CN110564818A - Capture probe, kit and library construction method for DNA sample containing INDEL region - Google Patents

Abstract

The invention provides a capture probe, a kit and a library construction method for a DNA sample containing an INDEL region. Wherein the capture probe comprises: a first probe sequence that complementarily pairs to the left side of the INDEL region; a second probe sequence that complementarily pairs with the right side of the INDEL region; and a third probe sequence that is perfectly complementary paired to the INDEL region. And designing three parts of probes aiming at the Indel region by taking the mutated gene sequence as a reference sequence according to the Indel (insertion deletion fragment) of the target region, wherein the three parts of probes are respectively a probe sequence on the left side of the Indel, a probe sequence on the right side of the Indel and a probe sequence covering the Indel mutation site. The three probes greatly improve the capture specificity and sensitivity of the target Indel sequence, and effectively solve the problems of low capture efficiency and low enrichment level of the Indel region of the current important gene.

Description

Capture probe, kit and library construction method for DNA sample containing INDEL region

Technical Field

the invention relates to the technical field of high-throughput sequencing, in particular to a capture probe, a kit and a library construction method for a DNA sample containing an INDEL region.

background

With the completion of the human genome project, human knowledge and mastery of genetic information has not been improved. Meanwhile, a gene detection technology platform at a molecular level is continuously developed and perfected, so that a gene detection technology is rapidly developed, and the gene detection efficiency is continuously improved. The second generation sequencing (NGS) technology has the advantages of large throughput, short time consumption, high accuracy, abundant information content and the like, can accurately locate genes in a short time, and mainly comprises Whole Genome Sequencing (WGS), whole exome sequencing (we) and Targeted Region Sequencing (TRS). Different sequencing technologies have great difference in sequencing range, data analysis amount, sequencing cost, sequencing time and the like, and the selection of a proper method plays a role in multiplying the scientific research with half the effort.

The whole genome sequencing and the whole exon sequencing are relatively expensive in cost, and more sequence information which is not concerned by a detector is often obtained, so that in order to reduce the cost and focus on the sequence information which is mainly interested by the detector, a 'targeted enrichment sequencing' strategy which focuses further than the whole exon can be adopted, and the application of the targeted sequencing is more and more extensive at present. Targeted sequencing, i.e., highly sequencing key genes or regions (500 x-1000 x or higher), to identify rare variations or to provide accurate and easily interpretable results for studies on disease-associated genes. The strategy effectively reduces the sequencing cost, improves the sequencing depth, and can more economically, efficiently and accurately discover the genetic variation information of a specific region. By researching the target area of a large number of samples, the method is helpful for discovering and verifying disease-related candidate genes or related sites, and has great application potential in clinical diagnosis, drug development and other aspects.

There are various strategies currently used for targeted enrichment, including PCR amplification-based methods, Molecular Inversion Probe (MIP) -based methods, and hybrid capture-based methods. The complete probe comprises a sequence of a target section and a universal sequence (index and a sequencing primer can be added) as a primer for constructing a subsequent library, an amplification product can be directly used as a second-generation sequencing library, and the constructed library can be directly subjected to machine sequencing. However, the method is rarely used at present because the library constructed by the method has poor uniformity and high probe cost. PCR amplification is widely applied at present and is suitable for large-scale samples. However, the multiplex PCR primer pool needs abundant primer design experience, and the method has significant defects on sequencing of regions with unstable amplification efficiency (such as a high GC content region and a high repetitive sequence region), large-fragment DNA sequencing and unknown fusion sequencing. The hybridized capture probe carries biotin, and after the probe is hybridized with the target segment, the probe is adsorbed by the streptavidin-modified magnetic bead, and the uncaptured fragments are thrown away. The probes can then be separated from the target segments by denaturation, and then all empty probes are discarded by adsorption with magnetic beads and target segment capture is complete. Compared with the amplicon library, the hybrid capture library has higher capture efficiency, good specificity and good repeatability, and the hybrid capture probe usually binds to the target region and simultaneously captures sequences on two sides of the target region, so the hybrid capture method can detect the target region which is generally difficult to capture by target sequencing.

The hybridization capture is based on the nucleic acid molecule base complementary hybridization principle to design the molecular probe. The probe may bind to the target region by base complementary pairing, thereby capturing the target segment. The probe-free region is eluted and discarded, and the captured fragment is subsequently subjected to in-silico sequencing by denaturing (typically by adjusting the pH to alkaline) to separate the probe and capture regions. The traditional capture probes are divided into RNA probes and DNA single-stranded probes, the basic design principle is a shingled design mode, the capture uniformity of the design method is good, but the capture specificity of the probes is poor, so that the capture efficiency of partial target segment fragments is low.

