Selection of inhibitory cyclization auxiliary sequences and their applications

Abstract

The present invention provides a selective inhibitory cyclization auxiliary sequence and application. The selective inhibitory cyclization auxiliary sequence is composed of an interfering sequence binding region a, a linker binding region a, a linker binding region b and an interfering sequence binding region b from the 5' end to the 3' end, and the 5' end of the interfering sequence binding region a has a phosphate group; the interfering sequence specifically bound to the 5' end of the interfering sequence binding region a is adjacent to the interfering sequence specifically bound to the 3' end of the interfering sequence binding region b, and the interfering sequence is an interfering sequence in a fragment to be sequenced; the linker binding region a and the linker binding region b are respectively used for specific binding to a double-end linker sequence. The problem that a large number of sequencing results for uninteresting sequences or interfering sequences exist in the sequencing data in the prior art can be solved, and the method is suitable for the field of high-throughput sequencing.

Description

Technical Field

[0001] The present invention relates to the field of high-throughput sequencing, and in particular to a selective inhibitory cyclization auxiliary sequence and application thereof. Background Art

[0002] High-throughput sequencing technology is one of the most widely used basic technologies in the fields of life science and medicine. Targeted enrichment sequencing greatly reduces the cost of sequencing and bioinformatics analysis by prioritizing the sequencing of sequences of interest, and improves the sensitivity of the analysis.

[0003] High-throughput sequencing technology is generally divided into two stages: library preparation and sequencing on the machine. Targeted enrichment is usually introduced in different steps of library preparation. For example, multiplex PCR targeted enrichment sequencing based on PCR technology is to amplify the library on the template nucleic acid through multiplex PCR; targeted enrichment sequencing based on hybridization capture is to capture the target area after preparing the pre-library by hybridizing the probe with the pre-library to obtain a targeted enriched library.

[0004] For DNA nanoball sequencing technology, circularization is required during library preparation, and then the circular single-stranded library molecules are used as templates for linear amplification to generate nanoballs, i.e., sequencing templates. However, it is difficult to eliminate uninteresting or interfering sequences in high-throughput sequencing, and a large amount of sequencing throughput is wasted in sequencing the uninteresting or interfering sequences, which reduces the sequencing depth of the sequences of interest and generates a large amount of useless sequencing data, affecting data analysis. Summary of the invention

[0005] The main purpose of the present invention is to provide a selective inhibitory cyclization auxiliary sequence and application to solve the problem that there are a large number of sequencing results for uninteresting sequences or interfering sequences in the sequencing data in the prior art.

[0006] In order to achieve the above-mentioned purpose, according to the first aspect of the present invention, a selective inhibitory cyclization auxiliary sequence is provided, which comprises, from the 5' end to the 3' end, an interfering sequence binding region a, a linker binding region a, a linker binding region b and an interfering sequence binding region b, wherein the 5' end of the interfering sequence binding region a has a phosphate group; the interfering sequence specifically bound to the 5' end of the interfering sequence binding region a is adjacent to the interfering sequence specifically bound to the 3' end of the interfering sequence binding region b, and the interfering sequence is an interfering sequence in the fragment to be sequenced; the linker binding region a and the linker binding region b are respectively used for specific binding to the double-ended linker sequence.

[0007] Further, the selection of inhibitory cyclization auxiliary sequences includes: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID Any one of the sequences shown in SEQ ID NO:65 or SEQ ID NO:66.

[0008] In order to achieve the above object, according to the second aspect of the present invention, a method for preparing a single-chain circularization library is provided, the preparation method comprising: circularizing the single-chain fragment to be sequenced using the above-mentioned selective inhibitory circularization auxiliary sequence to obtain a single-chain circularization library.

[0009] Furthermore, the single-stranded fragment to be sequenced is obtained by the following method: the fragment to be sequenced is end-repaired to obtain an end-repaired fragment; the end-repaired fragment is connector-connected to obtain a connector fragment with connector sequences connected at both ends; the connector fragment with connector sequences connected at both ends is denatured to obtain a single-stranded fragment to be sequenced.

