Probe combination for unbiased enrichment of nucleic acid as well as design method and application of probe combination

Abstract

The invention belongs to the technical field of hybrid capture and high-throughput sequencing, and particularly relates to a probe combination for unbiased enrichment of nucleic acid as well as a design method and application of the probe combination. The probe is only combined with sequences on the two sides of a polymorphic site and is not directly combined with the polymorphic site, so that the combination capacity of different allele fragments has no difference, hybridization capture bias is eliminated, and the allele frequency in an enriched product is closer to a true value. On the basis, the length of a region where the probes are combined with the target is set to be 20-40 basic groups, and sequences of which the two sides are combined with other probes are combined, so that a plurality of probes can quickly form a stable complex with the target fragment, the experiment time is shortened, and the experiment steps are simplified. Meanwhile, the area where the probe is combined with the target is 20-40 basic groups, and a sufficient number of probe combinations are provided for relatively short target fragments, so that the capture efficiency of the target fragments generated by different fracture sites around the polymorphic site is ensured.

Description

Technical Field

[0001] The present invention belongs to the technical fields of gene detection, hybridization capture and high-throughput sequencing, and specifically relates to a probe combination for unbiased enrichment of nucleic acids, a design method and an application thereof. Background Art

[0002] Considering the cost of sequencing and the difficulty of bioinformatics analysis, targeted enrichment technology is an important part of high-throughput sequencing (NGS) detection of sequence variations. Among them, the targeted enrichment technology based on hybridization capture has the characteristics of strong scalability and high fault tolerance, and has been widely used. The principle of hybridization capture technology is to achieve the enrichment of the target fragment by complementary pairing of probes with modified groups and target sequences, and then using the modified groups on the probes to capture the probe / target fragment complex with the corresponding medium. The complementary binding of the probe and the target fragment can allow a certain degree of sequence mismatch. Therefore, although the probe is usually designed according to the sequence of the reference genome, it can also capture allele sequences that differ from the reference genome sequence.

[0003] In some applications, although the probes can bind to different alleles, the capture bias caused by differences in binding strength cannot be ignored. Specifically, this bias refers to the increase in the proportion of alleles consistent with the reference genome sequence in the captured product, while the proportion of alleles inconsistent with the reference genome sequence decreases. Applications sensitive to bias include low-frequency mutations and accurate quantification of allele ratios. A typical application of low-frequency mutation detection is the detection of minimal residual lesions. When the binding ability of the lesion molecular marker sequence to the probe is weaker than that of the reference sequence, it is more likely to be missed because the proportion of lesion molecules is already very low. In non-invasive prenatal testing, accurate quantification of allele ratios is urgently needed because fetal free DNA is in the background of maternal free DNA, and the allele ratio offset caused by its copy number abnormality is diluted. Inaccurate allele ratio analysis results make its detection sensitivity low.

[0004] It can be seen that the existing hybridization capture technology often has the problem of capture bias, the enrichment efficiency between different alleles varies greatly, and the allele frequency differs greatly from the true value. Summary of the Invention

[0005] The object of the present invention is to provide a probe combination for unbiased enrichment of nucleic acids, a design method and application thereof, wherein the probe and probe combination eliminate hybridization capture bias, and the allele frequency in the enriched product is closer to the true value.

[0006] The present invention provides a probe, which is any one of multiple probes targeting the same target sequence. The probe includes a target binding sequence segment and a probe binding sequence segment. The target binding sequence segment does not cover a polymorphic site, and the probe binding sequence segment is complementary and paired with the probe binding sequence segment of an adjacent probe.

[0007] Preferably, the probe comprises two probe-binding sequence segments, and the two probe-binding sequence segments are respectively located at the 5' end and the 3' end of the target-binding sequence segment.

[0008] Preferably, the 5' end probe binding sequence segment complements the 3' end probe binding sequence segment of the adjacent probe, and the 3' end probe binding sequence segment complements the 5' end probe binding sequence segment of another adjacent probe.

[0009] Preferably, the target binding sequence segment is 20-40 nt in length.

[0010] Preferably, the length of the probe binding sequence segment is 8-30 nt.

[0011] Preferably, the probe is further connected with a modification group.