Compared with whole genome sequencing, capture sequencing can be used for separating and enriching interested regions, so that the detection sensitivity is higher, and the subsequent data analysis work is greatly reduced. However, the hybrid capture method has the disadvantages that the capture probe is very sensitive to the base composition of the sample, and the capture efficiency is very low for the sample with low DNA integrity or the target region containing a small fragment insertion deletion (INDEL, the size of the insertion deletion fragment is less than or equal to 50bp), and the INDEL cannot be detected. Therefore, there is still a need for improvements to existing capture probes to improve the capture efficiency of such samples.

Disclosure of Invention

The invention mainly aims to provide a capture probe, a kit and a library construction method for a DNA sample containing an INDEL region, so as to solve the problem of low capture efficiency of the DNA sample containing the INDEL region on a target fragment in the prior art.

To achieve the above object, according to one aspect of the present invention, there is provided a capture probe for a DNA sample containing an INDEL region, the capture probe comprising: a first probe sequence that complementarily pairs to the left side of the INDEL region; a second probe sequence that complementarily pairs with the right side of the INDEL region; and a third probe sequence that is perfectly complementary paired to the INDEL region.

Further, the third probe sequence overlaps with the first probe sequence and the second probe sequence.

Furthermore, the overlapping length of the third section of probe sequence and the first section of probe sequence is 40-80 nt, and the overlapping length of the third section of probe sequence and the second section of probe sequence is 40-80 nt.

Furthermore, the lengths of the first section of probe sequence, the second section of probe sequence and the third section of probe sequence are respectively 100-120 nt independently.

Further, in the capture probe, the first probe sequence, the second probe sequence and the third probe sequence are mixed in the same molar ratio.

Further, the working concentration of the capture probe is 0.1pM to 0.75 pM.

Furthermore, the first section of probe sequence, the second section of probe sequence and the third section of probe sequence are single-stranded DNA probes; preferably, the single-stranded DNA probe carries a biotin label at its 5' end.

Furthermore, the first probe sequence is shown as SEQ ID NO. 4, the second probe sequence is shown as SEQ ID NO. 5 and the third probe sequence is shown as SEQ ID NO. 6.

According to a second aspect of the present application, there is provided a library construction kit for a DNA sample comprising an INDEL region, the kit comprising a capture probe, the capture probe being any one of the capture probes described above.

According to a third aspect of the present application, there is provided a library construction method for a DNA sample containing an INDEL region, which is constructed by using the above library construction kit.

By applying the technical scheme of the invention, the improved capture probe takes a mutated gene sequence as a reference sequence according to Indel (insertion deletion fragment) of a target region, and three parts of probes are designed aiming at the Indel region, namely an Indel left side probe sequence, an Indel right side probe sequence and a probe sequence covering an Indel mutation site. The three probes greatly improve the capture specificity and sensitivity of the target Indel sequence, and effectively solve the problems of low capture efficiency and low enrichment level of the Indel region of the current important gene.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows the design principle of capture probes used in prior art capture libraries; and

FIG. 2 shows the design principle of the capture probe for the DNA sample containing the INDEL region improved by the present application.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

As mentioned in the background, the existing capture probes also have the defect of low capture efficiency, especially the INDEL mutation is difficult to detect for DNA samples containing INDEL region, and in order to further improve the capture efficiency of such samples, in an exemplary embodiment of the present application, a capture probe for DNA samples containing INDEL region is provided, the capture probe comprising: a first probe sequence that complementarily pairs to the left side of the INDEL region; a second probe sequence that complementarily pairs with the right side of the INDEL region; and a third probe sequence that is perfectly complementary paired to the INDEL region.

as shown in FIG. 1, in the conventional probe design, a CDS region (exon capture library) of a target gene or about 100bp of each upstream and downstream of a gene on the whole genome level is used as a target region to carry out full-coverage imbricated probe design, and the probe and a target sequence cannot be completely complementary paired in the probe hybridization process, so that a bulge appears in the middle of a double-stranded structure formed after hybridization, the hybridization capture efficiency is reduced, certain low-frequency or large-insertion-deletion-fragment target fragments cannot be captured, and part of important mutation sites are missed.

Compared with the traditional probe design, the improved capture probe disclosed by the application is shown in fig. 2, according to Indel (insertion deletion fragment) of a target region, a mutated gene sequence is taken as a reference sequence, and three parts of probes are designed for the Indel region, namely an Indel left side probe sequence, an Indel right side probe sequence and a probe sequence covering an Indel mutation site. The three probes greatly improve the capture specificity and sensitivity of the target Indel sequence, and effectively solve the problem of low Indel region enrichment level of the current important genes.

To further improve the efficiency of capture of the target INDEL region by the three-way probe sequence, in a preferred embodiment, the third-way probe sequence partially overlaps the first-way probe sequence and the second-way probe sequence. The mutual partial overlapping is beneficial to fully exerting the interaction between the three sections of probe sequences, thereby improving the capture efficiency.

in a preferred embodiment, the third probe sequence overlaps the first probe sequence by 40-80 nt, and the third probe sequence overlaps the second probe sequence by 40-80 nt. The length of the probes overlapped between every two probes is controlled within the length range, and the method has the advantage of improving the capture efficiency of the specific sites of the targeting probes.

