CN114774515A

CN114774515A - Capture probe, kit and detection method for detecting polycystic kidney disease gene mutation

Info

Publication number: CN114774515A
Application number: CN202210298474.3A
Authority: CN
Inventors: 曹彦东; 周洋; 杨鹏程; 王月星; 李晓天
Original assignee: Beijing Anzhiyin Biotechnology Co ltd
Current assignee: Beijing Anzhiyin Biotechnology Co ltd
Priority date: 2022-03-24
Filing date: 2022-03-24
Publication date: 2022-07-22

Abstract

The application relates to the field of gene detection, and particularly provides a capture probe, a kit and a detection method for detecting polycystic kidney disease gene mutation.

Description

Capture probe, kit and detection method for detecting polycystic kidney disease gene mutation

Technical Field

The application relates to the technical field of gene detection, and particularly provides a capture probe, a kit and a detection method for detecting polycystic kidney disease gene mutation.

Background

Polycystic Kidney Disease (PKD) is a common monogenic genetic disease characterized primarily by the formation of multiple progressive enlarged cysts in the kidneys. According to the genetic characteristics, it can be classified into Autosomal Dominant Polycystic Kidney (ADPKD) and Autosomal Recessive Polycystic Kidney (ARPKD). ADPKD is mainly caused by mutations of two genes, namely PKD1 and PKD2, and the gene products are Polycytin-1 (PC1) and Polycytin-2 (PC2) respectively; the ARPKD is caused by PKHD1 gene mutation, and the gene product is fibrolysin/polydectin (FPC). In addition to PKD1, PKD2, and PKHD1, a number of polycystic kidney-associated genes were found, including ALG8, ALG9, ANKS6, COL4A3, COL4a4, COL4a5, COL4a6, DNAJB11, DZIP1L, FLCN, GANAB, HNF1B, LRP5, MYH9, TSC1, TSC2, VHL, and the like.

Autosomal dominant hereditary polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, and patients usually have symptoms after adults but begin to form at the fetal stage, the size and shape of the kidney of the patients are normal or slightly bigger, the number and size of cysts gradually increase and grow with age, and symptoms appear only when the volume of the kidney grows to a certain extent in most cases to the age of 40-50 years, mainly manifested by bilateral kidney enlargement, pain in the kidney area, hematuria, hypertension and the like, and the incidence rate in the population is 1/400-1/1000. ADPKD is caused by mutations in protein kinase D1(PKD1) (about 85% of cases) or protein kinase D2(PKD2) (about 15% of cases). Compared with the PKD2 gene mutant, the PKD1 gene mutant has the end-stage kidney of patientsThe disease occurs 20 years earlier. The penetrance rate of ADPKD is nearly 100%, and the recurrence risk of children is 50%. The incidence of autosomal recessive hereditary polycystic kidney disease (ARPKD) is about 1/20000, while its mutation carriers (heterozygotes) are about 1/70. Abnormal proliferation of epithelial cells in a glomerular capsule of a polycystic kidney patient is one of the remarkable characteristics of the polycystic kidney, is in an immature state or a re-breeding state, and is highly prompted to cause disorder for the developmental maturation control of cells, so that the cells are in an immature state, and therefore, strong proliferation is displayed; epithelial cell transport abnormalities are another prominent feature of polycystic kidney cells, manifested by the closely related transport of Na⁺-K⁺-alterations in the subunit composition, distribution and expression of activity of atpases, abnormalities in cell signaling and alterations in ion transport channels; extracellular matrix dysplasia is the third prominent feature of polycystic kidneys. These abnormalities are involved in active factors involved in cell growth, and cell growth changes and matrix formation abnormalities caused by gene defects are important pathogenesis of the disease. The gene detection can be used for early diagnosis, the pathogenic variation is definite, and the family management (including the clinical screening and prenatal diagnosis of the family) can be guided in a targeted manner through genetic consultation.

