WO2023040723A1 - Sequencing linker, construction method, nanopore library construction kit and use - Google Patents

Abstract

A sequencing linker, a construction method, a nanopore library construction kit, and the use. The sequencing linker comprises a linker chain and a target linker, wherein the linker chain comprises a nanopore guiding chain and a first linking group arranged in the preset sequencing direction, and the nanopore guiding chain is used for guiding a to-be-tested sequence to pass through a nanopore sequencing channel; and the target linker comprises a second linking group that performs a click chemical reaction with the first linking group, and the second linking group is used for being linked to the to-be-tested sequence.

Description

Sequencing linker, construction method, nanopore library construction kit and application

Cross References to Related Applications

This application claims the priority of the Chinese patent application 202111076002.5 entitled "Sequencing adapter, construction method, nanopore library construction kit and application" submitted on September 14, 2021, the entire content of which is incorporated herein by reference.

technical field

The application belongs to the technical field of gene sequencing, and in particular relates to a sequencing linker, a construction method, a nanopore library construction kit and applications.

Background technique

In related technologies, regardless of second-generation high-throughput sequencing or nanopore sequencing, it is necessary to connect the sequencing linker and the sequence to be tested by DNA ligase during the library construction process, and then perform sequencing on the machine. Compared with the second-generation sequencing platform in the related art, the read length of the nanopore sequencing platform is longer, and the added DNA ligase will not only connect the sequencing adapter and the sequence to be tested, but also cause self-ligation of some sequences to be tested, seriously Interfering with the analysis of subsequent sequencing sequences. After the sequencing adapter and the sequence to be tested are ligated by DNA ligase, purification must be performed to remove the components in the ligation reaction, so as not to affect the subsequent sequencing on the machine. Purification processing steps will prolong the library preparation time and reduce the sequencing efficiency.

Contents of the invention

The embodiment of the present application provides a sequencing linker, a construction method, a nanopore library construction kit and its application, which can avoid the use of DNA ligase, thereby eliminating the need for purification, improving sequencing efficiency, and avoiding template self-ligation caused by the use of ligase. The artificial introduction of chimeric sequences affects the analysis of structural variation.

On the one hand, the embodiment of the present application provides a sequencing adapter, and the sequencing adapter includes:

Adapter top strand, including a nanopore guide strand and a first linking group arranged along a preset sequencing direction, the nanopore guide strand is used to guide the sequence to be tested through the nanopore sequencing channel;

The target linker includes a second linking group that performs a click chemical reaction with the first linking group, and the second linking group is used for linking with the sequence to be tested.

Optionally, the target linker also includes a transposase recognition sequence linked to the second linker.

Optionally, the target linker further includes a linking sequence connected to the second linking group, and along the predetermined sequencing direction, a protruding cohesive end is provided at the end of the linking sequence away from the second linking group.

Optionally, the target linker also includes a primer corresponding to the upstream or downstream of the sequence to be tested, and the second linking group is connected to the primer.

Optionally, when the target linker includes a linking sequence connected to the second linker, the target linker also includes a polymerase-resistant modification between the second linker and the primer; optionally, polymerase-resistant modification Decorated for dspacer.

Optionally, one of the first linking group and the second linking group is cyclooctene, and the other is tetrazine; Optionally, the first linking group is cyclooctene, and the second linking group is tetrazine Azinyl;

Or, one of the first linking group and the second linking group is diphenylcyclooctyne, and the other is an azido group.

Optionally, the molar amount of the adapter top strand is less than the molar amount of the target linker.

Optionally, the sequencing adapters also include:

The adapter bottom strand includes an adapter complementary strand that is partially complementary to the nanopore guide strand.

Optionally, the bottom strand of the adapter also includes a first sticky end sequence, and the first sticky end sequence and the complementary strand of the adapter are connected along the sequencing direction; the target linker also includes a second sticky end sequence complementary to the first sticky end sequence, and the second The linking group and the second cohesive end sequence are arranged along the sequencing direction.

Optionally, the molar quantity ratio of the adapter top strand and the adapter bottom strand is 1:1, and the molar quantity of the adapter top strand is less than the molar quantity of the target linker head.

On the other hand, the embodiment of the present application provides a method for constructing a library. For example, the above-mentioned sequencing linker is applied to the construction method, and the construction method includes:

Sequencing adapters are provided as above:

Connect the target connector to the sequence to be tested;

A click chemical reaction occurs between the first linking group and the second linking group to obtain a sample library.

On the other hand, the embodiment of the present application provides a nanopore library construction kit, which includes the above-mentioned sequencing adapter and polynucleotide binding protein.

In another aspect, the embodiment of the present application provides an application of the above-mentioned sequencing linker, method, or nanopore library construction kit in characterizing biopolymers or preparing products for characterizing biopolymers.

The sequencing linker, construction method, nanopore library construction kit and application of the embodiments of the present application, by setting the first linking group on the top strand of the linker, and setting the second linking group for connecting the sequence to be tested at the same time, so that the linker top strand The sequence to be tested is linked by a click chemical reaction through the first linking group and the second linking group, thereby eliminating the need to use T4 DNA ligase, and eliminating the need for purification before sequencing on the machine, improving sequencing efficiency; by setting nanopores The guide strand enables the sequencing connector connected with the sequence to be tested to accurately identify the nanopore, and guides the connected sequence to be tested to pass through the nanopore sequencing channel of the sequencer.

Description of drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the accompanying drawings that need to be used in the embodiments of the present application. Additional figures can be derived from these figures.