From the viewpoint of high capture efficiency, the specific length of the three-segment probe sequence can be obtained by reasonably adjusting the length of the existing probe with high capture efficiency. In a preferred embodiment of the present invention, the first probe sequence, the second probe sequence and the third probe sequence are each independently 100 to 120nt in length.

In order to achieve the capture of the target region fragments as uniformly and proportionally as possible, in a preferred embodiment, the capture probes are composed of a mixture of the first probe sequence, the second probe sequence, and the third probe sequence at the same molar ratio.

The working concentration of the capture probe formed by the three sequences can be reasonably selected according to actual needs, and in a preferred embodiment, the working concentration of the capture probe is 0.1 pM-0.75 pM, and the capture efficiency is higher at the concentration.

in a preferred embodiment, the first probe sequence, the second probe sequence and the third probe sequence are single-stranded DNA probes. To further facilitate subsequent manipulation, the single-stranded DNA probe is preferably labeled with biotin at its 5' end.

By utilizing the design thought and principle of the capture probe, the corresponding capture probe sequence can be reasonably designed according to different target genes captured according to actual needs. In a preferred embodiment, the first probe sequence is represented by SEQ ID NO. 4, the second probe sequence is represented by SEQ ID NO. 5 and the third probe sequence is represented by SEQ ID NO. 6. The three sections of probe sequences are capture probes for detecting the 19 th exon indel of the EGFR gene. Compared with the traditional shingled single-stranded DNA probe, the improved capture probe has the advantages that the original data volume and the sequencing effective depth of the constructed library are remarkably improved, particularly, the detection frequency of the INDEL locus is close to the true value, and the frequency of the INDEL locus detected by the traditional capture probe is only 50% of the theoretical value.

In a second exemplary embodiment of the present application, a library construction kit for a DNA sample comprising INDEL regions is provided, the kit comprising a capture probe, the capture probe being any one of the capture probes described above. The library constructed by the capture probe has higher original data quantity and sequencing effective depth than the traditional capture library, and particularly the detection frequency of the INDEL locus is close to the true value.

In a third exemplary embodiment of the present application, a library construction method for a DNA sample containing INDEL region is provided, which is constructed by using any one of the above library construction kits. The library constructed by the capture probe has higher original data quantity and sequencing effective depth than the traditional capture library, and particularly the detection frequency of the INDEL locus is close to the true value.

The advantageous effects of the present application will be further described with reference to specific examples.

The epidermal growth factor EGFR gene del19 mutation sites are del E746-A750 in more than 65%, and the design method of the capture probe provided by the invention is verified by taking the detection of the 19 th exon Indel of EGFR as an example. The HD753 standard containing a Delta E746-A750 fragment deletion was selected, with a mutation frequency of 5.3%. Two capture probes were designed for the del19 mutation site: a traditional shingled single-stranded DNA probe (control group) and a single-stranded DNA probe designed according to the present invention comprising a three-part probe combination (experimental group). And (3) performing hybrid capture on the target sequence after library construction of the HD753 standard substance, and completing on-machine sequencing analysis. And comparing the capture efficiency of the traditional probe and the novel design probe, the effective depth of library sequencing data, mutation frequency and the like, and selecting the capture probe with better specificity.

Firstly, designing a probe

Through IDT online Probe Design software (Target Capture Probe Design & Ordering Tool), a Probe covering the whole EGFR gene exon region is designed by adopting a ginseng reference genome Human (Feb.2009GRCh37/hg19) as a reference genome, and the Probe does not contain a UTR region. Each probe was 120bp in length and the multiplier was 2 × (indicating that the gene was covered with 2 probes, the same applies below). The EGFR gene has 28 exons with different lengths, and the designed imbricated single-stranded DNA probe sequence ensures that each region is completely covered. Probes were designed separately for the del19 mutation site of the EGFR gene, and the base sequence of the del19 deletion is underlined in table 1 below. The probe design by the traditional IDT software and the probe design scheme of the invention are respectively designed to form two types (panel), and the specific design scheme is as follows.

Traditional hybrid capture single-stranded DNA probe design. An IDT online probe design software is utilized to design a shingled single-stranded DNA probe sequence aiming at the del19 mutation site of the EGFR gene, and the sequence is shown as SEQ ID NO: 1 to SEQ ID NO: 3, respectively. The probes comprise base sequences (underlined) deleted by del19, the 5' ends of the probes are all marked by biotin, the length of the probes is 120nt, each probe is mixed with probes of the whole exon regions of other EGFR genes in equal proportion, the working concentration of the capture probe is 0.75pM, and the mixed probes are used as traditional target region capture probes and are named as panel I.