The PKD1 gene is located on the short arm of chromosome 16 (16p13.3), the total length of the gene is about 52kb, and contains 46 exons, the mRNA length is about 14.2kb, and the gene codes transmembrane protein consisting of 4303 amino acid residues, namely polycystic 1(PC1), and mediates the interaction between cells and a cell matrix, promotes the differentiation of epithelial cells, and maintains the polarity of the cells. A reduction in PC1 levels can lead to the clinical features of ADPKD. The PKD2 gene is located on chromosome 4 (4q22-23), the total length of the gene is about 68kb, it contains 15 exons, the mRNA length is about 3kb, it codes the membrane protein formed from 968 amino acid residues-polycystic protein 2(PC2), it can increase the permeability of cell membrane to calcium ion, and develop Ca²⁺The channel functions. The PKHD1 gene is located on chromosome 6 (6p12.2), and has a length of about 500kb, 86 exons, and a longest ORF consisting of at least 67 exons, and encodes a fibronectin/polycystin (FPC) single transmembrane receptor-like protein consisting of 4074 amino acids.

To date, more than 2000 mutations of PKD1 gene and more than 300 mutations of PKD2 gene have been reported in Human Gene Mutation Database (HGMD), and not only the mutations are of different types (including missense mutation, nonsense mutation, splicing abnormality mutation, insertion and/or deletion, complex rearrangement), but also are distributed at different positions of the gene, and no mutation hot spots are a big problem in gene analysis of ADPKD patients. Meanwhile, there are 6 pseudogenes with highly similar sequences in exons 1 to 33 of the PKD1 gene, the homology is as high as 97.7% (see fig. 1), and about 80% of pathogenic mutations occur in this region. In addition, the spatial structure of the gene is complex, and the GC content in a part of the region is as high as 70-80%, so that the mutation detection is difficult and challenging. Therefore, for genetic analysis of ADPKD patients, a rapid, efficient and accurate sequence analysis method is lacked due to the fact that the number of involved exons (46 exons of PKD1 gene and 15 exons of PKD2 gene), no mutation hot spots exist, complex regions exist in a genome and the like.

The existing products generally have certain defects in the detection of polycystic kidney disease genes, such as: 1) the number of detected genes is less, more genes are 6, and most genes can only be 2; 2) the interference of the pseudogene is difficult to avoid during detection, and even if LR-PCR is used, the problem caused by the pseudogene cannot be thoroughly solved; 3) the LR-PCR detection has a limited detection range, cannot detect structural variation, and has a limited detection efficiency.

In view of this, the present application is presented.

Disclosure of Invention

Aiming at the defects of the prior art, the method and the system for identifying the position of the classified sequencing sequence on the genome and efficiently capturing the relevant gene region of the polycystic kidney disease are sought, the product can effectively reduce the cost and the workload, and has comprehensive detection variation types, high accuracy and high flux.

In order to achieve the above purpose, the present application provides a method for detecting single-point mutation (SNP), Copy Number Variation (CNV) and Structural Variation (SV) of polycystic kidney disease-related genes by blocking a tel-Seq linker sequence with a blocker and capturing a target region with a probe to perform NGS sequencing, thereby achieving high-depth sequencing.

Specifically, the technical scheme adopted by the application is as follows:

the application firstly provides a library construction method for polycystic kidney disease gene sequencing, which comprises the following steps:

1) adding index to the sample DNA;

2) sealing the index by using a locker;

3) adding a capture probe to capture a target area;

4) library elution and PCR amplification;

5) library purification and quantification.

Further, 1) adds index to the sample DNA based on the TELL-Seq technique.

Further, the blocker in the step 2) is a blocker sequence capable of being specifically and complementarily combined with the 18bp adaptor sequence.

Further, the capture probe in 3) is a probe against the full length of the genes PKD1 and PKD2, and a probe against the full coding regions and variable splicing regions of the genes PKHD1, ALG8, ALG9, ANKS6, COL4A3, COL4A4, COL4A5, COL4A6, DNAJB11, DZIP1L, FLCN, GANAB, HNF1B, LRP5, MYH9, TSC1, TSC2 and VHL.

Furthermore, the probe sequence is shown in SEQ ID NO. 1-227.

Further, the method may specifically comprise the steps of:

1) adding index to the sample DNA based on the TELL-Seq technology:

2) and (3) sealing the index by using a blocker: adding a blocker into the library in the step 1, uniformly mixing, and carrying out hybridization reaction by a PCR hybridization program;

3) and (3) adding a capture probe to capture a target area: centrifuging and uniformly mixing the mixed DNA template and required reagents, and carrying out hybridization reaction by a PCR hybridization program;

4) library elution and PCR amplification: washing and capturing by using streptavidin magnetic beads, adding the streptavidin magnetic beads into a hybridization system, uniformly mixing, and incubating to complete elution; adding PCR reaction solution into the library, and carrying out PCR amplification according to standard PCR reaction conditions.