[0012] Preferably, the modification group is located at the 5' end of the probe; the modification group includes biotin.

[0013] The present invention also provides a probe combination for unbiased enrichment of nucleic acids, comprising a sense chain probe combination and / or an antisense chain probe combination, wherein the sense chain probe combination and the antisense chain probe combination respectively comprise a plurality of sense chain probes and a plurality of antisense chain probes;

[0014] The target sequence of the sense chain probe is a sense chain containing a polymorphic site, and the sense chain probe includes a target binding sequence segment and a probe binding sequence segment, wherein the target binding sequence segment does not cover the polymorphic site, and the probe binding sequence segment is complementary and paired with the probe binding sequence segment of the adjacent sense chain probe;

[0015] The target sequence of the antisense chain probe is the reverse complementary sequence of the sense chain containing the polymorphic site. The antisense chain probe includes a target binding sequence segment and a probe binding sequence segment. The target binding sequence segment does not cover the polymorphic site. The probe binding sequence segment is complementary and paired with the probe binding sequence segment of the adjacent antisense chain probe.

[0016] The present invention also provides a method for designing the probe combination described in the above technical solution, comprising the following steps:

[0017] Extract the nucleotide sequence of the region where the polymorphic site is located from the reference genome as the reference sequence for probe design;

[0018] The bases of the polymorphic sites in the reference sequence are replaced with symbols other than A, T, C, and G;

[0019] A target binding sequence segment is selected from one end of the reference sequence as a starting point according to a predetermined method, and the resulting probe combination is a sense chain probe combination;

[0020] The predetermined method includes: if the target binding sequence segment contains symbols other than A, T, C, and G, abandoning the target binding sequence segment, moving downstream to the next base of the special letter, and reselecting the target binding sequence segment; if the target binding sequence segment does not contain symbols other than A, T, C, and G, selecting the next target binding sequence segment downstream of the target binding sequence segment until reaching the other end of the reference sequence.

[0021] Preferably, the design method further comprises generating a reverse complementary sequence of the reference sequence, and designing an antisense chain probe combination for the reverse complementary sequence according to the predetermined method; the sense chain probe combination and the antisense chain probe combination are used individually or in combination.

[0022] Preferably, the reference sequence includes an upstream region and a downstream region of the polymorphic site; the upstream region includes at least 80 bases, and the downstream region includes at least 80 bases.

[0023] Preferably, the step of selecting the target binding sequence segment further comprises adjusting the length of the target binding sequence segment according to an annealing temperature of 55-65°C.

[0024] The present invention also provides a kit for unbiased enrichment of nucleic acids, which comprises the probe combination described in the above technical solution or the probe combination designed by the design method described in the above technical solution, and hybridization capture-related reagents.

[0025] The present invention also provides a hybridization capture method for unbiased enrichment of nucleic acids, comprising the following steps:

[0026] Performing a hybridization reaction on the library to be captured using the probe combination to obtain a hybridization reaction product;

[0027] Mixing the hybridization reaction product with hybridization capture magnetic beads for hybridization capture to obtain a probe-target complex;

[0028] eluting the probe-target complex with an elution buffer to obtain a captured product;

[0029] performing PCR amplification on the captured products, and purifying the obtained PCR amplification products to obtain an enriched library;

[0030] The probe combination is the probe combination described in the above technical solution or the probe combination designed by the design method described in the above technical solution.

[0031] The present invention also provides the use of the probe described in the above technical solution, the probe combination described in the above technical solution, the probe combination designed by the design method described in the above technical solution, the kit described in the above technical solution, or the hybridization capture method described in the above technical solution in preparing reagents for gene variation analysis.

[0032] Preferably, the genetic variation analysis includes low-frequency mutation detection and / or allele ratio quantification.

[0033] Beneficial effects:

[0034] The present invention provides a probe, which is any one of multiple probes targeting the same target sequence. The probe comprises a target-binding sequence segment and a probe-binding sequence segment. The target-binding sequence segment does not overlap the polymorphic site, and the probe-binding sequence segment forms a complementary pair with the probe-binding sequence segment of an adjacent probe. The probe of the present invention binds only to sequences flanking the polymorphic site, not directly to the polymorphic site. Therefore, its binding capacity for different allele fragments is consistent, eliminating hybridization capture bias and ensuring that the allele frequencies in the enriched product are closer to the true value.