Figure 1 is a schematic diagram of a partial chemical formula of the linker top chain in one embodiment of the present application;

Fig. 2 is a partial chemical formula schematic diagram of the target linker in one embodiment of the present application;

Fig. 3 is a partial chemical formula schematic diagram of the annealed product of the linker top chain shown in Fig. 1 and the target linker head shown in Fig. 2;

Fig. 4 is a schematic structural diagram of a sequencing linker in an embodiment of the present application;

Fig. 5 is a schematic structural diagram of a sequencing linker in another embodiment of the present application;

Fig. 6 is a schematic structural diagram of a sequencing linker in another embodiment of the present application;

Figure 7 is a polyacrylamide gel electrophoresis figure of an embodiment of the present application;

Fig. 8 is a nanopore sequencing signal diagram of an embodiment of the present application;

Figure 9 is a polyacrylamide gel electrophoresis diagram of another embodiment of the present application;

Figure 10 is a polyacrylamide gel electrophoresis figure of another embodiment of the present application;

Fig. 11 is a polyacrylamide gel electrophoresis image of another embodiment of the present application.

Detailed ways

The characteristics and exemplary embodiments of various aspects of the application will be described in detail below. In order to make the purpose, technical solution and advantages of the application clearer, the application will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described here are only intended to explain the present application rather than limit the present application. It will be apparent to one skilled in the art that the present application may be practiced without some of these specific details. The following description of the embodiments is only to provide a better understanding of the present application by showing examples of the present application.

It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is a relationship between these entities or operations. There is no such actual relationship or order between them. Furthermore, the term "comprises", "comprises" or any other variation thereof is intended to cover a non-exclusive inclusion such that a process, method, article, or apparatus comprising a set of elements includes not only those elements, but also includes elements not expressly listed. other elements of or also include elements inherent in such a process, method, article, or device. Without further limitations, an element defined by the statement "comprising..." does not exclude the presence of additional identical elements in the process, method, article or device that includes the element.

In order to solve related technical problems, the embodiments of the present application provide a sequencing linker, a construction method, a nanopore library construction kit and applications. The following firstly introduces the sequencing linker provided in the embodiment of the present application.

The sequencing adapter includes an adapter top strand and a target adapter, the adapter top strand includes a nanopore guide strand and a first linking group arranged along a preset sequencing direction, and the nanopore guide strand is used to guide the sequence to be tested through the nanopore sequencing channel; the target The linker includes a second linking group that performs a click chemical reaction with the first linking group, and the second linking group is used for linking with the sequence to be tested.

The sequencing linker provided in this application is applied to a nanopore sequencing platform. Specifically, polynucleotides can be sequenced, and polynucleotides include DNA and/or RNA. Nanopore sequencing includes a nanopore sequencing channel through which the template strand and/or complementary strand of the sequence to be tested passes, and detects changes in electrical signals such as currents caused when the template strand and/or complementary strand of the sequence to be tested pass through the nanopore sequencing channel, Characterize the sequence to be tested. For example, the size information, sequence information, identity information, modification information, etc. of the analyte can be obtained according to the current change information.

The preset sequencing direction is the direction set by those skilled in the art according to the requirements of the sequence to be tested or the nanopore sequencing platform. The preset sequencing direction can specifically include performing along the 5' end to the 3' end of the sequence to be tested, or along the from the 3' end to the 5' end of the sequence. When the preset sequencing direction is from the 3' end to the 5' end, the first linking group is connected to the 5' end of the nanopore guide strand; when the preset sequencing direction is from the 5' end to the 3' end, the first The linking group is attached to the 3' end of the nanopore guide strand. The sequencing adapter can also include an adapter bottom strand that is partially or completely complementary to the adapter top strand, and the adapter bottom strand can also include a sequence recognized by the nanopore sequencing channel to guide the sequence to be tested to reach the vicinity of the nanopore sequencing channel; the adapter bottom strand can also be Set up polynucleotide binding proteins, such as helicase binding sites, etc. as required, so as to facilitate each small molecule of the sequence to be tested to pass through the nanopore sequencing channel in sequence. In one embodiment, the adapter top strand and the adapter bottom strand can be composed of two partially complementary oligomers, forming a y-shaped structure after annealing; the adapter top strand and the adapter bottom strand can also be a single oligomer that is internally complementary, forming Issuer structure. Those skilled in the art can design the top strand of the adapter and the bottom strand of the adapter according to the nanopore sequencing platform, the type of nanopore sequencing channel, the annealing temperature requirement, and the design of the identification tag. The top strand of the adapter and the bottom strand of the adapter can also include a variety of marker sequences, such as the detection date marker sequence, the sample number marker sequence, etc., to distinguish different sequencing adapters through different sequencing of bases; of course, the adapter top strand and the adapter bottom strand can also Including other biologically functional sequences and modifications.

Click chemistry is to quickly complete the chemical synthesis of different molecules through the splicing of small units. In this application, the first linking group and the second linking group can be connected together based on a click chemical reaction, that is, by connecting the first linking group and the second linking group, the nanopore guide strand and the sequence to be tested are connected , so as to further realize that the nanopore guide strand pulls the sequence to be tested through the nanopore sequencing channel. The first linking group and the second linking group may be linked by carbon-carbon multi-bond addition reaction, linked by nucleophilic ring-opening reaction, click chemical reaction such as cycloaddition reaction. For example: the first linking group can be cyclooctene (TCO), dibenzocyclooctyne (DBCO), difluorinated cyclooctyne (DIFO), bicyclonononyne (BCN) or dibenzocyclooctyne Any one of (DICO). The second linking group can be azido (N3), tetrazine (TZ) and the like. Those skilled in the art can understand that this embodiment does not limit the first linking group and the second linking group to which kind of groups can undergo click chemical reaction.