[0010] Furthermore, circularizing the single-stranded fragment to be sequenced using the selective inhibitory circularization auxiliary sequence includes: mixing the single-stranded fragment to be sequenced and the selective inhibitory circularization auxiliary sequence, performing single-strand circularization, and obtaining a circular fragment bound to the selective inhibitory circularization auxiliary sequence; performing enzyme cutting on the circular fragment bound to the selective inhibitory circularization auxiliary sequence, digesting and removing the selective inhibitory circularization auxiliary sequence, and obtaining a single-stranded circularization library.

[0011] Furthermore, the single-stranded fragment to be sequenced includes a target fragment to be detected and / or an interfering sequence.

[0012] Furthermore, the single-stranded circularization includes: mixing the single-stranded fragment to be sequenced and the selected inhibitory circularization auxiliary sequence, selecting the adapter binding region a and the adapter binding region b of the inhibitory circularization auxiliary sequence to specifically bind to the adapter sequences at both ends of the single-stranded fragment to be sequenced to form a double-stranded adapter structure region, and under the action of a ligase, the adapter sequences at both ends of the single-stranded fragment to be sequenced are connected end to end to form a circular fragment bound to the selected inhibitory circularization auxiliary sequence.

[0013] Furthermore, for a single-stranded sequencing fragment containing an interfering sequence, the interfering sequence binding region a and the interfering sequence binding region b of the selective inhibitory cyclization auxiliary sequence are both specifically bound to the interfering sequence to form a double-stranded structure region of the interfering sequence, and the phosphate group at the 5' end of the interfering sequence binding region a is connected to the 3' end of the interfering sequence binding region b to connect the selective inhibitory cyclization auxiliary sequence into a ring; when there is a mutation in the interfering sequence, the phosphate group at the 5' end of the interfering sequence binding region a is not connected to the 3' end of the interfering sequence binding region b.

[0014] Furthermore, in the enzyme digestion step, if the circular fragment combines with the selective inhibitory cyclization auxiliary sequence to form a double-stranded structure region containing the interfering sequence and the selective inhibitory cyclization auxiliary sequence is connected to form a ring, the selective inhibitory cyclization auxiliary sequence is not digested and removed, if the circular fragment combines with the selective inhibitory cyclization auxiliary sequence to not form a double-stranded structure region containing the interfering sequence, the selective inhibitory cyclization auxiliary sequence is digested and removed by enzyme digestion, and / or if the circular fragment combines with the selective inhibitory cyclization auxiliary sequence to form a double-stranded structure region containing the interfering sequence, but the phosphate group at the 5' end of the interfering sequence binding region a is not connected to the 3' end of the interfering sequence binding region b, the selective inhibitory cyclization auxiliary sequence is digested and removed.

[0015] In order to achieve the above object, according to the third aspect of the present invention, a method for preparing a DNB sequencing library is provided, the preparation method comprising performing rolling circle replication on the above single-stranded circularized library to obtain a DNB sequencing library.

[0016] In order to achieve the above-mentioned purpose, according to the fourth aspect of the present invention, a sequencing method for a DNB sequencing library is provided, the sequencing method comprising: preparing a DNB sequencing library using the above-mentioned preparation method; sequencing the DNB sequencing library using a DNA nanoball sequencing platform to obtain sequencing data.

[0017] In order to achieve the above-mentioned purpose, according to the fifth aspect of the present invention, there is provided a method for selecting an inhibitory cyclization auxiliary sequence, or a preparation method, or a sequencing method, for use in preparing a library required for a DNA nanoball sequencing platform, sequencing using a DNA nanoball sequencing platform, or reducing the proportion of interfering sequences in sequencing data.

[0018] By applying the technical solution of the present invention, the selective inhibitory cyclization auxiliary sequence containing the interfering sequence binding region a, the linker binding region a, the linker binding region b and the interfering sequence binding region b can replace the splint oligo used in the existing DNA nanoball sequencing library construction, that is, it has the linker binding region a and the linker binding region b that can specifically bind to the linker sequence on the DNA to be tested and promote the DNA to form a ring, and also has the interfering sequence binding region a and the interfering sequence binding region b that can specifically bind to the interfering sequence. After specifically binding to the interfering sequence to form a double-stranded structure, this circular library molecule with a double-stranded structure is difficult to perform rolling circle replication (rolling circle amplification) in the subsequent DNA nanoball sequencing, and thus cannot be sequenced, that is, it achieves the reduction of sequencing results for interfering sequences in sequencing data, reduces sequencing costs and improves analysis sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0020] Figure 1 A schematic diagram of the structure of selecting an inhibitory cyclization auxiliary sequence according to an embodiment of the present invention is shown.