[0035] On this basis, the present invention sets the region where the probe binds to the target to be 20-40 bases in length, and combines the target binding sequences bound to other probes on both sides, so that multiple probes can quickly form a stable complex with the target fragment, shortening the experimental time and simplifying the experimental steps. At the same time, the region where the probe binds to the target is 20-40 bases, and there are also a sufficient number of probe combinations for shorter target fragments (such as extracellular free DNA), ensuring the capture efficiency of target fragments generated at different break sites around the polymorphic site, thereby ensuring the abundance of enriched products, and avoiding the situation where the target binding region is longer, resulting in incomplete complementarity with the target fragment, and reduced capture efficiency under corresponding hybridization conditions.

[0036] The present invention also provides a method for designing a probe combination consisting of multiple probes with the aforementioned characteristics. Compared to probe design processes that do not consider polymorphic sites, the present invention simply adds the step of replacing the sequence at the polymorphic site, making it more convenient and efficient. Furthermore, for different types of polymorphic sites, such as point mutations, insertions / deletions, and variations in sequence repeat count, only the relevant bases need to be replaced, significantly reducing the difficulty of probe design. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0038] Figure 1 Schematic diagram of the probe combination design mode of the present invention;

[0039] Figure 2 Schematic diagram of probe design for three different hybridization capture schemes in Example 1, Comparative Example 1 and Comparative Example 2 of the present invention;

[0040] Figure 3 1 is a comparison chart of the on-target rates of the three hybridization capture methods in Example 1, Comparative Example 1 and Comparative Example 2 of the present invention;

[0041] Figure 4 Graphs showing the allele frequency distribution of heterozygous sites in three hybridization capture schemes in Example 1, Comparative Example 1, and Comparative Example 2 of the present invention;

[0042] Figure 5 Graphs showing allele frequency distributions of the three hybridization capture schemes for simulated sample sites containing low-frequency variations in Example 1, Comparative Example 1, and Comparative Example 2 of the present invention;

[0043] exist Figures 2-5 Scheme 1, Scheme 2 and Scheme 3 represent the schemes corresponding to Comparative Example 1, Example 1 and Comparative Example 2, respectively. DETAILED DESCRIPTION

[0044] The present invention provides a probe, which is any one of multiple probes targeting the same target sequence, comprising a target binding sequence segment and a probe binding sequence segment, wherein the target binding sequence segment does not cover a polymorphic site, and the probe binding sequence segment is complementary and paired with the probe binding sequence segment of an adjacent probe, wherein the schematic diagram of the probe is shown as follows Figure 1 shown.

[0045] The probe described in the present invention covers the nucleotide sequence upstream of the polymorphic site or the nucleotide sequence downstream of the polymorphic site in the target sequence, but does not cover the polymorphic site. That is, the probe described in the present invention only binds to the sequences on both sides of the polymorphic site, and does not directly bind to the polymorphic site. Therefore, there is no difference in its binding ability for different allele fragments, thereby eliminating hybridization capture bias, and the allele frequency in the enriched product is closer to the true value.

[0046] In the present invention, the probe preferably includes two probe-binding sequence segments, and the two probe-binding sequence segments are preferably located at the 5' end and the 3' end of the target-binding sequence segment, respectively. More preferably, the probe-binding sequence segment at the 5' end is complementary paired with the probe-binding sequence segment at the 3' end of the adjacent probe, and the probe-binding sequence segment at the 3' end is complementary paired with the probe-binding sequence segment at the 5' end of another adjacent probe.

[0047] In the present invention, the target binding sequence segment is preferably 20-40 nt in length; the probe binding sequence segment is preferably 8-30 nt in length. The probe of the present invention is preferably further connected to a modifying group; the modifying group is preferably located at the 5' end of the probe; the modifying group preferably comprises biotin, which can be captured by magnetic beads carrying streptavidin.