The second linking group in this application can be linked to the sequence to be tested in various ways. For example, the second linking group is coupled to the primer, and the sequence to be tested is used as a template to amplify by PCR (polymerase chain reaction) to obtain a plurality of sequences to be tested connected with the second linking group; the second linking group can also be The group is coupled to the sequence with the transposon, and the sequence connected with the second linking group is connected with the sequence to be tested by a transposase, thereby obtaining the sequence to be tested connected with the second linking group; Ligation connects the second linking group with the sequence to be tested, phosphorylates the 5' end of the test sequence and adds adenine (A) to the 3' end, and at the same time connects the second linking group to the 5' end of the preset sequence, Thymine (T) is added to the 3' end of the predetermined sequence, and the second linking group and the sequence to be tested are complementary connected by adenine (A) and thymine (T). This embodiment does not limit the connection method between the second linking group and the sequence to be tested.

In this application, by setting the first linking group on the top strand of the adapter and setting the second linking group for connecting the sequence to be tested at the same time, the top strand of the adapter and the sequence to be tested are connected through the first linking group and the second linking group. The groups are linked by click chemical reaction, thereby eliminating the need for DNA ligase and purification before sequencing on the machine, improving sequencing efficiency; by setting the top strand of the adapter and the bottom strand of the adapter, the sequencing adapter with the sequence to be tested can be connected It can accurately identify the nanopore, and guide the sequence to be connected to pass through the nanopore sequencing channel of the sequencer; at the same time, it avoids the self-ligation of the template caused by the use of ligase, artificially introducing chimeric sequences, and affecting the analysis of structural variation.

When the second connecting group is connected to the sequence to be tested through a primer, the target linker also includes a primer corresponding to the upstream or downstream of the sequence to be tested, and the second connecting group is connected to the primer. The bottom strand of the adapter also includes a first sticky end sequence, and the first sticky end sequence and the complementary strand of the adapter are connected along the sequencing direction; the target linker also includes a second sticky end sequence complementary to the first sticky end sequence, a second linking group and The second sticky end sequence is set along the sequencing direction. In this application, the primer can be an upstream primer or a downstream primer, and the second cohesive end sequence and the second linking group are set together on the same primer. For the convenience of description, the following will be described by taking the second connecting group, the second cohesive end and the upstream primer connection, and the sequencing direction is from the 5' end to the 3' end as an example. Through the target linker and the corresponding downstream primers, PCR amplification can be performed using the sequence to be tested as a template, and the generated amplification product is the sequence to be tested carrying the target linker; after annealing, the first sticky end and the second sticky end Complementary ends occur, and the top strand of the adapter and the target linker are spatially close together, thereby improving the efficiency of the click chemical reaction between the first chemical group and the second chemical group. Further increase the stability of target junction, junction bottom strand and junction top strand junctions.

Further, the target linker also includes a polymerase resistance modification located between the second linking group and the sequence to be tested. Polymerase resistance modification is a chemical modification that prevents the polymerase from continuing to extend, such as: 3' phosphorylation modification, dideoxycytidine (3'ddC Dideoxy-C), etc. The resistance polymerase modification can also be modified by dspacer, dspacer is a nucleotide with only a phosphate backbone but no base, so its spatial structure is smaller than that of normal nucleotides, so that when DNA polymerase extends to the position modified by dspacer, because It cannot be recognized and slips off from the amplified test sequence, thereby stopping the polymerization.

In one embodiment, one of the first linking group and the second linking group is cyclooctene (TCO), and the other is tetrazine (Tz). In another embodiment, one of the first linking group and the second linking group is diphenylcyclooctyne (DBCO), and the other is azido (N3). The chemical formula of TCO can be shown in Figure 1, and the chemical formula of Tz can be shown in Figure 2. Under the interaction of Tco and Tz, the following chemical formula is formed as shown in Figure 3, wherein, R1 and R2 are the nanopore guide chain and the to-be-measured sequence. Those skilled in the art can understand that R1 and R2 can also include other sequences or modifications, so that TCO or Tz is connected to R1 to form the top chain of the linker in each embodiment of the application, and TCO or Tz is connected to R2 to form each embodiment of the application. The target connection header in the example. Figure 2 shows 1,2,4,5-tetrazine, those skilled in the art can understand that, in the present application, tetrazine (Tz) can also be 1,2,3,4-tetrazine, 1,2 , 3,5-tetrazine or its derivatives to achieve connection through click chemical reaction, similarly the cyclooctene (TCO) in this application is not limited to the group shown in Figure 1, it can also be the group shown in Figure 1 group derivatives.

Further, the molar ratio of the adapter top strand and the adapter bottom strand is 1:1, so as to ensure that the adapter top strand and the adapter bottom strand can all complement each other one by one, avoiding the formation of free single strands, which will affect the subsequent detection on the machine. Since not all adapter top strands can be connected to the target connector one-to-one, the molar quantity of the adapter top strand is set to be smaller than the molar quantity of the target connector to ensure that all the added adapter top strands can be connected to the target connector to avoid The free joint top chain improves the accuracy and speed of subsequent on-board testing. The molar ratio of the linker top strand to the target linker can be 1:1-2.

