instrument according to the following settings: denaturation at 95°C for 3 min, followed by 13 cycles of 95°C for 30 s, 62°C for 30 s, and 72°C for 1.5 min, followed by extension at 72°C for 5 min and standby at 4°C.

[0103] After the amplification reaction is completed, purify and recover the product using 1-fold magnetic beads, and dissolve it in 25 μL of nuclease-free water. Take 1 μL of the purified product and use the Qubit fluorescence quantification system for quantification. The library concentration is 41.2 ng / μL, so the total library yield is about 1030 ng. Take another 1 μL and use the Qsep 100 NGS biological fragment analyzer of Guangding Biology for fragment analysis. The results are shown in Figure 2.

[0104] The results of Figure 2 show that the main peak of the PCR library fragments of serum free DNA is obvious, and the maximum main peak is about 295 bp, which is the PCR product when the insert is one nucleosome; the second peak is about 488 bp, which is the PCR product when the insert is two nucleosomes, and the results are consistent with the expectation.

[0105] The PCR amplification product is reacted at 95°C for 5 minutes, then immediately placed on ice for 5 minutes to denature the DNA into a single-stranded state, and then the PCR product is subjected to circularization treatment using a single-stranded circularization bridging oligonucleotide (Splint oligo) under the action of a ligase, and after circularization, a digestion enzyme is used to remove the DNA that has not been circularized, and the obtained single-stranded circular library can be used for MGI platform sequencing.

[0106] Example 2

[0107] In this example, the free DNA extracted from human serum is subjected to methylation detection, the sample is the same as in Example 1, the adapters, primer pairs and circularization bridging oligonucleotides used are the same as in Example 1, and the only difference is that in the primer pair of this example, the tag sequences used in the two primers are different from those in Example 1. Specifically as follows:

[0108] In the primer pair of this example, the first primer is the sequence shown in Seq ID No. 21, and the second primer is the sequence shown in Seq ID No. 22, and the 5' end has a phosphorylation modification. The underlined part in the primer sequence is the tag sequence.

[0109] Seq ID No. 21:

[0110] Seq ID No. 22:

[0111] The steps of constructing a methylation detection library and performing detection analysis are as follows:

[0112] 1) Bisulfite treatment

[0113] Take 5 ng of free DNA extracted from serum and add 0.05 ng of fragmented unmethylated lambda DNA, use EZ DNA Methylation-Gold TM Kit according to the instructions in the kit, add 130 μL of prepared transformation solution for methylation transformation, and the transformation reaction is completed in a PCR instrument. Purify the solution after transformation according to the instructions and dissolve in 11 μL of TE buffer.

[0114] 2) Pretreatment of DNA after transformation

[0115] Prepare the DNA pretreatment solution according to the following reaction system: 10.5 μL of the DNA sample dissolved in TE buffer obtained in the previous step, 1.2 μL of 10×T4 PNK buffer, 0.3 μL of T4 PNK, for a total of 12 μL.

[0116] Place the above reaction system in a PCR instrument and react at 37°C for 15 minutes, and then at 95°C for 5 minutes. After the reaction at 95°C is completed, immediately transfer to ice and stand for 5 minutes. The purpose of this step is to phosphorylate the 5' end nucleotides of the DNA fragment by using the polynucleotide kinase and dephosphorylate the 3' end phosphorylated nucleotides, and denature the DNA fragment into a single-stranded state.

[0117] 3) Linker ligation

[0118] Prepare the reaction system in the following manner: 12 μL of the single-stranded DNA-containing solution obtained in the previous reaction, 1 μL of the first double-stranded linker (20 μM), 1 μL of the second double-stranded linker (20 μM), and mix well; then continue to add 2 μL of 10×T4 ligase buffer, 2 μL of 50% PEG 4000, 0.6 μL of nuclease-free water, and 1.4 μL of T4 DNA ligase (600 U / μL), for a total of 20 μL.

[0119] After mixing well, place the above reaction system in a PCR instrument and react at 20°C for 60 minutes. After the reaction is completed, purify and recover the product using 1-fold volume of magnetic beads, and dissolve the purified product in 23 μL of nuclease-free water.

[0120] 4) PCR amplification

[0121] Prepare the PCR reaction system in the following manner in the purified DNA ligation product: 22 μL of the DNA ligation product, 1.5 μL of the first primer (50 μM), 1.5 μL of the second primer (50 μM), and 25 μL of KAPA HiFi HotStart Uracil+ReadyMix (2×), for a total of 50 μL.