5) Library purification and quantification.

The application also provides a capture probe set for constructing the polycystic kidney disease gene sequencing library, and the probe sequence is shown in SEQ ID NO. 1-227.

The application also provides a product for constructing a polycystic kidney disease gene sequencing library, which is characterized by comprising the capture probe set.

The application also provides application of the capture probe set in preparation of a kit for detecting polycystic kidney diseases.

The application also provides a polycystic kidney disease detection method, which is characterized by comprising the following steps:

a sequencing and data analysis step and a variation result annotation and interpretation step;

the sequencing and data analysis steps comprise:

1) and (4) sequencing by using an Illumina platform sequencer.

2) Splitting and correcting a sequencing result, and performing sequencing data quality control;

3) comparing the sequencing result file with a human genome reference sequence hg19/GRCh37, analyzing the coverage condition of a data target region, and performing quality control on the sequencing data;

4) detecting SNP, CNV and SV of a target region;

the variant result annotation and interpretation step comprises the following steps:

1) according to ClinVar, 1000G, HGMD, ClinGen, dbVar and gnomaD databases, annotating the detected SNP, INDEL, CNV and SV, and analyzing the pathogenicity of the mutation to polycystic kidney diseases;

2) and (3) manually interpreting the mutation according to ACMG guideline standards, screening out pathogenic mutation, and issuing a detection report according to pathogenic results.

The application also provides a polycystic kidney disease detection system which is characterized by comprising a library construction module, a sequencing and data analysis module and a variation result annotation and interpretation module;

the library construction module is used for executing the library construction method;

the sequencing and data analysis module is configured to perform the following:

1) and (4) sequencing by using an Illumina platform sequencer.

4) detecting SNP, CNV and SV of a target region;

the variant result annotation and interpretation module is used for executing the following steps:

2) and (3) manually reading the mutation according to the ACMG guideline standard, screening pathogenic mutation, and issuing a detection report according to a pathogenic result.

The application also provides a polycystic kidney disease detection system, which is characterized by comprising a library building module, wherein the library building module is used for executing the library building step and also comprises a sequencing and analysis module, and the analysis module is used for analyzing as follows:

1) the pathogenicity of the mutations to polycystic kidney disease was analyzed by annotating detected SNPs and INDEL, CNV and SV using vep v96, transvar v2.4.0 and AnnotSV v2.2, respectively, according to ClinVar, 1000G, HGMD, ClinGen, dbVar, gnomaD databases and the like.

2) And (3) manually interpreting the mutation according to guidelines such as ACMG and the like, screening out pathogenic mutation, and issuing a detection report according to pathogenic results.

Compared with the prior art, the method has at least the following advantages:

1) the effective sequencing detection of polycystic kidney diseases is realized by skillfully combining the TELL-Seq technology and the probe capture technology for the first time. In the TELL-Seq technology, interference of a homologous region on a target sequence is avoided through an index sequence, and meanwhile, a specific blocker closed joint is used for improving the hybridization capture efficiency and reducing the sequencing cost;

2) the present application has selected, through exploration, the capture of the full length of the PKD1 and PKD2 genes, the capture of PKHD1,

The total coding region and the variable splicing region (exon-intron extension is 20bp) of ALG8, ALG9, ANKS6, COL4A3, COL4A4, COL4A5, COL4A6, DNAJB11, DZIP1L, FLCN, GANAB, HNF1B, LRP5, MYH9, TSC1, TSC2 and VHL genes ensure the detection comprehensiveness; the probe sequence is optimized, and experiments prove that the detection is comprehensive and high in accuracy, and the effective sequencing detection of polycystic kidney diseases can be realized;

3) the application uses the TELL-Seq technology together with the blocking of specific blockers and the capture of a target region, solves the problem of sequencing analysis difficulty caused by high homology and high GC content in PKD1 and PKD2 gene regions related to polycystic kidney diseases, reduces the cost and improves the detection efficiency and accuracy.