[0048] The present invention sets the region where the probe binds to the target to be 20-40 bases in length, and combines the target binding sequences bound to other probes on both sides. Multiple probes can quickly form a stable complex with the target fragment, shortening the experimental time and simplifying the experimental steps. At the same time, the region where the probe binds to the target is 20-40 bases, and there are a sufficient number of probe combinations for shorter target fragments (such as extracellular free DNA), ensuring the capture efficiency of target fragments generated at different break sites around the polymorphic site, thereby ensuring the abundance of enriched products and avoiding the situation where the target binding region is longer, resulting in incomplete complementarity with the target fragment and reduced capture efficiency under corresponding hybridization conditions.

[0049] The present invention also provides a probe combination for unbiased enrichment of nucleic acids, including a sense chain probe combination and / or an antisense chain probe combination, wherein the sense chain probe combination and the antisense chain probe combination respectively include multiple sense chain probes and multiple antisense chain probes; the target sequence of the sense chain probe is a sense chain containing a polymorphic site, the sense chain probe includes a target binding sequence segment and a probe binding sequence segment, the target binding sequence segment does not cover the polymorphic site, and the probe binding sequence segment is complementary and paired with the probe binding sequence segment of an adjacent sense chain probe; the target sequence of the antisense chain probe is the reverse complementary sequence of the sense chain containing the polymorphic site, the antisense chain probe includes a target binding sequence segment and a probe binding sequence segment, the target binding sequence segment does not cover the polymorphic site, and the probe binding sequence segment is complementary and paired with the probe binding sequence segment of an adjacent antisense chain probe. The sense chain probe combination or the antisense chain probe combination of the present invention respectively includes a probe covering the upstream of the polymorphic site and a probe covering the downstream of the polymorphic site.

[0050] The present invention also provides a method for designing the probe combination described in the above technical solution, comprising the following steps:

[0051] Extract the nucleotide sequence of the region where the polymorphic site is located from the reference genome as the reference sequence for probe design;

[0052] The bases at the polymorphic sites in the reference sequence are replaced with symbols other than A, T, C, or G, such as N;

[0053] A target binding sequence segment is selected from one end of the reference sequence as a starting point according to a predetermined method, and the resulting probe combination is a sense chain probe combination;

[0054] The predetermined method includes: if the target binding sequence segment contains a symbol other than A, T, C, or G, abandoning the target binding sequence segment, moving downstream to the next base of the symbol other than A, T, C, or G, and reselecting the target binding sequence segment; if the target binding sequence segment does not contain a symbol other than A, T, C, or G, selecting the next target binding sequence segment downstream of the target binding sequence segment until reaching the other end of the reference sequence.

[0055] The design method of the present invention preferably further includes generating a reverse complementary sequence of the reference sequence, and designing an antisense chain probe combination for the reverse complementary sequence according to the predetermined method; the sense chain probe combination and the antisense chain probe combination are preferably used separately or in combination, more preferably used in combination.

[0056] The reference sequence of the present invention includes an upstream region and a downstream region of the polymorphic site; the upstream region preferably includes at least 80 bases, and the downstream region preferably includes at least 80 bases; the total number of bases in the upstream and downstream regions can cover the length of the target sequence, including extracellular free DNA fragments (approximately 160 bases). The present invention preferably adjusts the length of the target binding sequence segment based on an annealing temperature of 55-65°C, thereby simultaneously meeting the target binding sequence segment length of 20-40 nt and the annealing temperature of 55-65°C.

[0057] Preferably, the probe-binding sequence segments of multiple probes targeting the same target sequence of the present invention have the same nucleotide sequence. More preferably, the nucleotide sequence of the probe-binding sequence segments at the 5' ends of the multiple probes is the same, and the nucleotide sequence of the probe-binding sequence segments at the 3' ends of the multiple probes is the same. The probe-binding sequence segments of the present invention are preferably sequences that appear less frequently in the background sequence.

[0058] After obtaining the target binding sequence segment and the probe binding sequence segment, the present invention combines the target binding sequence segment with the probe binding sequence segment, preferably connecting the probe binding sequence segment or its reverse complementary sequence at the 5' end and 3' end of the target binding sequence segment, respectively, such as connecting the probe binding sequence segment at the 5' end of the target binding sequence segment and connecting the reverse complementary sequence of the probe binding sequence segment at the 3' end, or connecting the probe binding sequence segment at the 3' end of the target binding sequence segment and connecting the reverse complementary sequence of the probe binding sequence segment at the 5' end, so that two probes bound to adjacent intervals on the target sequence can further bind to each other through the probe binding sequence segment.