Those skilled in the art can design the primers in the target linker by themselves according to the structure of the sequence to be tested, and determine the amplification system according to the length of the sequence to be tested, the CG content, etc., so that the amplified product has a second linking group sequence to be tested. Please refer to Figure 4. In one embodiment, the sequencing direction is from 5' to 3', and the top strand 11 of the adapter is: 5'- AATGT ACTTC GTTC AGTTA CGTAT TGC - TCO -3';

The linker bottom chain 12 is: 5'- GCCG - GCAAT ACGT AACTG AACGA AGTAC ATT -GAGGC GAGCG GTCAA T-3';

The upstream primer is 228-F: 5'- Tz - CGGC - dspacer -ATCGGCATCAGAGCAGAT TGTA-3';

The downstream primer is 228-R:5'-AACGTCGTGACTGGGAAAAC-3'.

Among them, "CGGC" in the upstream primer 228-F is the second sticky end, " GCCG " at the 5' end of the adapter bottom strand 12 is the first sticky end, and the "CGGC" sequence and the " GCCG " sequence are connected by base complementary pairing ; " AATGTACTTCGTTCAGTTACGTATTGC " in the top strand 11 of the adapter is a polynucleotide that simulates the structure of the nanopore guide strand, and " GCAATACGTAACTGAACGAAGTA CATT " in the base strand 12 of the adapter is a complementary sequence to the simulated nanopore guide strand. There is also a sequence at the ' end that is not complementary to the top strand of the adapter, so that when the top strand 11 of the adapter is paired with the bottom strand 12 of the adapter, the top strand 11 of the adapter and the bottom strand 12 of the adapter form a Y-shaped structure. Tz in the upstream primer 228-F is the second linking group, TCO in the top strand of the adapter is the first linking group, the upstream primer 228-F and the downstream primer 228-R use the sequence to be tested as a template, and the amplified product obtained is 228-F-the sequence to be tested, the Tz group in the 228-F-the sequence to be tested and the TCO group in the top chain of the linker are linked by click chemistry, so as to realize the connection between the nanopore guide strand and the sequence to be tested. Those skilled in the art can understand that the length and base type of the first sticky end and the second sticky end can be designed according to the spatial structure, sequence length, CG content, etc. of the upstream primer and the top strand of the adapter. It is sufficient to ensure that the first cohesive end and the second cohesive end can be paired through complementary bases.

Please refer to FIG. 5 , when the second linking group is linked to the sequence to be tested by a transposase, the target linker also includes a transposase recognition sequence linked to the second linking group. The transposase recognition sequence is a sequence that can be recognized by the transposase, and the transposase and the transposase recognition sequence can combine to form a transposase complex. Under the action of transposase, the second linking group is introduced into the end of the sequence to be tested. Those skilled in the art can understand that the target linker not only includes the second linking group connected to the transposase recognition sequence, but also includes the first cohesive end or other sequences that need to be introduced or bound proteins. In one embodiment, the sequencing direction is from the 5' end to the 3' end, and the adapter top strand 11 is: 5'- AATGT ACTTC GTTC AGTTA CGTAT TGC - TCO -3';

The linker bottom chain 12 is: 5'- GCCG - GCAAT ACGT AACTG AACGA AGTAC ATT -GAGGC GAGCG GTCAA T-3';

Transposase recognition sequences include complementary:

Tn5-Top: TZ-CGGC AGATGTGTATAAGAGACAG ;

Tn5-bottom: CTGTCTCTTATACACATCT.

Among them, "CGGC" in the transposase recognition sequence Tn5-Top is the second sticky end, " GCCG " at the 5' end of the adapter bottom strand is the first sticky end, and the "CGGC" sequence and the " GCCG " sequence are complemented by base Paired connection; " AATGTACTTCGTTCAGTTACGTATTGC " in the top strand of the adapter is a polynucleotide that simulates the structure of the nanopore guide strand, and " GCAATACGTAACTGAACGAAGTA CATT " in the bottom strand of the adapter is a complementary sequence to the nanopore guide strand. Tz in the transposase recognition sequence Tn5-Top is the second linking group, and TCO in the top strand of the linker is the first linking group. After the transposase recognition sequence Tn5-Top and Tn5-bottom are annealed and incubated with Tn5 transposase, the transposase complex can be obtained. After the transposase complex is incubated with the sequence to be tested, "TZ-CGGC" and the sequence to be tested Connection, so as to realize the connection between the nanopore guide strand and the sequence to be tested. In another embodiment, the top strand of the adapter is "5'-(iSpC3) ₃₀ -GCGTG ACTAT CGGAC TCGTG GTC TTTTT TTTTT-(iSp18) ₄ -GTCAG TTCGC TTCTT ACGCA -TCO-3'", and the bottom strand of the adapter is "5 '-GCCG TGCGT AAGAA GCGAA CTGAC AGTCC AGCAC CGACC T-3'", in this embodiment, the top strand of the linker also includes (iSpC3) ₃₀ modification, (iSp18) ₄ modification, and "TTTTT TTTTT" flexible sequence, etc.