The invention relates to a hybridization capture single-stranded DNA probe design. Aiming at the del19 mutation site of the EGFR gene, single-stranded probe sequences at the left end of the mutation site are respectively designed, and the sequences are shown as SEQ ID NO in the following table 1: 4 is shown in the specification; the right single-stranded probe sequence of the mutation site is shown as SEQ ID NO:5 is shown in the specification; and single-stranded probe sequences with mutation sites in the middle, as shown in table 1 of SEQ ID NOs: and 6. The 5' ends of the probes are all marked by biotin, the length of the probes is 120nt, each probe is mixed with other EGFR gene full exon region probes in equal proportion, the working concentration of the capture probe is 0.75pM, and the mixed probe is used as a novel target region capture probe and is named as panel II.

TABLE 1 EGFR _ Exon 19_ del Probe sequences

second, library preparation

The HD753 standard DNA was fragmented using a Covaris S2 instrument and finally fragmented into a 200bp mixture of double stranded DNA fragments. The cleaved DNA mixture was taken in 6 portions of 1ug each, and subjected to conventional library preparation (using KAPA's library-constructing reagent). The operation method comprises the following steps:

(1) End repair & add a: the melted reagents were placed on ice, a terminal repair & Add A reaction system was placed in a PCR tube as shown in Table 2, and the mixture was gently mixed by blowing up and down with a gun. The reaction system was placed in a PCR apparatus, and the reaction was carried out according to the condition parameter setting program shown in Table 3.

TABLE 2 end repair & addition A reaction System

TABLE 3 end repair & Add A reaction procedure

(2) Connecting a joint: the ligase was shaken and mixed for 5-10s at room temperature, the reaction system was placed in the PCR tube as shown in Table 4, and the mixture was gently mixed by up-and-down blowing with a gun. The prepared reaction system is divided into 2 tubes, placed on a PCR instrument and reacted for 15min at 22 ℃.

TABLE 4 linker ligation reaction System

(3) AMPure XP magnetic bead purification DNA sample. And (3) preparing AMPure XP magnetic beads, balancing for 30min at room temperature, adding 100 mu L of AMPure XP magnetic beads into each sample, and shaking and mixing uniformly. Incubate at room temperature for 5min, place the PCR tube on a magnetic rack for 5min of adsorption, remove the supernatant. Add 200. mu.L of 80% ethanol solution to each sample, incubate for 30s at room temperature, carefully remove the supernatant. The ethanol washing process was repeated for 2 total washes. And (3) airing the magnetic beads at room temperature, adding 22 mu L of nuclease-free water, shaking and uniformly mixing, incubating at room temperature for 5min, placing the PCR tube on a magnetic frame for adsorption for 5min, sucking 20 mu L of supernatant, and placing in a new PCR tube to obtain a purified DNA sample.

(4) And (3) PCR amplification: the reaction system was placed in the PCR tube as shown in Table 5, and the mixture was gently mixed by up-and-down blowing with a gun. The reaction system was placed in a PCR apparatus, and the reaction was carried out according to the condition parameter setting program shown in Table 6.

TABLE 5 PCR amplification reaction System

TABLE 6 PCR amplification reaction procedure

(5) AMPure XP magnetic bead purification DNA sample. And (3) preparing AMPure XP magnetic beads, balancing for 30min at room temperature, adding 50 mu L of AMPure XP magnetic beads into each sample, and shaking and mixing uniformly. Incubate at room temperature for 5min, place the PCR tube on a magnetic rack for 5min of adsorption, remove the supernatant. Add 200. mu.L of 80% ethanol solution to each sample, incubate for 30s at room temperature, carefully remove the supernatant. The ethanol washing process was repeated for 2 total washes. And (3) airing the magnetic beads at room temperature, adding 22 mu L of nuclease-free water, shaking and uniformly mixing, incubating at room temperature for 5min, placing the PCR tube on a magnetic frame for adsorption for 5min, sucking 20 mu L of supernatant, and placing in a new PCR tube to obtain a purified DNA sample. The resulting 6 libraries were numbered S1-S6, respectively.

Third, library hybrid Capture

500ng of the S1-S6 library constructed above was placed in a new centrifuge tube and concentrated using a vacuum extractor until the library was evaporated to dryness. Using IDT hybrid capture kit, hybrid capture and elution were performed according to the methods described in the specification. During the hybridization process, the tight cover of the tube must be ensured, the evaporation of the volume of the hybridization mixed solution is minimized, otherwise, the hybridization effect is influenced. The S1, S2 and S3 libraries are subjected to hybridization capture by using single-stranded probes (SEQ ID NO: 1-SEQ ID NO: 3) synthesized by a traditional method, and the S4, S5 and S6 libraries are subjected to hybridization capture by using probes (SEQ ID NO: 4-SEQ ID NO: 6) synthesized by the invention. The reaction procedure for hybrid capture is shown in table 7.