[0021] Figure 2 A schematic diagram of a method for preparing different fragments to be sequenced according to an embodiment of the present invention is shown.

[0022] Figure 3 A graph showing the result of how the selection of inhibitory cyclization auxiliary sequences affects the coverage depth of interfering sequences according to Example 1 of the present invention.

[0023] Figure 4 A schematic diagram of gene frequencies under different cyclization modes according to Example 3 of the present invention is shown. DETAILED DESCRIPTION

[0024] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0025] Interference sequence: refers to a sequence that exists in the sequence to be sequenced, occupies a certain proportion in the sequencing results, but does not provide valid information.

[0026] As mentioned in the background technology, it is difficult to remove uninteresting sequences or interfering sequences in high-throughput sequencing, and a large amount of sequencing flux is wasted in sequencing the uninteresting sequences or interfering sequences, which reduces the sequencing depth of the sequences of interest and generates a large amount of useless sequencing data, which has an adverse effect on data analysis. Therefore, in the present application, the inventors have attempted to develop a selective inhibitory cyclization auxiliary sequence, which can not only play the role of assisting the cyclization of the conventional splint oligo single-stranded library, but also reduce the sequencing depth of the interfering sequences on the DNA nanoball sequencing platform, and on this basis, a series of protection schemes of the present application are proposed.

[0027] In a first typical embodiment of the present application, a selective inhibitory cyclization auxiliary sequence is provided, which comprises, from the 5' end to the 3' end, an interfering sequence binding region a, a linker binding region a, a linker binding region b and an interfering sequence binding region b, wherein the 5' end of the interfering sequence binding region a has a phosphate group (pho); the interfering sequence specifically bound to the 5' end of the interfering sequence binding region a is adjacent to the interfering sequence specifically bound to the 3' end of the interfering sequence binding region b, and the interfering sequence is an interfering sequence in the fragment to be sequenced; the linker binding region a and the linker binding region b are respectively used for specific binding to the double-ended linker sequence.

[0028] The functions of the above-mentioned adapter binding region a and adapter binding region b are the same as those of the splint oligo used in the construction of the DNA nanoball sequencing library (including the sequence shown in SEQ ID NO 67), and the sequences of the adapter binding region a and adapter binding region b can be the same as the sequence of the splint oligo. In the prior art, such splint oligo can guide the cyclization of the single-stranded DNA fragment to be detected to form a circular DNA single strand. The structure of the inhibitory cyclization auxiliary sequence is selected as Figure 1 This selection of inhibitory cyclization auxiliary sequences is used for DNA nanoball sequencing library construction.

[0029] SEQ ID NO 67: aagctgacgtactgagaggcatggcgacctaa.

[0030] The double-ended adapter sequence that specifically binds to the adapter binding region a and the adapter binding region b is the adapter sequence connected to the two ends of the fragment to be sequenced, which helps to complete the circularization of the single-stranded DNA fragment and is also the amplification primer binding site for the subsequent rolling circle replication to prepare DNA nanoballs.

[0031] The above-mentioned fragments to be sequenced are fragmented samples to be sequenced by DNA nanoballs, and have multiple DNA sequences. For the original sample used for DNA nanoball sequencing, it can be a DNA or RNA sample, and the DNA obtained after reverse transcription of the RNA sample can be sequenced. In the fragments to be sequenced, some fragments include interfering sequences, and such interfering sequences include but are not limited to cDNA sequences of ribosomal RNA, wild-type genes and other sequences that researchers are not interested in. The above-mentioned selective inhibitory cyclization auxiliary sequence can specifically bind to the interfering sequence, and the "single-stranded circular DNA" generated after cyclization is actually a circular DNA with a partial double-stranded structure, with a double-cyclized (single-stranded DNA circular and selective inhibitory cyclization auxiliary sequence into a ring) structure. This kind of circular DNA is difficult to perform rolling circle replication and be prepared as a DNA nanoball, so it cannot be sequenced by DNA nanoballs, thereby reducing the sequencing depth of the interfering sequence in the sequencing data.