Please refer to Figure 6, when the second linking group is connected to the sequence to be tested by TA connection, the target linker also includes the linking sequence connected to the second linking group, and the target linker also includes the linking sequence connected to the second linking group. As for the connection sequence, along the preset sequencing direction, the end of the connection sequence away from the second connection group is provided with a protruding sticky end, and the connection sequence is connected to the sequence to be tested by TA connection through the protruding sticky end. For example, a protruding thymidine nucleotide terminus can be provided at the 3' end of the linking sequence. Those skilled in the art can understand that the end of the linking sequence away from the second linking group is not a blunt end, and the protruding thymine nucleotide end can be connected with the sequence to be tested by adding A treatment through base pairing, or The protruding adenine nucleotide terminal can be connected with the sequence to be tested by adding T through base pairing. Likewise, those skilled in the art can understand that the target linker not only includes the second linker gene, but also includes the first cohesive end or other sequences that need to be introduced. The design of the connecting sequence is based on the principles of not being complementary to the sequence to be tested, the top strand of the adapter, etc., and the appropriate CG content. In one embodiment, the sequencing direction is from the 5' end to the 3' end, and the adapter top strand 11 is: 5'-(iSpC3) ₃₀ -GCGTG ACTAT CGGAC TCGTG GTC TTTTT TTTTT-(iSp18) ₄ -GTCAG TTCGC TTCTT ACGCA -TCO -3';

The linker bottom chain 12 is: 5'-GCCG TGCGT AAGAA GCGAA CTGAC AGTCC AGCAC CGACC T-3';

Linker sequences include complementary:

TA-Top: TZ-CGGC AGATGTGTATAAGAGACAG T;

TA-bottom: 5p- CTGTCTCTTATACACATCT .

Among them, "CGGC" in the connection sequence TA-Top is the second cohesive end, " GCCG " at the 5' end of the adapter bottom strand is the first cohesive end, and the "CGGC" sequence and the " GCCG " sequence are connected by base complementary pairing; Tz in the linking sequence TA-Top is the second linking group, and TCO in the linker top chain is the first linking group. The connection sequence TA-Top and TA-bottom are annealed and mixed with the test sequence treated with adenine nucleotide (A), the connection sequence is connected with the test sequence, and "TZ-CGGC" is connected with the top strand of the adapter, so as to realize The nanopore guide strand is connected to the sequence to be tested.

In addition, in combination with the sequencing adapters in the above embodiments, the embodiments of the present application also provide a method for constructing a library. For example, the above sequencing adapters are applied to the construction method, and the construction methods include:

Step S1, providing the sequencing adapters as described above;

Step S2, connecting the target connector with the sequence to be tested;

Step S3, making a click chemical reaction between the first linking group and the second linking group to obtain a sample library.

In step S1, various methods can be used to prepare the above-mentioned sequencing adapters. Similarly, in step S2, various methods can be used to connect the second linking group to the sequence to be tested. For details, please refer to the methods listed in the above examples, which will not be repeated here. A repeat.

Step S3 of the present application specifically includes: annealing the top strand of the adapter and the bottom strand of the adapter to obtain an annealed product; contacting the annealed product with the sequence to be tested to which the second linking group is attached, so that the first linking group and the second linking group The two linking groups undergo a click chemical reaction to obtain a sample library. It is only necessary to mix the annealed product prepared in step S2 with the sequence to be tested prepared in step S1 connected with the second linking group, and a click chemical reaction can occur between the first linking group and the second linking group to realize the target sequence. The sequence is connected to the top strand of the adapter to prepare a sample library. In step S3, there is no need to add DNA ligase, so that step S3 does not need to be purified, and a sample library that can be used for further biomolecular characterization can be prepared.

The sequencing adapters provided in the embodiments of the present application have the same technical effect due to the use of the sequencing adapters provided in any of the above embodiments, and will not be repeated here.

In addition, in combination with the sequencing adapters in the above embodiments, the embodiments of the present application also provide a nanopore library construction kit. The nanopore library construction kit includes the above-mentioned sequencing adapter and polynucleotide binding protein. The adapter top strand and target adapter in the sequencing adapter can be stored in a freeze-dried manner, or in a buffer storage method. Those skilled in the art can choose an appropriate way to store the sequencing adapters according to reasons such as operation convenience, transportation convenience, or storage stability. Polynucleic acid binding protein can comprise: one or more in helicase, exonuclease, telomerase, topoisomerase, reverse transcriptase, translocase and/or polymerase, for making The translocation rate of the sequence to be tested through the nanopore sequencing channel is slower than that of nucleic acid binding proteins, helicases, exonucleases, telomerases, topoisomerases, reverse transcriptases, translocases, and/or polymerases Indexing speed when present.

In one embodiment, the nanopore library construction kit includes a first solution and a second solution, the first solution includes a first buffer, and an adapter top strand and an adapter bottom strand placed in the first buffer; the second solution includes a second buffer and the target adapter placed in the second buffer. The nanopore library construction kit provided in the embodiment of the present application has the same technical effect due to the use of the sequencing linker provided in any of the above embodiments, and will not be repeated here.

The first buffer solution can be a buffer solution such as phosphate buffer solution, TE buffer solution (Tris-EDTA buffer solution), etc. that can stably preserve the structure of the adapter top strand and the adapter bottom strand. The first buffer can be a DNA oligonucleotide annealing buffer (Annealing Buffer), thereby stabilizing the structure of the adapter top strand and the adapter bottom strand, while ensuring that the adapter top strand and the adapter bottom strand structure can effectively hybridize. For example: the first buffer includes 10mM Tris, 50mM NaCl and 1mM EDTA, pH 7.5-8.0. By storing the adapter top strand and the adapter bottom strand in the first buffer solution, the stability of the structure of the adapter top strand and the adapter bottom strand can be improved.