[0059] Compared to probe design processes that don't consider polymorphic sites, this method simply adds the step of replacing the sequence at the polymorphic site, making it quick and convenient. Furthermore, for different forms of polymorphic sites at the same location, such as point mutations, insertions / deletions, and variations in sequence repeat count, only the relevant bases need to be replaced, significantly reducing the difficulty of probe design.

[0060] The present invention also provides a kit for unbiased nucleic acid enrichment, comprising the probe combination described in the above technical solution or the probe combination designed using the design method described in the above technical solution, and hybridization capture-related reagents. The present invention does not particularly limit the source and type of the hybridization capture-related reagents; conventional commercially available hybridization capture-related reagents in the art can be used.

[0061] The present invention also provides a hybridization capture method for unbiased enrichment of nucleic acids, comprising the following steps:

[0062] Performing a hybridization reaction on the library to be captured using the probe combination to obtain a hybridization reaction product;

[0063] Mixing the hybridization reaction product with hybridization capture magnetic beads for hybridization capture to obtain a probe-target complex;

[0064] eluting the probe-target complex with an elution buffer to obtain a captured product;

[0065] performing PCR amplification on the captured products, and purifying the obtained PCR amplification products to obtain an enriched library;

[0066] The probe combination is the probe combination described in the above technical solution or the probe combination designed by the design method described in the above technical solution.

[0067] The present invention uses a probe combination to perform a hybridization reaction on the library to be captured to obtain a hybridization reaction product. The present invention does not specifically limit the preparation method of the library to be captured. After selecting the sample to be tested, it can be prepared using the conventional capture library preparation method in the art; the sample to be tested preferably includes a DNA sample or an RNA sample, and more preferably includes but is not limited to an extracellular free DNA (cfDNA) sample. The conditions of the hybridization reaction of the present invention preferably include: denaturation at 94-98°C for 2-5 minutes, and then hybridization at 55-65°C for 1-2 hours; the temperature of the denaturation is preferably 95°C, and the time is preferably 2 minutes; the temperature of the hybridization is preferably 60°C, and the time is preferably 1 hour. The hybridization system of the hybridization reaction of the present invention preferably includes 2-10 fmol of probe, 1×HybBuffer, 1×Enhance, 1μg Human Cot-1 and 100pmmol of Blocker.

[0068] After obtaining the hybridization reaction product, the present invention mixes the hybridization reaction product with hybridization capture magnetic beads for hybridization capture to obtain a probe-target complex. The hybridization capture magnetic beads of the present invention preferably include streptavidin magnetic beads. The hybridization capture temperature of the present invention is preferably 55-65°C, more preferably 60°C, and the time is preferably 15-30 minutes, more preferably 20 minutes. The amount of the hybridization capture magnetic beads used is not particularly limited and can be used in the amount of hybridization capture magnetic beads conventionally used in the art.

[0069] After obtaining the probe-target complex, the present invention elutes the probe-target complex with an elution buffer to obtain a captured product. The elution buffer of the present invention preferably includes elution buffer I, elution buffer II, and elution buffer III. The elution buffer I preferably includes 5×SSPE and 0.5%-5% SDS, the elution buffer II preferably includes 2×SSPE, 0.05%-0.5% SDS, and the elution buffer III preferably includes 0.1×SSPE and 0.005%-0.05% SDS. Specifically, the present invention preferably washes the probe-target complex (captured streptavidin magnetic beads) once each with elution buffer I, elution buffer II, and elution buffer III.

[0070] After obtaining the captured product, the present invention performs PCR amplification on the captured product and purifies the obtained PCR amplification product to obtain an enriched library. The present invention does not specifically limit the PCR amplification and purification processes, and the PCR amplification and purification steps in conventional hybridization capture methods in the art can be followed.