TABLE 7 hybrid Capture reaction procedure

The capture library was PCR amplified. After the preparation of a PCR reaction solution on ice in accordance with the reaction system shown in Table 8 and the confirmation of the mixing of the reaction solution containing magnetic beads, the tube was put into a PCR apparatus for amplification, the reaction procedure is shown in Table 9:

TABLE 8 Capture library PCR amplification reaction System

TABLE 9 Capture library PCR amplification reaction procedure

And (3) purifying the library obtained by PCR amplification by using AMPure XP magnetic beads. And (3) preparing AMPure XP magnetic beads, balancing for 30min at room temperature, adding 90 mu L of AMPure XP magnetic beads into each sample, and shaking and mixing uniformly. Incubate at room temperature for 5min, place the PCR tube on a magnetic rack for 5min of adsorption, remove the supernatant. Add 200. mu.L of 80% ethanol solution to each sample, incubate for 30s at room temperature, carefully remove the supernatant. The ethanol washing process was repeated for 2 total washes. Air-drying the magnetic beads at room temperature, adding 22 mu L of nuclease-free water, shaking and uniformly mixing, incubating at room temperature for 5min, placing the PCR tube on a magnetic frame for adsorption for 5min, sucking 20 mu L of supernatant, placing the supernatant in a new PCR tube, and respectively naming the final library as Z1-Z6 (respectively corresponding to S1-S6 libraries).

The library sequencing of the Z1-Z6 library was performed using the nextseq 500 sequencer from the Illumina platform, and the bioinformatic sequencing results are shown in Table 10.

TABLE 10 analysis of library sequencing data

As is clear from the data in Table 10, the amount of the original data and the effective depth of the Z3-Z6 library using the probe of the present invention were stable; the capture specificity of the probe is high; there was no significant difference in the repetition rate of the two probe capture libraries; the mutation frequency of the site del19 of the EGFR gene is detected to be remarkably different. The frequency of detected Indel sites of the target capture probe designed by the traditional method is only about 2/5 of the theoretical value, the standard true value of mutation frequency of the sites is 5.3%, the frequency of the original probe after capture is only about 2%, only about 40% of the original Indel mutation can be detected, and the capture frequency of the improved probe is about 5.1%, close to the actual value and can reach 91%. Compared with the prior probe, the capture efficiency is improved by about 50 percent.

example two

Through IDT online Probe Design software (Target Capture Probe Design & Ordering Tool), a Probe covering the whole EGFR gene exon region is designed by adopting a ginseng reference genome Human (Feb.2009GRCh37/hg19) as a reference genome, and the Probe does not contain a UTR region. Each probe was 120nt in length and the multiplier was 2X. The EGFR gene has 28 exons with different lengths, and the designed imbricated single-stranded DNA probe sequence ensures that each region is completely covered. For the EGFR gene chromosome 20 insertion, the insertion sequence is underlined. The probe design by the traditional IDT software and the probe design scheme of the invention are respectively designed to form two types (panel), and the specific design scheme is as follows.

Traditional hybrid capture single-stranded DNA probe design. An IDT online probe design software is utilized to design a shingled single-stranded DNA probe sequence aiming at the 20 th chromosome mutation site of the EGFR gene, and the sequence is shown as SEQ ID NO: 7 to SEQ ID NO: shown at 9. The 5' ends of the probes are all marked by biotin, the length of the probes is 120nt, each probe is mixed with other EGFR gene full exon region probes in equal proportion, the working concentration of the capture probe is 0.75pM, and the mixed probe is used as a traditional target region capture probe and is named as panel III.

The invention relates to a hybridization capture single-stranded DNA probe design. Aiming at an EGFR gene insertion site V769_ D770insASV, single-stranded probe sequences at the left end of a mutation site are respectively designed, and SEQ ID NO: 10 is shown in the figure; the right single-stranded probe sequence of the mutation site is shown as SEQ ID NO: 11 is shown in the figure; and single-stranded probe sequences with mutation sites in the middle, as shown in table 11, SEQ ID NOs: shown at 12. The 5' ends of the probes are all marked by biotin, the length of the probes is 120nt, each probe is mixed with other EGFR gene full exon region probes in equal proportion, the working concentration of the capture probe is 0.75pM, and the mixed probe is used as a novel target region capture probe and is named as panel IV.