Likewise, the second buffer is a buffer for stabilizing the linker of interest. Those skilled in the art can select an appropriate buffer according to the structure of the target linker. When the target connector includes upstream sequencing primers or downstream sequencing primers corresponding to the sequence to be tested, the second buffer is a buffer that is conducive to DNA structure stability, such as: TE buffer; when the target connector includes transposon sequences , the second buffer is a buffer that is beneficial to the stability of the DNA structure and the reaction of the transposase.

Those skilled in the art can understand that the nanopore library construction kit provided by the application can also include independently packaged Primer-index complex F (forward primer), Primer-index complex R (reverse primer), Amplify enzyme (amplification Enzyme) and Amplifybuffer (amplification buffer), PCR tube (PCR tube), EP tube (EP tube), etc. are the same or different, and are not limited to this, so that users can prepare samples that can be directly on the machine.

In addition, in combination with the nanopore library construction kit in the above embodiments, the embodiment of the present application also provides an application of the nanopore library construction kit in nucleotide sequencing. The application of the nanopore library construction kit provided in the embodiments of the present application to nucleotide sequencing has the same technical effect due to the use of the nanopore library construction kit provided in any of the above embodiments, and will not be repeated here.

When the target connector also includes an upstream primer or a downstream primer corresponding to the sequence to be tested, the second linking group is located at the 5' end of the primer; the sequencing method includes:

Step S21, taking the second solution to amplify the sequence to be tested to obtain an amplified product;

Specifically, mix the second solution, PCR amplification buffer, dNTP, sequence to be tested, polymerase, primers corresponding to the primers of the target linker, etc., and set up a suitable amplification system and Amplification parameters for PCR amplification.

Step S22, mixing the amplified product with the first solution, so that the first linking group and the second linking group undergo a click chemical reaction, and the amplified product is connected to the top strand of the adapter to generate an annealed product;

According to the copy number of the amplified product, an appropriate amount of the first solution can be added, so that the molar quantity of the first linking group is less than that of the second linking group. Stand at room temperature for a period of time, so that the first linking group and the second linking group have sufficient time for the click chemical reaction to occur.

In step S23, the annealed product is sequenced by a nanopore sequencer to obtain nucleotide sequence information corresponding to the sequence to be tested.

The present application does not limit the nanopore sequencer of the nanopore sequencing platform, it is only necessary to ensure that the nanopore sequencer used is compatible with the adapter top strand and the adapter bottom strand in the first solution. When it is necessary to detect and sequence the sequence to be tested that may exist in multiple samples, an amplification primer with a second linking group can be designed and synthesized in advance according to the sequence to be tested, and after mixing the second buffer, it is prepared into the first Second solution. The inspectors extract nucleotides from each sample in advance, and then amplify the extracted nucleotides as a template, and complete the detection and sequencing of each sample by referring to the above steps S21-S23.

Compared with existing sequencing methods, this example does not require end-filling, phosphorylation at the 5' end, and A treatment at the 3' end of the sequence to be tested, and also does not require the use of DNA ligase, thereby eliminating the need for on-machine sequencing The previous purification step improves the sequencing efficiency; since no DNA ligase is used, the self-ligation of the sequence to be tested is avoided, thereby improving the accuracy of the sequencing result and facilitating subsequent sequence analysis.

The application also provides the following validation tests to illustrate the effect of the sequencing linker, construction method, nanopore library construction kit and application provided by the application.

Example 1:

(1) Design and synthesize the simulated joint top chain of the simulated joint top chain structure and the simulated joint bottom chain of the simulated joint bottom chain, wherein,

Simulate the adapter-top chain as adapter-top:

5'- AATGTACTTCGTTCAGTTACGTATTGC -TCO-3';

The bottom chain of the simulated connector is adapter-bottom:

5'- GCCGGCAATACGTAACTGAACGAAGTACATTGAGGCGAGCGGTCAAT -3'.

The annealing buffer was used to dissolve the synthesized simulated adapter top strand and simulated adapter bottom strand respectively to prepare 20uM stock solution A and stock solution B. Mix stock solution A and stock solution B in equal proportions and perform annealing treatment to obtain annealed products.

(2) According to the known sequence of the pUC19 plasmid, design a pair of primers with a product size of 228bp, wherein the 5' end of the upstream primer is appropriately modified as follows:

228-F: 5'-Tz-CGGC-dspacer-ATCGGCATCAGAGCAGATTGTA-3';

The downstream primer 228-R is: 5'-AACGTCGTGACTGGGAAAAC-3'.

(3) Using HiFi DNA polymerase prepared by KAPA Biosystems, and the above-mentioned 228-F and 228-R primers, amplify with reference to the following amplification system and reaction parameters, and purify with 1x Ampure xp beads after amplification, qubit4 .0 After quantification, the copy number was converted according to the length.

Enzyme hot start at 95°C for 2-5min; denaturation at 98°C for 120s, annealing at 55°C for 15s, extension at 72°C for 9s, 30 cycles; extension at 72°C for 5min.

(4) According to the converted copy number, take 150 fmol of the amplified product into a new centrifuge tube, take 300 fmol of the annealed product of the top strand of the simulated adapter and the bottom strand of the simulated adapter and put it into the centrifuge tube. Make up to 10 μl, and after standing at room temperature for 10 minutes, the annealed product was obtained.