Drawings

In order to more clearly illustrate the detailed description of the present application or the technical solutions in the prior art, the drawings needed to be used in the detailed description of the present application or the prior art description will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 homology between the PKD1 gene and its pseudogene;

FIG. 2 is a flow chart of the overall analysis of the present application;

FIG. 3 shows the first verification result of the first sample PKD1 gene Insert site;

FIG. 4 shows the first generation verification result of SNP sites of PKD1 gene in the second sample;

FIG. 5 shows the first generation verification result of the first SNP site of the PKD1 gene in the third example;

FIG. 6 shows the first generation verification result of the second SNP site of the PKD1 gene in the third example;

FIG. 7 is a visualization result of original data of a pathogenic site of a LRP5 gene of a first sample;

FIG. 8, a second example sample PKHD1 gene pathogenic site original data visualization result;

FIG. 9, visualization results of original data of pathogenic sites of PKHD1 gene in the third example;

FIG. 10 shows the PKD1 gene MLPA validation results for the PKD-1 sample;

FIG. 11 shows the PKD1 gene MLPA validation results for the PKD-5 sample.

Detailed Description

The technical solutions of the present application will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The following terms or definitions are provided solely to aid in the understanding of the present application. These definitions should not be construed to have a scope less than understood by those skilled in the art.

Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present application are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.

As used in this application, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.

The terms "about" and "substantially" in this application denote the interval of accuracy that a person skilled in the art can understand while still guaranteeing the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments described herein are capable of operation in other sequences than described or illustrated herein.

The library construction method for polycystic kidney disease gene sequencing is shown as a module 1 in an attached figure 2, and the method is characterized in that a blocker is used for closing a TELL-Seq connector sequence, and a target region is captured by a probe for performing NGS sequencing, so that high-depth sequencing is achieved to identify single-point mutation, copy number variation and structural variation detection of polycystic kidney disease related genes. In order to avoid the influence of pseudogenes in a PKD1 gene high homology region and ensure that a target region is completely covered, the method is based on TELL-Seq, an Index sequence is added into a DNA sequence, and then breaking and using a probe set to capture the full length of PKD1 and PKD2 genes and PKHD1, ALG8, ALG9, ANKS6, COL4A3, COL4A4, COL4A5, COL4A6, DNAJB11, DZIP1L, FLCN, GANAB, HNF1B, LRP5, MYH9, TSC1, TSC2 and CDs regions of VHL genes are captured, a specific blocker blocking linker sequence is added during capturing to improve capturing efficiency, an Illumina sequencing platform is used for comparison to a reference sequence by using a method capable of identifying the Index, SNP, INSV, DELSV and CNV mutation conditions are obtained through bioinformation analysis, and the pathogenic information of the SNPs and CNV information is annotated.

The blocker is combined with a joint sequence based on a base complementary pairing principle, so that non-specific combination of a probe and a joint is avoided, specific combination of the probe and a target fragment is ensured, and accordingly, hybridization capture efficiency of the target fragment is improved. Based on the longer index sequence (18bp) in the application, some general purpose blockers cannot play an effective blocking effect (only aiming at the index of 6-8 bp), so that the capture efficiency is low. The special blocker is designed for a longer index sequence, and the blocker sequence which can be combined with the long index sequence in a complementary pairing mode is designed, so that an effective sealing effect is achieved, nonspecific combination among joints is reduced, the sequencing reading long targeting rate is improved, and the enrichment depth is increased.

In some embodiments, the library construction method for polycystic kidney disease gene sequencing employed herein is as follows:

1) adding index to the sample DNA; 2) sealing the index by using a locker; 3) adding a capture probe to capture a target area; 4) library elution and PCR amplification; 5) library purification and quantification. In some embodiments, the 1) is based on a TELL-Seq technique to add an index to the sample DNA.

Without limitation, in some embodiments, the blocker in 2) is a blocker sequence capable of specifically and complementarily binding to an 18bp linker sequence. In some embodiments, the capture probes in 3) are probes directed to the full length of genes PKD1, PKD2, and probes directed to the full coding and variable splicing regions of genes PKHD1, ALG8, ALG9, ANKS6, COL4A3, COL4a4, COL4a5, COL4a6, DNAJB11, DZIP1L, FLCN, GANAB, HNF1B, LRP5, MYH9, TSC1, TSC2, and VHL. In some embodiments, the probe sequence is set forth in SEQ ID NO. 1-227. In some embodiments, the method may specifically comprise the steps of: 1) adding index to the sample DNA based on TELL-Seq technique: 2) and (3) sealing the index by using the blocks: adding a blocker into the library in the step 1, uniformly mixing, and carrying out hybridization reaction by a PCR hybridization program; 3) and (3) adding a capture probe to capture a target area: centrifuging and uniformly mixing the mixed DNA template and the required reagent, and carrying out hybridization reaction by a PCR hybridization program; 4) library elution and PCR amplification: washing and capturing by using streptavidin magnetic beads, adding the streptavidin magnetic beads into a hybridization system, uniformly mixing, and incubating to complete elution; adding PCR reaction solution into the library, and carrying out PCR amplification according to standard PCR reaction conditions. 5) Library purification and quantification.