[0071] The present invention also provides the use of the probes described in the above technical solutions, the probe combinations described in the above technical solutions, the probe combinations designed using the design methods described in the above technical solutions, the kits described in the above technical solutions, or the hybridization capture methods described in the above technical solutions in preparing reagents for genetic variation analysis. The genetic variation analysis described in the present invention preferably includes low-frequency mutation detection and / or allele ratio quantification; the low-frequency mutation detection preferably includes minimal residual disease detection, and the allele ratio quantification preferably includes non-invasive prenatal testing.

[0072] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0073] Example 1

[0074] A method for unbiased enrichment of sequence polymorphisms, comprising the following steps:

[0075] 32 SNP sites were selected from the human genome, as shown in Table 1.

[0076] Table 1 Information of 32 SNP loci

[0077]

[0078]

[0079] For the regions where the 32 SNP sites in Table 1 are located, unbiased probe combinations were designed according to the design method provided by the present invention. The design pattern is shown in the figure below: Figure 2 The schematic diagram of Scheme 2 described in is shown in Table 2.

[0080] Table 2 Unbiased probe combinations

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Perform hybridization capture using the unbiased probe combination in Table 2 according to the following steps:

[0089] 1) Prepare a hybridization reaction system consisting of the capture library, probe, and hybridization buffer. The hybridization reaction system contains 10 fmol of probe, 1× Hyb Buffer, 1× Enhance, 1 μg of HumanCot-1, and 100 pmmol of Blocker. Place the hybridization reaction system in a temperature controller and perform the reaction at 95°C for 2 minutes and 60°C for 1 hour.

[0090] 2) After the hybridization reaction is completed, the supernatant is transferred to a new PCR tube, and 10 μL M270 beads are added to the PCR reaction tube for hybridization capture. The capture is carried out at 60°C for 20 minutes to obtain the probe-target complex;

[0091] 3) After 20 minutes of capture, the captured probe-target complex was washed once each with Wash Buffer I, Wash Buffer II, and Wash Buffer III;

[0092] The elution buffer I is 5×SSPE, 1% SDS; the elution buffer II is 2×SSPE, 0.1% SDS; and the elution buffer III is 0.1×SSPE, 0.01% SDS.

[0093] 4) After washing, add the PCR reaction system to the M270 beads. The PCR reaction system consists of 2× HiFi PCR Master Mix, 5 μL Index Primer Mix, and 20 μL TE. Start the PCR amplification program on a PCR temperature controller. After the reaction is completed, use 1 volume of magnetic beads for purification to obtain the captured library.

[0094] The above steps were used to perform capture sequencing on the sample 1 library, the sample 2 library, and a library containing 1% of sample 2 mixed with a simulated low-frequency mutation in sample 1. Both samples 1 and 2 were plasma-free DNA provided by healthy individuals. Sequencing reads were mapped to the hg19 genome for analysis.

[0095] Comparative Example 1

[0096] Conventional 120-base hybridization capture method, the steps are as follows:

[0097] For the 32 SNP sites in Table 1, 5' biotin probes with a length of 120 bases were designed according to the conventional hybridization capture method. One probe was designed for each polymorphic site, and the polymorphic site was located in the middle of the probe coverage area, such as Figure 2 The probes designed according to the design pattern indicated in Scheme 1 are shown in Table 3.

[0098] use Hybrid Capture Reagents are used for hybrid capture enrichment to obtain the post-capture library.

[0099] Table 3 120 base hybridization capture probe

[0100]

[0101]

[0102]

[0103] Capture sequencing was performed using the above steps and the probe sequences in Table 3 for the sample 1 library, the sample 2 library, and a simulated low-frequency mutation library containing 1% sample 2 mixed with sample 1. Both samples 1 and 2 were prepared from plasma cell-free DNA provided by healthy individuals. Sequencing reads were mapped to the hg19 genome for analysis.

[0104] Comparative Example 2

[0105] Regardless of whether the probe covers the polymorphic site, the steps of the hybridization capture method are as follows:

[0106] For the 32 SNP sites in Table 1, the probe combination with the same structural features as in Example 1 was designed, with the only difference being that whether the probe covered the polymorphic site was not considered, e.g. Figure 2 The probe combinations designed according to the design pattern indicated in Scheme 3 are shown in Table 4.