The above annealed products, amplification products and annealed products were detected by polyacrylamide gel electrophoresis. As shown in Figure 7, it is the electrophoretic image of the simulated sequencing adapter connected to the sequence to be tested provided by this application. Among them, the corresponding samples of the gel holes are, from left to right, maker; hole 1: adapter top strand; hole 2: amplified product ; Holes 3 and 4 are sequences to be tested connected with simulated sequencing adapters. As can be seen from Figure 7, there is a band of about 200 bp corresponding to the 2 holes, 3 holes and 4 holes, which is consistent with the target product size corresponding to the 228-F primer and the 228-R primer; There is also a band of about 300bp in the 4 wells, the size of this band is consistent with the size of the sequence to be tested connected with the simulated sequencing adapter, which proves that the amplified product is successfully connected to the top strand of the simulated adapter; through 2 holes, 3 holes and The brightness of the 4 wells shows that at least half of the amplified products are connected to the mock sequencing adapters.

Example 2:

(1) Design and synthesize the joint top chain and the joint bottom chain, wherein,

The adapter top chain is adapter-top:

5'-(iSpC3) ₃₀ -GCGTG ACTAT CGGAC TCGTG GTC TTTTT TTTTT-(iSp18) ₄ - GTCAGTTCGCTTCTTACGCA -TCO-3';

The bottom chain of the connector is adapter-bottom:

5'-GCCG TGCGT AAGAA GCGAA CTGAC AGTCC AGCAC CGACC T-3';

The annealing buffer was used to dissolve the synthesized adapter top strand and adapter bottom strand respectively to prepare 20 μM stock solution A and stock solution B, and stock solution A and stock solution B were mixed in equal proportions before annealing.

The T4 Dda helicase is loaded onto the Y adapter, resulting in a complex of enzyme and adapter. The sequence of T4 Dda helicase is as SEQ ID NO.1.

(2) According to the known sequence of phage (phage), design a pair of PCR amplification primers with a product of 502bp, and the upstream primers are modified as follows:

phage-502-F: 5'-Tz-CGGC-dspacer-AATAACGTCGGCAACTTTGG-3';

The downstream primer is: phage-502-R: 5'-GTTACGCCACCAGTCATCCT-3'.

(3) Perform PCR amplification, purification, and copy number calculation with reference to the amplification system and amplification parameters of Example 1.

(4) According to the converted copy number, take 100 fmol of the amplified product into a new centrifuge tube, take 100 fmol of the complex of the enzyme and the linker and put it into the centrifuge tube, make up the total volume to 10ul with annealing buffer, and obtain the connection A sequence to be tested with a sequencing adapter;

(5) Use the gene sequencer QNome-9604 of Qitan Technology Co., Ltd., the sequencing chip Qcell-3841 and the sequencing kit Qeagen-8 to sequence the sequence to be tested, and combine the reaction product of the sequencing kit Qeagen-8 with the sequencing adapter. The sequences to be tested are mixed, and the mixture is dropped onto the sequencing chip to obtain the via signal map.

Please refer to Figure 8, which is a diagram of the through-hole signal for on-machine sequencing of the sequence to be tested connected with the sequencing adapter. The circled part shown in Figure 8 is the via signal of Tco-Tz-CGGC-dspacer, and the right side of the via signal is the via signal of different bases, which proves that the sequencing linker provided in this example can be applied to nanopore sequencing.

Example 3:

Refer to the scheme of Example 2 to prepare the test sequence connected with the sequencing adapter, the difference is that the Tco group in the top strand of the adapter is replaced by the DBCO group, and the Tz group in the upstream primer is replaced by the N3 group; And set up 3 control groups, in the 3 control groups, the mixture of annealed product and amplified product was left standing for 25min, 2hour and 6hour respectively, to verify the influence of different standing time on the connection rate of annealed product and amplified product.

The above products were detected by polyacrylamide gel electrophoresis. Please refer to Figure 9 to Figure 11. Figure 9 is the electrophoresis diagram of the product after standing for 25 minutes. Among them, the corresponding samples of the gel holes are from left to right: maker; holes 1-3: the annealed product and amplification after standing for 25 minutes Mixture of products; well 4: amplification product; well 5: adapter top strand. Figure 10 is the electrophoresis diagram of the product after standing for 2 hours, in which the corresponding samples of the gel holes are from left to right: maker; 1 hole-3 holes: a mixture of annealed products and amplification products that have been left standing for 2 hours; 4 holes: Amplification product; well 5: Adapter top strand. Figure 11 is the electrophoresis image of the product after standing for 6 hours, in which, the corresponding samples of the gel holes are from left to right: maker; hole 1-3: the mixture of annealed product and amplification product left standing for 6 hours; hole 4: Amplification product; well 5: Adapter top strand.

It can be seen from Figure 9, Figure 10 and Figure 11 that there are 2 obvious bands corresponding to the mixture, which proves that the sequence to be tested is successfully connected with the sequencing adapter; and comparing Figure 9, Figure 10 and Figure 11, it can be seen that, In the case of standing for 25 minutes, 2 hours and 6 hours, the sequence to be tested connected with the sequencing adapter gradually increased with time.

Nanopore sequencing: the method is the same as in Example 2 step (5), and the result: the via signal of DBCO-N3-CGGC-dspacer is similar to the via signal of Tco-Tz-CGGC-dspacer in Example 2 step (5), That is, the through-hole signal is significantly different from the base to be detected, which proves that the sequencing linker provided in this example can be applied to nanopore sequencing.