In order to further achieve the purpose of detecting polycystic kidney diseases, the specific polycystic kidney disease detection method comprises the library construction method and further comprises the following steps:

in some embodiments, the sequencing and data analysis step comprises:

1) and (4) sequencing by using an Illumina platform sequencer.

4) detecting SNP, CNV and SV of a target region;

in some embodiments, the variant outcome annotation and interpretation step comprises:

Experimental example, establishment of Gene detection method for polycystic kidney disease in the application

The overall experimental process of the gene detection of polycystic kidney disease of the application is shown in fig. 2, and specifically comprises the following steps:

firstly, library construction and probe capture sequencing:

1. library construction with addition of index was performed according to TELL-Seq technical standard:

1) code marking DNA:

a. using 5ng of DNA, strongly shaking TELL Bead for at least 30 seconds, centrifuging for no more than 1 second to enable the solution to return to the bottom, and repeatedly blowing the solution for 5 times by using 200ul of wall heads to ensure that all the magnetic beads are uniformly mixed;

b. adding 8ul of 5x Reaction Buffer, 30ul of nuclease-free water, 8ul of Cofactor and 12ul of TELL Bead into a 0.2ml PCR microtube, blowing, beating and mixing uniformly, then centrifuging for 1 second, adding 4ul of Barcoding Enzyme, blowing, beating and mixing uniformly;

c. adding Suspension Buffer 6ul by using a wide-hole gun head, slowly blowing, uniformly mixing, and slowly rotating and culturing for 15 minutes at 35 ℃;

2) DNA stabilization: adding 2ul of stabilizer, slowly blowing, beating and uniformly mixing, and continuously carrying out slow rotary culture at 35 ℃ for 30 minutes;

3) enzymatic fragmentation of DNA: adding 2ul of each of the marker enzyme and the exonuclease, blowing, beating and uniformly mixing, and continuously culturing for 10 minutes at 35 ℃;

4) cleaning the microbeads:

a. placing the PCR on a magnetic bracket for 1 minute, and sucking out and discarding supernatant after the solution is clarified;

b. adding 125ul of washing solution for cleaning, clarifying the solution again and discarding the supernatant;

c. adding 80ul of stop solution, blowing to suspend, clarifying again, and incubating for 5 minutes at room temperature;

d. the PCR was returned to the magnetic holder for 1 minute, the solution was clarified and the supernatant discarded;

e. adding 125ul of washing solution, blowing, beating and mixing uniformly, transferring to a new PCR tube, and incubating for 3 minutes at 63 ℃;

f. the PCR was returned to the magnetic holder for 1 minute, the solution was clarified and the supernatant discarded;

g. repeating the steps d, e and f once;

h. adding 20ul of washing solution, and resuspending the magnetic beads;

5) amplifying the library:

a. violently shaking the magnetic beads for 10 seconds to suspend the magnetic beads, quickly centrifuging for 1 second, and then blowing to make the magnetic beads completely suspended;

b. placing the PCR on a magnetic bracket for 1 minute, clarifying the solution, sucking out supernatant, and reserving 2 ul;

c. adding 10ul of nuclease-free water, 25ul of 2x PCR Master Mix, 5ul of 10x Primer I, 5ul of 10x Primer II and 3ul of Enhancer, blowing, beating and mixing uniformly, and then centrifuging quickly for 1 second;

d. performing PCR amplification, and storing 2ul of product for quality inspection after amplification;

6) cleaning the library:

a. placing the PCR on a magnetic bracket for 1 minute, and transferring the supernatant to a 0.2ml PCR tube after the solution is clarified;

b. adding nuclease-free water to 100 ul;

c. adding 78ul AMPure XP, beating and mixing evenly, and incubating for 5 minutes at room temperature;

d. placing the PCR on a magnetic bracket for 1 minute, sucking out the supernatant after the solution is clarified, adding 200ul of 75% ethanol, standing for 30 seconds, sucking out and discarding the supernatant;

e. repeating the step d once, and opening the cover for 1-2 minutes to volatilize the ethanol;

f. adding 25ul of TE buffer solution, blowing, stirring uniformly, and standing for 5 minutes;

g. placing the PCR on a magnetic bracket for 1 minute, and transferring 23ul of supernatant to a new tube after the solution is clarified;

7) quality and quantity library: the reserved 2ul products were tested for quality.