[0122] After mixing well, perform the amplification reaction in a PCR instrument according to the following settings: denaturation at 95°C for 3 minutes, followed by 13 cycles of 95°C for 30 seconds, 62°C for 30 seconds, and 72°C for 1.5 minutes, and then extension at 72°C for 5 minutes after the cycles are completed, and standby at 4°C.

[0123] After the amplification reaction, 1 times of magnetic beads were used to purify and recover the product, and then dissolved in 25 μL of nuclease-free water. 1 μL of purified product was used for quantification using Qubit fluorescence quantification system. The library concentration was 40 ng / μL, and the total yield of the library was about 1000 ng. Another 1 μL was used for fragment analysis using Qsep 100 NGS biological fragment analyzer of Guangding Biology. The results are shown in Figure 3.

[0124] The results of Figure 3 show that the main peak of the PCR library of serum free DNA is obvious, and the maximum main peak is about 295 bp, which is the PCR product when the insert is one nucleosome; the second peak is about 481 bp, which is the PCR product when the insert is two nucleosomes, and the results are consistent with the expectation.

[0125] 5) Circularization

[0126] Before high-throughput sequencing using the MGI platform, the PCR product was treated with circularization under the action of a circularization oligonucleotide and a ligase, and then the uncircularized nucleic acid molecules were removed using a digestion enzyme. After purification, a library that can be used for high-throughput sequencing was obtained. The specific steps are as follows:

[0127] Take 100 ng of PCR amplification product into a 0.2 mL PCR tube, supplement with TE Buffer to a total volume of 48 μL, place the solution on ice for 2 minutes, 95°C for 5 minutes, then immediately place on ice for 5 minutes. The purpose of this step is to denature the DNA into a single-stranded state.

[0128] Prepare the single-stranded circularization reaction solution as follows: Splint Buffer 11.6 μL, DNA Rapid Ligase 0.5 μL, total 12.1 μL. Add 12.1 μL of circularization reaction solution to the denatured single-stranded PCR amplification product, mix well, and then place in a PCR instrument at 37°C for 30 minutes. After the reaction is completed, take out the PCR tube and immediately proceed to the enzyme digestion reaction.

[0129] Prepare the enzyme digestion reaction solution as follows: Digestion Buffer 1.4 μL, Digestion Enzyme 2.6 μL, total 4 μL. Add 4 μL of enzyme digestion reaction solution to the solution after circularization reaction, mix well, and then place in a PCR instrument at 37°C for 30 minutes. After the reaction is completed, add 7.5 μL of Digestion Stop Buffer to the reaction solution, mix well, and then purify using 2.5 times of magnetic beads, and dissolve in 30 μL of TE Buffer.

[0130] 6) Sequencing and data analysis

[0131] The single-stranded circular library was sequenced using the MGI platform. Specifically, 7 ng of the DNA single-stranded circle obtained in the previous reaction was taken as a template for rolling circle amplification to prepare DNA nanoballs. The DNA nanoballs were subjected to double-end 100 bp sequencing (PE100) using a DNBSEQ-T7 sequencer. Each sample yielded about 386M read pairs and 77G bp of raw data.

[0132] The raw data was subjected to a quality control process to remove adapter sequences and low-quality reads to obtain data that can be used for alignment. Then, the sequences passing the quality control were aligned to the reference genome using bitmapperBS software to obtain basic analysis results. The methylation pattern correlation between repeated samples was calculated using methylkit. The quality control and basic analysis results of the 7 repeated samples are shown in Table 4, and the methylation pattern correlation results between the 7 repeated samples are shown in FIG. 4.

[0133] Table 4: Quality control analysis data of repeated samples

[0134] The results in Table 4 show that the effective data rate is 90.9±1.59%, the proportion of bases reaching Q20 is 96.5±0.18%, the proportion of bases reaching Q30 is 89.3±0.53%, the unique alignment rate is 87.7±1.02%, the PCR repeat rate is 23.4±5.36%, the data utilization rate after deduplication is 61.1±3.97%, the genome coverage and effective depth are 90.8±0.52% and 15.3±2.59×, respectively, the CpG coverage and average depth are 83.3±1.36% and 7.2±1.16×, respectively, and the CT conversion rate of lambda DNA is 99.4±0.05%. These indicators show that the adapter and library construction method in this example can effectively capture free DNA fragments after bisulfite conversion, and the quality of the raw data is high, with more than 89% reaching the Q30 level. The data utilization rate after removing PCR repeats can reach 60%. FIG. 4 shows the methylation pattern correlation (R 2 ) between the 7 samples. The correlation between each two samples is high, all above 0.91.