TABLE 11 EGFR _ Exon 20_ ins Probe sequences

Library preparation procedure was the same as in example one, and 500ng of each of the 6 libraries constructed (S7-S12) were placed in a fresh centrifuge tube and concentrated using a vacuum extractor until the library was evaporated to dryness. Using IDT hybrid capture kit, hybrid capture and elution were performed according to the methods described in the specification. During the hybridization process, the tight cover of the tube must be ensured, the evaporation of the volume of the hybridization mixed solution is minimized, otherwise, the hybridization effect is influenced. The S7, S8 and S9 libraries are subjected to hybridization capture by using single-stranded probes (SEQ ID NO: 7-SEQ ID NO: 9) synthesized by a traditional method, and the S10, S11 and S12 libraries are subjected to hybridization capture by using probes (SEQ ID NO: 10-SEQ ID NO: 12) synthesized by the invention. The final library of hybrid capture was designated as Z7-Z12 (corresponding to the S7-S12 libraries, respectively). Library sequencing was performed using the nextseq 500 sequencer from the Illumina platform, and the bioinformatic sequencing results are shown in table 12.

TABLE 12 results of analysis of library sequencing data

The result shows that the mutation frequency for detecting the EGFR gene No. 20 chromosome locus has obvious difference, the frequency for detecting Indel locus by adopting the target capture probe designed by the traditional method is only about 1/2 of the theoretical value, while the frequency for detecting the Indel locus by adopting the probe capture method disclosed by the invention is close to the true value by 5.6%, and the allele mutation frequency is improved by about 50%.

EXAMPLE III

Through IDT online Probe Design software (Target Capture Probe Design & Ordering Tool), a Probe covering the whole EGFR gene exon region is designed by adopting a ginseng reference genome Human (Feb.2009GRCh37/hg19) as a reference genome, and the Probe does not contain a UTR region. Aiming at the deletion of A1689 in the short fragment of the BRCA2 gene, the deleted base sequence is GCA. The probe design by the traditional IDT software and the probe design scheme of the invention are respectively designed to form two types (panel), and the specific design scheme is as follows.

Traditional hybrid capture single-stranded DNA probe design. An IDT online probe design software is utilized to design a shingled single-stranded DNA probe sequence aiming at BRCA2 gene mutation sites, as shown in Table 13 below, wherein SEQ ID NO 13-SEQ ID NO15 show that each probe is 120bp in length and 60nt in overlapping region, the 5' ends of the probes are all marked by biotin, each probe is mixed with probes in the whole exon region of other EGFR genes in equal proportion, the working concentration of the capture probes is 0.75pM, and the mixed probes are used as traditional target region capture probes and named as panel V. Wherein, as shown in SEQ ID NO 13-SEQ ID NO15, each probe has the length of 100bp, the overlapping region is 50nt, the 5' ends of the probes are all marked by biotin, each probe is mixed with probes of the whole exon regions of other EGFR genes in equal proportion, the working concentration of the capture probe is 0.1pM, and the mixed probe is used as a traditional target region capture probe and is named as panel VI.

The invention relates to a hybridization capture single-stranded DNA probe design. Aiming at the deletion site A1689fs of the BRCA2 gene, single-stranded probe sequences at the left end of the mutation site are respectively designed, and the sequences are shown as SEQ ID NO: 16 is shown in the figure; the right single-stranded probe sequence of the mutation site is shown as SEQ ID NO: 17 is shown; and single-stranded probe sequences with mutation sites in the middle, as shown in table 13 of seq id NOs: 18, respectively. The 5' ends of the probes are all marked by biotin, the length of the probes is 120nt, the overlapping interval is 60nt, each probe is mixed with probes of all exon regions of other EGFR genes in equal proportion, the working concentration of the capture probe is 0.75pM, and the mixed probes are used as novel target region capture probes and are named as panel VII. Wherein, as shown in SEQ ID NO 13-SEQ ID NO15, each probe has the length of 100bp, the overlapping region is 50nt, the 5' ends of the probes are all marked by biotin, each probe is mixed with probes of the whole exon regions of other EGFR genes in equal proportion, the working concentration of the capture probe is 0.1pM, and the mixed probe is used as a traditional capture probe of the target region named as panel VIII.

TABLE 13 BRCA2_ del Probe sequences

Library preparation procedure was the same as in example one, and 500ng of each of the 12 constructed libraries (S13-S24) were placed in a new centrifuge tube and concentrated using a vacuum extractor until the library was evaporated to dryness. Using IDT hybrid capture kit, hybrid capture and elution were performed according to the methods described in the specification. Wherein, the S13, S14 and S15 long probe libraries and the S16, S17 and S18 short probe libraries are respectively subjected to hybridization capture by using single-stranded probes (SEQ ID NO: 13-SEQ ID NO: 18) synthesized by a traditional method, and the S19, S20 and S21 long probe libraries and the S22, S23 and S24 short probe libraries are respectively subjected to hybridization capture by using probes (SEQ ID NO: 19-SEQ ID NO: 24) synthesized by the invention. The final library of hybrid capture was designated as Z13-Z24 (corresponding to the S13-S24 libraries, respectively). Library sequencing was performed using the nextseq 500 sequencer from the Illumina platform, and the bioinformatic sequencing results are shown in table 14.