2. And (3) closing the index by using a specific blocker: and (3) adding a blocker into the library in the step 1, uniformly mixing by blowing, and carrying out hybridization reaction by a PCR hybridization program.

3. Adding a probe to capture a target area: the DNA template and the required reagents are mixed, centrifuged and mixed well, and subjected to hybridization reaction by a PCR hybridization procedure.

4. Library elution: and washing and capturing by using streptavidin magnetic beads, adding the streptavidin magnetic beads into the hybridization system, uniformly mixing, incubating and then completing elution.

5. And (3) PCR amplification: adding PCR reaction solution into the library, and carrying out PCR amplification according to standard PCR reaction conditions.

6. Library purification and quantification: after the PCR amplification is finished, a purification reagent is added into the PCR tube for purification, the library is subjected to quantitative and fragment distribution detection, and the purified PCR product can be stored at the temperature of-20 ℃ for one week.

Secondly, sequencing and data analysis:

5) and after the library is prepared and the quality inspection is qualified, sequencing by using an Illumina platform sequencer, wherein the sequencing step is carried out according to the existing operation protocol.

6) And (4) splitting and correcting the sequencing result by using tellread v1.0.3 software, and performing sequencing data quality control.

7) And (3) using EMA v0.6.2 software to align the sequencing result file to a human genome reference sequence (hg19/GRCh37) to obtain a BAM file, and using bamdst software to analyze the coverage condition of a data target region to perform quality control on the sequencing data.

8) SNP, CNV and SV of the target region are detected by using a sentienon software driver module.

Third, annotation and interpretation of variation results

3) The pathogenicity of the mutations to polycystic kidney disease was analyzed by annotating detected SNPs and INDEL, CNV and SV respectively with vepv96, transvar v2.4.0 and AnnotSV 2.2 from databases such as ClinVar, 1000G, HGMD, ClinGen, dbVar, gnomAD, etc.

4) And (3) manually reading the mutation according to guidelines such as ACMG and the like, screening pathogenic mutation, and issuing a detection report according to a pathogenic result.

Example 1 design and optimization procedure for Capture Probe set of the present application

1. Design of Probe-Targeted genes and Targeted regions

It is clear in the art that PKD1 and PKD2 gene mutations vary in type, including missense mutations, nonsense mutations, splice abnormalities mutations, insertions and/or deletions, complex rearrangements, etc.; and the gene is distributed at different positions of the gene, and has no mutation hot spots; meanwhile, 6 pseudogenes with highly similar sequences exist in the 1 st to 33 rd exons of the PKD1 gene, the homology is as high as 97.7 percent (see figure 1), and about 80 percent of pathogenic mutations occur in the region; in addition, the spatial structure of the gene is complex, and the GC content of a part of the region is up to 70-80%. These all present significant difficulties and challenges to the genetic analysis of ADPKD patients. Through the exploration, the full-length of PKD1 and PKD2 genes is selected to carry out targeted capture probe set design, and the full-coding region and the variable splicing region (20 bp extending from an exon to an intron) of PKHD1, ALG8, ALG9, ANKS6, COL4A3, COL4A4, COL4A5, COL4A6, DNAJB11, DZIP1L, FLCN, GANAB, HNF1B, LRP5, MYH9, TSC1, TSC2 and VHL genes are also targeted capture probe set design, so that the detection problems of a large number of exons, no mutation hot spots, a complex region in a genome and the like are effectively solved.

2. Design and optimization of capture probe sequences

Further, the present implementation selects a clinically diagnosed polycystic kidney positive sample, uses a preliminarily designed capture probe set to capture a target region, performs sequencing by an Illumina sequencing platform, analyzes the full length of PKD1 and PKD2 genes and the coverage of PKHD1, ALG8, ALG9, ANKS6, COL4A3, COL4a4, COL4a5, COL4a6, jb11, DZIP1L, FLCN, GANAB, HNF1B, LRP5, MYH9, 1, TSC2, the full coding region of VHL genes and the variable splicing region (exon-intron extension of 20bp) in the sequencing result, and evaluates the capture efficiency of the capture probe set. The operation flow is as follows:

1) extracting sample DNA according to a conventional process and fragmenting, wherein the fragmentation length is 150 bp;

2) capturing the DNA obtained in the first step according to the probe capturing and library building sequencing process specification, and sequencing by using an Illumina platform;

3) and after splitting the sequencing result, counting data coverage information by using the existing letter generation analysis tool to obtain a target area coverage result.