[0032] In a preferred embodiment, the inhibitory cyclization auxiliary sequence is selected to include but is not limited to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 Any one of the sequences shown in SEQ ID NO:65 or SEQ ID NO:66.

[0033] In a second typical embodiment of the present application, a method for preparing a single-chain circularization library is provided, the preparation method comprising: circularizing the single-chain fragment to be sequenced using the above-mentioned selective inhibitory circularization auxiliary sequence to obtain a single-chain circularization library.

[0034] In the above preparation method, the fragment to be sequenced is used to replace the splint oligo in the prior art, so that the target inhibition library can be prepared.

[0035] In a preferred embodiment, the single-stranded fragment to be sequenced is obtained by the following method: the fragment to be sequenced is end-repaired to obtain an end-repaired fragment; the end-repaired fragment is connector-connected to obtain a connector fragment with connector sequences connected at both ends; the connector fragment with connector sequences connected at both ends is denatured to obtain a single-stranded fragment to be sequenced.

[0036] In a preferred embodiment, circularizing the single-stranded fragment to be sequenced using the selective inhibitory circularization auxiliary sequence includes: mixing the single-stranded fragment to be sequenced and the selective inhibitory circularization auxiliary sequence, performing single-strand circularization, and obtaining a circular fragment bound to the selective inhibitory circularization auxiliary sequence; performing enzyme cutting on the circular fragment bound to the selective inhibitory circularization auxiliary sequence, digesting and removing the selective inhibitory circularization auxiliary sequence, and obtaining a single-stranded circularization library.

[0037] In the above-mentioned enzyme cutting process, exonucleases are used to cut and digest DNA from the exposed DNA ends, and circular DNA has no exposed ends and is therefore not affected. In the prior art, conventional splint oligos do not cyclize themselves, and are therefore digested and removed in this step. The selective inhibitory cyclization auxiliary sequence with an interfering sequence binding region used in the present method also cyclizes itself when the fragment contains an interfering sequence, and thus cannot be removed.

[0038] In a preferred embodiment, the single-stranded fragment to be sequenced includes the target fragment to be detected and / or an interfering sequence.

[0039] In a preferred embodiment, the single-stranded circularization includes: mixing the single-stranded fragment to be sequenced and the selected inhibitory circularization auxiliary sequence, selecting the adapter binding region a and the adapter binding region b of the inhibitory circularization auxiliary sequence to specifically bind to the adapter sequences at both ends of the single-stranded fragment to be sequenced to form a double-stranded adapter structure region, and under the action of a ligase, the adapter sequences at both ends of the single-stranded fragment to be sequenced are connected end to end to form a circular fragment bound to the selected inhibitory circularization auxiliary sequence.

[0040] In a preferred embodiment, for a single-stranded sequencing fragment containing an interfering sequence, the interfering sequence binding region a and the interfering sequence binding region b of the selective inhibitory cyclization auxiliary sequence are both specifically bound to the interfering sequence to form a double-stranded structure region of the interfering sequence, and the phosphate group at the 5' end of the interfering sequence binding region a is connected to the 3' end of the interfering sequence binding region b to connect the selective inhibitory cyclization auxiliary sequence into a ring; when there is a mutation in the interfering sequence, the phosphate group at the 5' end of the interfering sequence binding region a is not connected to the 3' end of the interfering sequence binding region b.

[0041] In a preferred embodiment, in the enzyme digestion step, if the circular fragment combines with the selective inhibitory cyclization auxiliary sequence to form a double-stranded structure region containing the interfering sequence and the selective inhibitory cyclization auxiliary sequence is connected to form a ring, the selective inhibitory cyclization auxiliary sequence is not digested and removed, if the circular fragment combines with the selective inhibitory cyclization auxiliary sequence to not form a double-stranded structure region containing the interfering sequence, the selective inhibitory cyclization auxiliary sequence is digested and removed by enzyme digestion, and / or if the circular fragment combines with the selective inhibitory cyclization auxiliary sequence to form a double-stranded structure region containing the interfering sequence, but the phosphate group at the 5' end of the interfering sequence binding region a is not connected to the 3' end of the interfering sequence binding region b, the selective inhibitory cyclization auxiliary sequence is digested and removed.