[0107] Table 4 Hybridization capture probe combinations regardless of whether the probe covers the polymorphic site

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] Capture sequencing was performed on the sample 1 library, the sample 2 library, and a simulated low-frequency mutation library containing 1% sample 2 mixed with sample 1 using the same hybridization capture steps as in Example 1 and the probe combinations listed in Table 4. Both samples 1 and 2 were prepared from plasma cell-free DNA provided by healthy individuals. Sequencing reads were mapped to the hg19 genome for analysis.

[0115] Test Example 1

[0116] 1) Calculate the on-target rate in Example 1 and Comparative Examples 1-2. The on-target rate is the ratio of sequencing reads in the target SNP region to the total reads. The results are as follows: Figure 3 shown.

[0117] Comparing the on-target rates measured in Example 1 and Comparative Examples 1-2, it can be seen that the on-target rate in Comparative Example 2 is only about 20%, which is expected for a small capture area with only 32 sites. The on-target rates in Example 1 and Comparative Example 2 are greater than 70%.

[0118] 2) Sample 2 contains both homozygous and heterozygous genotypes, with the theoretical frequency of the heterozygous genotype being 0.5. Specifically, 10 SNPs in Sample 2 are heterozygous. In a healthy individual, the two different genotypes at these heterozygous sites come from one set of chromosomes, the father, and the mother, respectively, in a 1:1 ratio. Therefore, the frequency of both alleles should be 0.5.

[0119] By comparing the allele frequencies of heterozygous sites in the data of sample 2 captured and sequenced using the probes and hybridization capture methods in Example 1 and Comparative Examples 1-2, the differences in the true allele ratios, i.e., the difference from 0.5, can be compared. Figure 4 shown.

[0120] The average value of the conventional method in Comparative Example 1 is 0.4877, and the average value of Comparative Example 2 is 0.4868, both of which are significantly lower than 0.5, indicating that their capture has a preference for the reference genome genotype. In contrast, Example 1 is the method of the present invention, with an average value of 0.4987, which deviates much less from 0.5 than Comparative Examples 1 and 2. It can be seen that in conventional hybridization capture methods, due to the different abilities of probes to bind to different genotype sequences, the final captured gene frequency will deviate from 0.5. The method of the present invention solves the problem of differences in the ability of probes to bind to different allele sequences, so the final measured gene frequency is closer to 0.5.

[0121] 3) After mixing 1% of sample 2 into sample 1 with all 32 SNPs, since sample 2 has a different genotype, low-frequency alleles are formed. Depending on whether the alleles in sample 2 are homozygous or heterozygous, the theoretical frequency of the mixed sample is 1% or 0.5%.

[0122] For example, sample 1 and sample 2 are mixed at a ratio of 99:1. Sample 1 has homozygous genotypes at all loci, designated A / A. Sample 2 has a genotype that differs from sample 1, designated a. If sample 2 has a homozygous a / a genotype, the genotype frequencies in the mixed sample are 99% A and 1% a. If sample 2 has a heterozygous A / a genotype, the genotype frequencies in the mixed sample are 99.5% A and 0.5% a.

[0123] For low-frequency variants, Example 1 also showed an allele frequency distribution that was more consistent with expectations. Both the 1% group and the 0.5% group were closer to the expected values ​​and the distribution was more concentrated ( Figure 5 ). Both Comparative Examples 1 and 2 showed lower than expected average allele frequencies, and Comparative Example 1 also had a missed detection. The expected C allele frequency of rs2285144 was 1%, but no C allele appeared in the captured data.

[0124] It can be concluded from the above examples that the probe combination and capture method of the present invention can eliminate hybridization capture bias, and the allele frequencies in the enriched products are closer to the true values.

[0125] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A probe, wherein the probe is any one of multiple probes targeting the same target sequence, characterized in that: The probe comprises a target binding sequence segment and a probe binding sequence segment, wherein the target binding sequence segment does not cover the polymorphic site, and the probe binding sequence segment is complementary and paired with the probe binding sequence segment of an adjacent probe.

2. The probe according to claim 1, characterized in that The probe comprises two probe-binding sequence segments, and the two probe-binding sequence segments are respectively located at the 5' end and the 3' end of the target-binding sequence segment.