TABLE 14 results of analysis of library sequencing data

The result shows that the difference of the detection BRCA2 mutation frequency is obvious, the frequency of Indel sites detected by the probe capture method adopted by the invention is close to the true value of 5.6%, and the allele mutation frequency is improved by about 40%. The above results indicate that the capture effect is better when the probe length is 120nt, the overlap region is 60nt, and the working concentration of the capture probe is 0.75 pM.

From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects:

Compared with a capture probe designed by a imbricated probe in the prior art, the capture probe is covered with a small fragment insertion deletion (Indel) region, and the probe is mismatched or not combined, so that allele loss is caused, and the overall coverage of a target sequence is reduced; and Indel greatly influences the length matching of the capture probe, and the capture efficiency is greatly reduced, so that Indel defects cannot be detected.

The invention improves the design principle of the hybrid capture probe according to the characteristics of the gene mutation of the sample to be detected, and the verification result of the improved capture probe shows that the library capacity of the constructed library is obviously increased. And sequencing analysis results show that the capture advantages of the improved probe are improved, particularly the depth of a captured target region, the capture specificity and the mutation frequency of a capture site are obviously improved, an effective capture scheme is provided for detecting the low mutation frequency of the insertion deletion site of the gene fragment at present, Indel missing detection of important genes can be avoided, and the richness of a hybridization capture library is improved.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

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<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> EGFR_exon 19_del_3

<400> 3

cctcgatgtg agtttctgct ttgctgtgtg ggggtccatg gctctgaacc tcaggcccac 60

cttttctcat gtctggcagc tgctctgctc tagaccctgc tcatctccac atcctaaatg 120

<210> 4

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> EGFR_exon 19_del_4

<400> 4

tcactgggca gcatgtggca ccatctcaca attgccagtt aacgtcttcc ttctctctct 60

gtcataggga ctctggatcc cagaaggtga gaaagttaaa attcccgtcg ctatcaagga 120

<210> 5

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> EGFR_exon 19_del_5

<400> 5

atctccgaaa gccaacaagg aaatcctcga tgtgagtttc tgctttgctg tgtgggggtc 60

catggctctg aacctcaggc ccaccttttc tcatgtctgg cagctgctct gctctagacc 120

<210> 6

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> EGFR_exon 19_del_6

<400> 6

gtcataggga ctctggatcc cagaaggtga gaaagttaaa attcccgtcg ctatcaagga 60

atctccgaaa gccaacaagg aaatcctcga tgtgagtttc tgctttgctg tgtgggggtc 120

<210> 7

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> EGFR_exon 20_ins_1

<400> 7

tgaaactcaa gatcgcattc atgcgtcttc acctggaagg ggtccatgtg cccctccttc 60

tggccaccat gcgaagccac actgacgtgc ctctccctcc ctccaggaag cctacgtgat 120

<210> 8

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> EGFR_exon 20_ins_2

<400> 8

tggccaccat gcgaagccac actgacgtgc ctctccctcc ctccaggaag cctacgtgat 60

ggccagcgtg gacaaccccc acgtgtgccg cctgctgggc atctgcctca cctccaccgt 120

<210> 9

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> egfr_exon 20_ins_3

<400> 9

ggccagcgtg gacaaccccc acgtgtgccg cctgctgggc atctgcctca cctccaccgt 60

gcagctcatc acgcagctca tgcccttcgg ctgcctcctg gactatgtcc gggaacacaa 120

<210> 10

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> egfr_exon 20_ins_4

<400> 10

gatcgcattc atgcgtcttc acctggaagg ggtccatgtg cccctccttc tggccaccat 60

gcgaagccac actgacgtgc ctctccctcc ctccaggaag cctacgtgat ggccagcgtg 120

<210> 11

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> egfr_exon 20_ins_5

<400> 11

gacaaccccc acgtgtgccg cctgctgggc atctgcctca cctccaccgt gcagctcatc 60

acgcagctca tgcccttcgg ctgcctcctg gactatgtcc gggaacacaa agacaatatt 120

<210> 12

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> egfr_exon 20_ins_6

<220>

<221> misc_feature

<222> (56)..(64)

<223> insertion sequence

<400> 12

gccacactga cgtgcctctc cctccctcca ggaagcctac gtgatggcca gcgtggccag 60

cgtggacaac ccccacgtgt gccgcctgct gggcatctgc ctcacctcca ccgtgcagct 120

<210> 13

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> brca2_del_1

<220>

<221> misc_feature

<222> (101)..(103)

<223> deletion sequence

<400> 13

cacaaatcag tccccttatt cagtcattga aaattcagcc ttagcttttt acacaagttg 60

tagtagaaaa acttctgtga gtcagacttc attacttgaa gcaaaaaaat ggcttagaga 120

<210> 14

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> brca2_del_2

<220>

<221> misc_feature

<222> (41)..(43)