[0042] In the above preparation method, a selective inhibitory cyclization auxiliary sequence is used to cyclize the fragment to be sequenced. If the fragment to be sequenced does not contain an interfering sequence, the end of the fragment to be sequenced is single-stranded cyclized under the guidance of the adapter binding region a and the adapter binding region b to obtain a circular fragment. If the fragment to be sequenced contains an interfering sequence, in the obtained circular fragment, in addition to the end of the fragment to be sequenced being cyclized under the guidance of the adapter, the interfering sequence binding region a and the interfering sequence binding region b can specifically bind to the interfering sequence on the sequencing fragment, and the phosphate group at the 5' end of the interfering sequence binding region a is connected to the 3' end of the interfering sequence binding region b.

[0043] However, for the fragments to be sequenced containing mutations in the interfering sequence, the interfering sequence binding region a and the interfering sequence binding region b cannot specifically bind to the fragments to be sequenced containing mutations, and the phosphate group at the 5' end of the interfering sequence binding region a is difficult to connect with the 3' end of the interfering sequence binding region b.

[0044] The closer the mutation position is to the connection point between the interfering sequence binding region a and the interfering sequence binding region b, the greater the effect on the connection efficiency. Preferably, the mutation position is ≤2 bp from the connection point. For a specific mutation, by setting the connection point between the interfering sequence binding regions a and b near the mutation position, preferably at the mutation position, the mutation fragment can be undisturbed; while the non-mutated fragment produces a double-cyclized structure and is disturbed.

[0045] After enzymatic cutting (digestion) of the circular fragments, circular library molecules are obtained. The circular library molecules formed by the fragments to be sequenced without interfering sequences or containing mutations are circular single-stranded DNA; in the circular library molecules formed by the fragments to be sequenced containing interfering sequences, the interfering sequences specifically bind to the interfering sequence binding region a and the interfering sequence binding region b to form double-stranded DNA. The schematic diagram of the preparation method of different fragments to be sequenced is shown in FIG. Figure 2 shown.

[0046] In a third typical embodiment of the present application, a method for preparing a DNB sequencing library is provided, the preparation method comprising performing rolling circle replication on the above-mentioned single-stranded circularized library to obtain a DNB sequencing library.

[0047] In a fourth typical embodiment of the present application, a sequencing method for a DNB sequencing library is provided, the sequencing method comprising: preparing a DNB sequencing library using the above-mentioned preparation method; sequencing the DNB sequencing library using a DNA nanoball sequencing platform to obtain sequencing data.

[0048] In the preparation method and sequencing method of the above-mentioned DNB sequencing library, the circular library molecules are amplified to form DNA nanoballs (DNBs) and sequenced. For the circular library molecules without double-stranded DNA, that is, single-stranded circular DNA, rolling circle replication (rolling circle amplification) can be performed to amplify DNA nanoballs to complete sequencing. For the circular library molecules with double-stranded DNA, it is difficult to complete rolling circle replication and amplification to DNA nanoballs. Therefore, the circular library molecules carrying interfering sequences cannot be sequenced in the DNA nanoball sequencing platform. The above-mentioned preparation method is used to prepare the interfering sequence suppression library, and the DNA nanoball sequencing platform is used to sequence the interfering sequence suppression library, which can reduce the sequencing of the interfering sequence, thereby reducing the sequencing depth of the interfering sequence.

[0049] In a fifth typical embodiment of the present application, there is provided an application of the above-mentioned selective inhibitory cyclization auxiliary sequence, or preparation method, or sequencing method in preparing the library required for the DNA nanoball sequencing platform, sequencing using the DNA nanoball sequencing platform, or reducing the proportion of interfering sequences in sequencing data.

[0050] The above-mentioned selective inhibitory cyclization auxiliary sequence, on the one hand, assumes the function of assisting the cyclization of library fragments (through the adapter binding region), and on the other hand, when the sequence to be sequenced contains a specific sequence (herein called an interfering sequence, which needs to be inhibited, reduced, or excluded), the selective inhibitory cyclization auxiliary sequence itself will also cyclize to produce a double-cyclized structure. In the subsequent digestion process, the double ring cannot be digested, maintains its state, and cannot further generate nanospheres and cannot be sequenced. This ultimately reduces the proportion of interfering sequences in the sequencing data.