3. The probe according to claim 2, characterized in that The 5' end probe binding sequence segment is complementary to the 3' end probe binding sequence segment of the adjacent probe, and the 3' end probe binding sequence segment is complementary to the 5' end probe binding sequence segment of another adjacent probe.

4. The probe according to claim 1, wherein The target binding sequence segment has a length of 20-40 nt.

5. The probe according to any one of claims 1 to 3, characterized in that: The length of the probe binding sequence segment is 8-30 nt.

6. The probe according to claim 1 or 2, characterized in that The probe is further connected with a modification group.

7. The probe according to claim 6, characterized in that The modification group is located at the 5' end of the probe; the modification group includes biotin.

8. A probe combination for unbiased enrichment of nucleic acids, characterized in that: It includes a sense chain probe combination and / or an antisense chain probe combination, wherein the sense chain probe combination and the antisense chain probe combination respectively include multiple sense chain probes and multiple antisense chain probes; The target sequence of the sense chain probe is a sense chain containing a polymorphic site, and the sense chain probe includes a target binding sequence segment and a probe binding sequence segment, wherein the target binding sequence segment does not cover the polymorphic site, and the probe binding sequence segment is complementary and paired with the probe binding sequence segment of the adjacent sense chain probe; The target sequence of the antisense chain probe is the reverse complementary sequence of the sense chain containing the polymorphic site. The antisense chain probe includes a target binding sequence segment and a probe binding sequence segment. The target binding sequence segment does not cover the polymorphic site. The probe binding sequence segment is complementary and paired with the probe binding sequence segment of the adjacent antisense chain probe.

9. The method for designing a probe combination according to claim 8, characterized in that: The steps include: Extract the nucleotide sequence of the region where the polymorphic site is located from the reference genome as the reference sequence for probe design; The bases of the polymorphic sites in the reference sequence are replaced with symbols other than A, T, C, and G; A target binding sequence segment is selected from one end of the reference sequence as a starting point according to a predetermined method, and the resulting probe combination is a sense chain probe combination; The predetermined method includes: if the target binding sequence segment contains a symbol other than A, T, C, or G, abandoning the target binding sequence segment, moving downstream to the next base of the symbol other than A, T, C, or G, and reselecting the target binding sequence segment; if the target binding sequence segment does not contain a symbol other than A, T, C, or G, selecting the next target binding sequence segment downstream of the target binding sequence segment until reaching the other end of the reference sequence.

10. The design method according to claim 9, characterized in that: The design method further includes generating a reverse complementary sequence of the reference sequence, and designing an antisense strand probe combination targeting the reverse complementary sequence according to the predetermined method.

11. The design method according to claim 9 or 10, characterized in that: The reference sequence includes an upstream region and a downstream region of the polymorphic site; the upstream region includes at least 80 bases, and the downstream region includes at least 80 bases.

12. The design method according to claim 9 or 10, characterized in that: The step of selecting the target binding sequence segment further includes adjusting the length of the target binding sequence segment according to an annealing temperature of 55-65°C.

13. A kit for unbiased enrichment of nucleic acids, characterized in that: The kit comprises the probe combination according to claim 8 or the probe combination designed by the design method according to any one of claims 9 to 12, and hybridization capture-related reagents.

14. A hybridization capture method for unbiased enrichment of nucleic acids, characterized in that: The steps include: Performing a hybridization reaction on the library to be captured using the probe combination to obtain a hybridization reaction product; Mixing the hybridization reaction product with hybridization capture magnetic beads for hybridization capture to obtain a probe-target complex; eluting the probe-target complex with an elution buffer to obtain a captured product; performing PCR amplification on the captured products, and purifying the obtained PCR amplification products to obtain an enriched library; The probe combination is the probe combination according to claim 8 or the probe combination designed by the design method according to any one of claims 9 to 12.

15. Use of the probe according to any one of claims 1 to 7, the probe combination according to claim 8, the probe combination designed by the design method according to any one of claims 9 to 12, the kit according to claim 13, or the hybridization capture method according to claim 14 in preparing reagents for gene variation analysis.

16. The use according to claim 15, characterized in that The genetic variation analysis includes low-frequency mutation detection and / or allele ratio quantification.

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