<223> deletion sequence

<400> 14

tagtagaaaa acttctgtga gtcagacttc attacttgaa gcaaaaaaat ggcttagaga 60

aggaatattt gatggtcaac cagaaagaat aaatactgca gattatgtag gaaattattt 120

<210> 15

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> brca2_del_3

<400> 15

aggaatattt gatggtcaac cagaaagaat aaatactgca gattatgtag gaaattattt 60

gtatgaaaat aattcaaaca gtactatagc tgaaaatgac aaaaatcatc tctccgaaaa 120

<210> 16

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> brca2_del_4

<400> 16

aaaagtcctg caacttgtta cacaaatcag tccccttatt cagtcattga aaattcagcc 60

ttagcttttt acacaagttg tagtagaaaa acttctgtga gtcagacttc attacttgaa 120

<210> 17

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> brca2_del_5

<400> 17

aaaaaatggc ttagagaagg aatatttgat ggtcaaccag aaagaataaa tactgcagat 60

tatgtaggaa attatttgta tgaaaataat tcaaacagta ctatagctga aaatgacaaa 120

<210> 18

<211> 120

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(120)

<223> brca2_del_7

<400> 18

cttagctttt tacacaagtt gtagtagaaa aacttctgtg agtcagactt cattacttga 60

aaaaaaatgg cttagagaag gaatatttga tggtcaacca gaaagaataa atactgcaga 120

<210> 19

<211> 100

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(100)

<223> brca2_del_8

<220>

<221> misc_feature

<222> (88)..(90)

<223> deletion sequence

<400> 19

ccttattcag tcattgaaaa ttcagcctta gctttttaca caagttgtag tagaaaaact 60

tctgtgagtc agacttcatt acttgaagca aaaaaatggc 100

<210> 20

<211> 100

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(100)

<223> brca2_del_9

<220>

<221> misc_feature

<222> (38)..(40)

<223> deletion sequence

<400> 20

tagaaaaact tctgtgagtc agacttcatt acttgaagca aaaaaatggc ttagagaagg 60

aatatttgat ggtcaaccag aaagaataaa tactgcagat 100

<210> 21

<211> 100

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(100)

<223> brca2_del_10

<400> 21

ttagagaagg aatatttgat ggtcaaccag aaagaataaa tactgcagat tatgtaggaa 60

attatttgta tgaaaataat tcaaacagta ctatagctga 100

<210> 22

<211> 100

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(100)

<223> brca2_del_11

<400> 22

cacaaatcag tccccttatt cagtcattga aaattcagcc ttagcttttt acacaagttg 60

tagtagaaaa acttctgtga gtcagacttc attacttgaa 100

<210> 23

<211> 100

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(100)

<223> brca2_del_12

<400> 23

aaaaaatggc ttagagaagg aatatttgat ggtcaaccag aaagaataaa tactgcagat 60

tatgtaggaa attatttgta tgaaaataat tcaaacagta 100

<210> 24

<211> 100

<212> DNA

<213> Homo sapiens

<220>

<221> misc_feature

<222> (1)..(100)

<223> brca2_del_13

<400> 24

acacaagttg tagtagaaaa acttctgtga gtcagacttc attacttgaa aaaaaatggc 60

ttagagaagg aatatttgat ggtcaaccag aaagaataaa 100

Claims (10)

1. A capture probe for a DNA sample containing an INDEL region, the capture probe comprising:

A first probe sequence that complementarily pairs to the left side of the INDEL region;

A second probe sequence that complementarily pairs with the right side of the INDEL region; and

A third segment of probe sequence that is perfectly complementary paired to the INDEL region.

2. The capture probe of claim 1, wherein the third probe sequence partially overlaps the first probe sequence and the second probe sequence.

3. The capture probe of claim 1, wherein the third probe sequence overlaps the first probe sequence by 40-80 nt, and the third probe sequence overlaps the second probe sequence by 40-80 nt.

4. The capture probe of claim 1, wherein the first, second, and third probe sequences are each independently 100 to 120nt in length.

5. The capture probe of any one of claims 1 to 4, wherein the first, second, and third probe sequences are mixed in the same molar ratio in the capture probe.

6. The capture probe of claim 5, wherein the capture probe has a working concentration of 0.1pM to 0.75 pM.

7. The capture probe of any one of claims 1 to 4, wherein the first, second, and third probe sequences are single-stranded DNA probes; preferably, the 5' end of the single-stranded DNA probe carries a biotin label.

8. The capture probe of claim 1, wherein the first probe sequence is represented by SEQ ID NO. 4, the second probe sequence is represented by SEQ ID NO. 5, and the third probe sequence is represented by SEQ ID NO. 6.

9. A library construction kit for a DNA sample containing INDEL regions, the kit comprising a capture probe, wherein the capture probe is a capture probe according to any one of claims 1 to 8.

10. A method of constructing a library from a DNA sample comprising INDEL regions, wherein the method is constructed using the library construction kit of claim 9.