[0051] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0052] Example 1

[0053] In RNA samples, ribosomal RNA usually occupies the majority and is usually considered an interfering sequence. It needs to be removed before or during the preparation of RNA sequencing libraries. The sequencing cost saved is usually higher than the cost of mRNA enrichment or rRNA removal steps. Targeted sequencing can exclude ribosomal RNA to a certain extent. For example, hybrid capture RNA sequencing usually does not require the preparation of sequencing libraries that remove ribosomal RNA. The proportion of ribosomal RNA sequences in the total RNA library decreases after hybrid capture, but there are usually still residues.

[0054] Based on the human ribosome sequence, a selective inhibitory cyclization auxiliary sequence Probe1 was designed according to the present method, and the sequence was shown in SEQ ID NO: 1, and its effect of reducing the proportion of interfering sequences was tested.

[0055] Probe1: 5'pho-gtccaagaatttcacctctaaaaagctgacgtactgagaggcatggcgaccttaactctggtccgtcttgcgccg-3' (SEQ ID NO: 1).

[0056] This selective inhibitory circularization auxiliary sequence can bind to the 18S ribosomal RNA sequence, and the corresponding region on the reference genome hg19 is chrUn_gl000220:110031-110070 (and chrUn_gl000220:154003-154042). RNA sequencing library, using Universal cyclization kit v2 (Naonda, 1002241) was used for cyclization, using the original splint oligo or replacing the original splint oligo with the above-mentioned selective inhibitory cyclization auxiliary sequence. The cyclized library was sequenced by MGI2000. The sequencing data was aligned to the hg19 reference genome using STAR software. After adding the above-mentioned selective inhibitory cyclization auxiliary sequence, the coverage depth of this region was significantly reduced, but since only the library containing the forward sequence was inhibited, the reduction was less than 50%.

[0057] Further, a selection inhibition cyclization auxiliary sequence Probe2 shown in SEQ ID NO: 2 is added. This selection inhibition cyclization auxiliary sequence targets the reverse complementary sequence, and the corresponding regions on the reference genome hg19 are chrUn_gl000220:110071-110110 and chrUn_gl000220:154043-154082, avoiding overlapping complementarity with the previous selection inhibition cyclization auxiliary sequence. The selection inhibition cyclization auxiliary sequence also replaces the function of splint oligo, so its total concentration is equal to that of the original splint oligo.

[0058] Probe2: 5'pho-gttttcattaatcaagaacgaaaagctgacgtactgagaggcatggcgaccttaacgaaagcatttgccaagaat-3' (SEQ ID NO: 2).

[0059] After adding two selective inhibitory cyclization auxiliary sequences at the same time, the coverage depth of the corresponding region (interference sequence) further decreased (e.g. Figure 3 If further inhibition of all rRNAs is desired, an inhibitory cyclization auxiliary sequence needs to be further supplemented.

[0060] Example 2

[0061] Low-frequency mutation detection is a basic technology in the fields of tumor liquid biopsy, micro-residual lesion monitoring, and early cancer screening. For low-frequency mutations, high-throughput sequencing is performed, and most of the sequences detected are wild-type sequences. This part of the sequence accounts for a large amount of sequencing costs and also increases the difficulty of improving sensitivity by increasing the sequencing amount. Through this method, selective inhibitory cyclization auxiliary sequences that selectively inhibit wild-type sequences are designed, which can greatly increase the proportion of rare mutation sequences in sequencing data.

[0062] Sixteen single-base mutation sites that differ between two healthy individuals, A and B, were selected. The genomic DNA of A and B was mixed at a ratio of 99 to 1. The 16 sites were amplified by multiplex PCR to prepare the MGI platform sequencing library. The library was divided into two parts, Universal cyclization kit v2 (Naonda, 1002241) and customized cyclization kit with splint oligo replaced by 16 selection inhibitory cyclization auxiliary sequences were used for cyclization. MGI2000 sequencer was used for sequencing. The genomic DNA of A and B was mixed at a ratio of 99:1. When the differential site of the genomic DNA of B was homozygous, the gene frequency of B was 1%; when the differential site of the genomic DNA of B was heterozygous, the gene frequency of B was 0.5%.

[0063] The multiplex PCR primers and the selection of inhibitory cyclization auxiliary sequences are shown in Table 1 below.