[0135] The above content is a further detailed description of the present application in combination with specific embodiments, and cannot be regarded as limiting the specific implementation of the present application to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, some simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

A linker for single-stranded DNA library construction, characterized in that: consists of a first double-stranded adaptor and a second double-stranded adaptor; the first double-stranded adaptor is a double-stranded adaptor with complementary base pairs and containing a single-end overhang, wherein the 5' end of the first strand has a phosphorylation modification for connecting the 3' end of the single-stranded DNA, and the 3' end of the second strand has an overhang consisting of a random base sequence; the second double-stranded adaptor is a double-stranded adaptor with complementary base pairs and containing a single-end overhang, wherein the 3' end of the first strand is for connecting the 5' end of the single-stranded DNA, and the 5' end of the second strand has an overhang consisting of a random base sequence. The linker for single-stranded DNA library construction according to claim 1, characterized in that: of the four single-stranded oligonucleotides of the first double-stranded adaptor and the second double-stranded adaptor, except for the two end points connected to the single-stranded DNA, the remaining six end points are modified by blocking to prevent the 5' end phosphate group and the 3' end hydroxyl group of the adaptors from connecting to cause self-ligation between the adaptors; preferably, the blocking modification is a C6spacer modification or a NH2-C6modification at the 5' end, a C6spacer modification, a NH2-C6modification or a dideoxy modification at the 3' end. The linker for single-stranded DNA library construction according to claim 1 or 2, characterized in that: the length of the complementary base pairs in the first double-stranded adaptor and the second double-stranded adaptor is 18-58 bp; preferably, the length of the complementary base pairs in the first double-stranded adaptor and the second double-stranded adaptor is 25-30 bp; preferably, the length of the overhang consisting of a random base sequence in the first double-stranded adaptor and the second double-stranded adaptor is 2-12 bp; preferably, the length of the overhang consisting of a random base sequence in the first double-stranded adaptor and the second double-stranded adaptor is 6-10 bp. A kit for single-stranded DNA library construction, characterized by: the kit further comprises a primer pair, wherein the primer pair comprises two single-stranded primers for PCR amplification corresponding to the first double-stranded adaptor and the second double-stranded adaptor, respectively; The kit according to claim 4, characterized in that: preferably, at least one of the two single-stranded primers in the primer pair comprises a tag sequence; preferably, the 5' end of one of the two single-stranded primers in the primer pair has a phosphorylation modification. the kit further comprises a bridging oligonucleotide for circularizing one of the single-stranded products of the primer pair by PCR amplification. The kit according to claim 5, characterized in that: the adaptor for single-stranded DNA library construction according to any one of claims 1-3 or the kit according to any one of claims 4-6 is used in library construction of a low starting amount or a low quality sample, high-throughput sequencing of a low starting amount or a low quality sample, methylation library construction, or methylation high-throughput sequencing. comprises the following steps, in one reaction system, the adaptor for single-stranded DNA library construction according to any one of claims 1-3 is connected to the two ends of the single-stranded DNA of the sample to be tested using a ligase, respectively; A method of library construction, characterized in that: the primer pair in the kit according to claim 5 is used to perform PCR amplification on the adaptor connection product of the single-stranded DNA of the sample to be tested, to obtain a sequencing library. comprises performing methylation conversion on the DNA sample to be tested, to obtain single-stranded DNA of the methylation-converted DNA sample to be tested; ​ A method of methylation library construction, characterized in that: ​ In a reaction system, the adapters for single-stranded DNA library construction according to any one of claims 1-3 are respectively connected to two ends of the single-stranded DNA of the methylation-transformed DNA sample to be tested by using a ligase; The product connected by the adapters is subjected to PCR amplification by using the primer pair in the kit according to claim 5, so as to obtain a methylation library. The method according to claim 8 or 9, characterized in that Further comprising that one single strand in the PCR amplification product is circularized by using the bridging oligonucleotide in the kit according to claim 6, so as to obtain a single-stranded circular library; Preferably, in the reaction system for adapter connection, the ratio of the molar concentration of the adapters to the molar concentration of the single-stranded DNA is 100-1000; Preferably, in the reaction system for adapter connection, a macromolecular crowding agent is further added; Preferably, the macromolecular crowding agent is polyethylene glycol.

Images