Example 4:

(1) Refer to Example 2 step (1) to prepare the complex of enzyme and linker;

(2) designing and synthesizing the connecting sequence, the connecting sequence includes two partially complementary sequences,

Tn5-Top: 5'-TZ-CGGC AGATGTGTATAAGAGACAG -3',

Tn5-bottom: 5'- CTGTCTCTTATACACATCT- 3';

(3) Dilute Tn5-Top and Tn5-bottom with anneal buffer to a concentration of 40uM, then mix them in equal proportions, and perform annealing of the two sequences to obtain annealed products;

(4) taking a certain amount of the annealed product prepared in step (3) and incubating with Tn5 transposase at an appropriate concentration to prepare a transposase complex;

(5) Take a certain amount of long fragment DNA of known sequence, and use the above-mentioned transposase complex to fragment, so that "Tz-CGGC" is introduced into the end of the fragmented DNA;

(6) Referring to steps (4) and (5) of Example 2, the sequence to be tested connected with the sequencing adapter was sequenced to obtain the through-hole signal, and the sequence obtained by the through-hole signal analysis was consistent with the known sequence.

Example 5:

(1) Refer to Example 2 step (1) to prepare the complex of enzyme and linker;

(2) designing and synthesizing the connecting sequence, the connecting sequence includes two partially complementary sequences,

TA-Top: 5'-TZ-CGGC AGATGTGTATAAGAGACAG T-3',

TA-bottom: 5'-5p- CTGTCTCTTATACACATCT -3';

(3) Dilute TA-Top and TA-bottom with anneal buffer to a concentration of 40uM, then mix them in equal proportions, and anneal the two sequences to obtain the annealed product;

(4) Perform end repair on the test sequence of the known sequence, and add adenine nucleotide (A) to make it phosphorylate at the 5' end, and protrude an A base at the 3' end;

(5) Use T4 DNA ligase to connect the annealed product in step (3) and the sequence to be tested after processing in step (4). After the connection, Tz-CGGC will be introduced into the end of the sequence to be tested, and the annealed product Purify;

(6) Referring to steps (4) and (5) of Example 2, the sequence to be tested connected with the sequencing adapter was sequenced to obtain the through-hole signal, and the sequence obtained by the through-hole signal analysis was consistent with the known sequence.

In addition, the term "and/or" in this article is only an association relationship describing associated objects, which means that there may be three relationships, for example, A and/or B, which may mean: A exists alone, A and B exist at the same time, There are three cases of B alone. In addition, the character "/" in this article generally indicates that the contextual objects are an "or" relationship. It should be understood that in this embodiment of the present application, "B corresponding to A" means that B is associated with A, and B can be determined according to A. However, it should also be understood that determining B according to A does not mean determining B only according to A, and B may also be determined according to A and/or other information.

The above is only the specific implementation of the application, but the protection scope of the application is not limited thereto. Any person familiar with the technical field can easily think of various equivalent modifications or modifications within the technical scope disclosed in the application. Replacement, these modifications or replacements should be covered within the scope of protection of this application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims (10)

A sequencing connector, the sequencing connector is used for nanopore sequencing, the sequencing connector includes: Adapter top strand, including a nanopore guide strand and a first linking group arranged along a preset sequencing direction, the nanopore guide strand is used to guide the sequence to be tested through the nanopore sequencing channel; The target linker includes a second linking group that performs a click chemical reaction with the first linking group, and the second linking group is used for linking with the sequence to be tested. The sequencing connector according to claim 1, wherein the target connector further comprises a transposase recognition sequence connected to the second linking group; And/or, the target linker also includes a linking sequence connected to the second linking group, along the predetermined sequencing direction, the end of the linking sequence away from the second linking group is provided with a protruding cohesive end; And/or, the target connector further includes a primer corresponding to the upstream or downstream of the sequence to be tested, and the second linking group is connected to the primer. The sequencing linker according to claim 2, wherein when the target linker includes primers corresponding to the upstream or downstream of the sequence to be tested, the target linker also includes primers located between the second linking group and the The resistance polymerase modification between the primers; optionally, the resistance polymerase modification is dspacer modification. The sequencing linker according to claim 1 or 2, wherein one of the first linking group and the second linking group is cyclooctene, and the other is tetrazine; optionally, the second linking group One linking group is cyclooctene, and the second linking group is tetrazine; Or, one of the first linking group and the second linking group is diphenylcyclooctyne, and the other is azido. The sequencing adapter according to claim 1 or 2, wherein the molar quantity of the top strand of the adapter is less than the molar quantity of the target linker. The sequencing adapter according to claim 1 or 2, wherein the sequencing adapter further comprises: The adapter bottom strand includes an adapter complementary strand partially complementary to the nanopore guide strand. The sequencing adapter according to claim 6, wherein the bottom strand of the adapter further comprises a first sticky end sequence, and the first sticky end sequence and the complementary strand of the adapter are arranged along the sequencing direction; the target linker A second cohesive end sequence complementary to the first cohesive end sequence is also included, and the second linking group and the second cohesive end sequence are arranged along the sequencing direction. A method for constructing a library, comprising: providing the sequencing linker according to any one of claims 1 to 7: connecting the target connector to the sequence to be tested; A click chemical reaction occurs between the first linking group and the second linking group to obtain a sample library. A nanopore library construction kit, comprising the sequencing linker and polynucleotide binding protein according to any one of claims 1 to 7. The application of the sequencing linker according to any one of claims 1 to 7, the method according to claim 8, or the nanopore library construction kit according to claim 9 in characterizing biopolymers or preparing products for characterizing biopolymers.

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