[0064] Table 1

[0065]

[0066]

[0067]

[0068] In the artificial mixed sample, the theoretical gene frequencies of the 16 loci are shown in Table 2. The gene frequencies were consistent with the theoretical values ​​by circularization sequencing with the standard kit. The gene frequencies were increased by at least 4 times by using the above-mentioned selective inhibitory circularization to assist sequence circularization. The experimental results are shown in Table 2.

[0069] Table 2

[0070]

[0071] Example 3

[0072] The amplicon sequencing library does not contain the break position information of the original fragment, while the hybrid capture library contains this important information. Combined with the double-end molecular tag, the original fragment can be traced to improve the accuracy of the analysis. For the 16 sites described in Example 2, a hybrid capture probe (NAD Probes custom probe, Nanoda) was customized. The genomic DNA of A and B was mixed at a ratio of 99 to 1. dsDNA Fragmentation. Plasma free DNA double-end molecular tag library construction kit (for MGI) was used to prepare a double-end molecular tag library with an input of 10 ng. HybridCapture Reagents were used for hybridization capture. Universal Circularization Kit v2 (Naonda, 1002241) and a customized circularization kit in which the splint oligo was replaced with 16 sets of selective inhibitory circularization auxiliary sequences (selective inhibitory circularization auxiliary sequences shown in Table 1) were used for circularization. Sequencing was performed using an MGI2000 sequencer.

[0073] The sequencing data was deduplicated and traced using double-end molecular tags to generate consensus sequences. The consensus sequences were analyzed for variation, and the B gene frequencies obtained by the two cyclization methods are shown in the following table. The A / B genotype consensus sequence coverage depth and original sequence coverage depth contained in the sequencing data are shown in Tables 3 and 4.

[0074] Table 3

[0075]

[0076] Table 4

[0077]

[0078] like Figure 4As shown in the figure, in the original sequence analysis, replacing the splint oligo with 16 sets of selective inhibitory cyclization auxiliary sequences significantly increased the frequency of gene B. However, in the consensus sequence analysis, the magnitude of this increase was reduced, because for each A genotype sequencing sequence generated by the original fragment, the selective inhibitory cyclization auxiliary sequence was inhibited at a certain ratio, and each original fragment still had the opportunity to be sequenced.

[0079] From the above description, it can be seen that the above embodiment of the present invention achieves the following technical effects: the splint oligo used in the existing DNA nanoball sequencing library construction can be replaced by the selective inhibitory cyclization auxiliary sequence containing the interfering sequence binding region a, the joint binding region a, the joint binding region b and the interfering sequence binding region b, that is, the joint binding region a and the joint binding region b that can specifically bind to the joint sequence on the DNA to be tested and promote the DNA to form a ring, and the interfering sequence binding region a and the interfering sequence binding region b that can specifically bind to the interfering sequence are also added. The screening of the target area and the non-target area is added in the cyclization step. After the double-stranded structure is formed by specifically binding to the interfering sequence, the circular library molecule with the double-stranded structure is difficult to perform rolling circle replication in the subsequent DNA nanoball sequencing, so that it cannot be sequenced, that is, the sequencing results for the interfering sequence in the sequencing data are reduced, the sequencing cost is reduced and the analysis sensitivity is improved. And this screening process for the interfering sequence is integrated with the library cyclization step, without adding experimental steps, which is convenient for the normal conduct of the experiment.

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A selective inhibitory cyclization auxiliary sequence, characterized in that The selective inhibitory cyclization auxiliary sequence is composed of an interfering sequence binding region a, a linker binding region a, a linker binding region b and an interfering sequence binding region b from the 5' end to the 3' end, and the 5' end of the interfering sequence binding region a has a phosphate group; The interfering sequence specifically bound to the 5' end of the interfering sequence binding region a is adjacent to the interfering sequence specifically bound to the 3' end of the interfering sequence binding region b, and the interfering sequence is an interfering sequence in the fragment to be sequenced; The linker binding region a and the linker binding region b are respectively used for specifically binding to the double-end linker sequence.

2. The selective inhibitory cyclization auxiliary sequence according to claim 1, characterized in that The selective inhibitory cyclization auxiliary sequence comprises: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ Any one of the sequences shown in IDNO: 58, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 65 or SEQ ID NO:

66.

3. A method for preparing a single-chain circularized library, characterized in that: The preparation method comprises: The single-stranded fragment to be sequenced is circularized using the selective inhibitory circularization auxiliary sequence described in claim 1 or 2 to obtain a single-stranded circularized library.

4. The preparation method according to claim 3, characterized in that: The single-stranded fragment to be sequenced is obtained as follows: Performing end repair on the fragment to be sequenced to obtain an end repair fragment; Performing adapter ligation on the end repair fragment to obtain a connection fragment with adapter sequences connected to both ends; The connection fragment with adapter sequences connected to both ends is denatured to obtain the single-stranded fragment to be sequenced.

5. The preparation method according to claim 3, characterized in that: Circularizing the single-stranded fragment to be sequenced using the selective inhibitory circularization auxiliary sequence comprises: Mixing the single-stranded fragment to be sequenced and the selective inhibitory cyclization auxiliary sequence, performing single-strand cyclization, and obtaining a circular fragment bound to the selective inhibitory cyclization auxiliary sequence; The circular fragment bound to the selection inhibitory cyclization auxiliary sequence is digested to remove the selection inhibitory cyclization auxiliary sequence, thereby obtaining the single-chain cyclization library.

6. The preparation method according to claim 5, characterized in that: The single-stranded fragment to be sequenced includes the target fragment to be detected and / or the interfering sequence.

7. The preparation method according to claim 5, characterized in that: The single-stranded circularization comprises: mixing the single-stranded fragment to be sequenced and the selective inhibitory circularization auxiliary sequence, the adapter binding region a and the adapter binding region b of the selective inhibitory circularization auxiliary sequence specifically binding to the adapter sequences at both ends of the single-stranded fragment to be sequenced to form a double-stranded adapter structure region, and under the action of a ligase, the adapter sequences at both ends of the single-stranded fragment to be sequenced are connected end to end to form the circular fragment bound to the selective inhibitory circularization auxiliary sequence.

8. The preparation method according to claim 6, characterized in that: For the single-stranded sequencing fragment containing the interfering sequence, the interfering sequence binding region a and the interfering sequence binding region b of the selective inhibitory cyclization auxiliary sequence are both specifically bound to the interfering sequence to form an interfering sequence double-stranded structure region, and the phosphate group at the 5' end of the interfering sequence binding region a is connected to the 3' end of the interfering sequence binding region b to connect the selective inhibitory cyclization auxiliary sequence into a ring; When the interfering sequence is mutated, the phosphate group at the 5' end of the interfering sequence binding region a is not connected to the 3' end of the interfering sequence binding region b.

9. The preparation method according to claim 8, characterized in that: In the enzyme digestion step, the circular fragment combines with the selective inhibitory cyclization auxiliary sequence to form a double-stranded structure region containing the interfering sequence and the selective inhibitory cyclization auxiliary sequence is connected to form a ring, and the selective inhibitory cyclization auxiliary sequence is not digested and removed. The circular fragment binds to the selective inhibitory cyclization auxiliary sequence and does not form a double-stranded structure region containing the interfering sequence, and the selective inhibitory cyclization auxiliary sequence is removed by enzyme digestion, and / or The circular fragment combines with the selective inhibitory cyclization auxiliary sequence to form a double-stranded structure region containing the interfering sequence, but the phosphate group at the 5' end of the interfering sequence binding region a is not connected to the 3' end of the interfering sequence binding region b, and the selective inhibitory cyclization auxiliary sequence is digested and removed.

10. A method for preparing a DNB sequencing library, characterized in that: The preparation method comprises performing rolling circle replication on the single-chain circularization library according to any one of claims 3 to 9 to obtain the DNB sequencing library.

11. A method for sequencing a DNB sequencing library, characterized in that: The sequencing method comprises: The DNB sequencing library is prepared by the preparation method according to any one of claims 3 to 9; The DNB sequencing library is sequenced using a DNA nanoball sequencing platform to obtain sequencing data.

12. Use of the selective inhibitory cyclization auxiliary sequence according to claim 1 or 2, or the preparation method according to any one of claims 3 to 9, or the sequencing method according to claim 11 in preparing a library required for a DNA nanoball sequencing platform, sequencing using a DNA nanoball sequencing platform, or reducing the proportion of interfering sequences in sequencing data.

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