Results of gene coverage in the target region: the PKD1 gene 4X coverage was 53%, the PKD2 gene was not covered, and all of ALG9, ALG8, COL4a6, LRP5, and COL4a4 genes were not completely covered, see in particular table 1.

TABLE 1 detection of coverage during optimization of capture probe sets

The results show that the capture efficiency of the probe is low, and the design of the probe needs to be further optimized. The optimization process comprises the following steps: redesigning capture probes for uncovered gene regions, adjusting the proportion of the probes according to the binding capacity of the probes, and compensating the probes with weak binding capacity so as to improve the coverage and uniformity of capture.

Illustratively, probes with weak binding capacity are compensated for: aiming at the regions from exon1 to exon5 of the ALG9 gene in the table 1, the probe capture efficiency is low, and the part without coverage is adjusted below, and the probe sequence SEQ ID NO.44-47 is added to realize comprehensive coverage; aiming at the exon13 region of the gene ALG8, a probe sequence SEQ ID NO.32 is added so as to realize comprehensive coverage; aiming at the exon24 region of the gene COL4A6, a probe sequence SEQ ID NO.170 is added, so that comprehensive coverage is realized; aiming at the exon8 region of the gene LRP5, a probe sequence SEQ ID NO.30 is added so as to realize comprehensive coverage; aiming at the exon43 region of the gene COL4A4, a probe sequence SEQ ID NO.63 is added, so that the comprehensive coverage is realized.

After adjustment, the optimized capture probe set is used for target region capture and sequencing through an Illumina sequencing platform, the full length of PKD1 and PKD2 genes and the coverage of PKHD1, ALG8, ALG9, ANKS6, COL4A3, COL4A4, COL4A5, COL4A6, DNA 11, DZIP1L, FLCN, GANAB, HNF1B, LRP5, MYH9, TSC1, TSC2 and the region of a variable splicing region (exon-intron extension of 20bp) in the sequencing result are analyzed, and the capture efficiency of the capture probe set is evaluated, and the specific operation flow is the same as the above.

Table 2 shows that the results after optimization show that the PKD1 gene exon1 region has homology as high as 100%, 4X coverage is 54%, and the intro region has no coverage, and the overall coverage is 99%; the partial intro region of the PKD2 gene is not covered, the overall coverage is 99%, and the coverage of the other genes, 4X, is all 100%, so that the probe set has better capture efficiency.

Table 2 capture probe sets of the present application capture sample target area coverage results.

This example finally resulted in a probe set for use in the present application, the specific sequence of which is shown in SEQ ID NO.1-227, see Table 3 below.

TABLE 3 optimized Probe sequences

Example 2 TELL-Seq/Blocker-based optimization of targeted capture technology

1. Exploration based on TELL-Seq targeted capture

TELL-Seq is a technique used for whole genome library preparation, specifically using a transposase to fragment DNA and add barcodes to help reassemble the reads after sequencing. The TELL-Seq kit can be used for connecting pseudo-long reads, the average length is 50kb, the maximum length is 100kb, and the interference of homologous sequences can be effectively avoided. However, the whole genome sequencing is high in cost, in order to save cost and complete detection quickly and efficiently, the application improves a TELL-Seq library and performs probe capture on a target region, namely the whole genome sequencing is specifically applied to probe capture detection of polycystic kidney disease gene mutation.

2. Optimization based on blocker seal index

Considering that the blocking effect of index when a probe is used for capturing is greatly related to the capturing efficiency, the length of the index sequence of the TELL-Seq technology reaches 18bp, which is far longer than the conventional index sequence of 6-8bp, so that the blocking technology using the conventional blocker cannot effectively block the index sequence of the TELL-Seq, and the capturing efficiency is low. The embodiment searches for the long index sequence which can be effectively identified by using a specific blocker, and realizes an effective sealing effect on the index sequence of the TELL-Seq. And then, capturing the target region by utilizing a probe capture technology according to the base hybridization complementary pairing principle, thereby realizing the high-efficiency capture of the target region. Specifically, the method comprises the following steps:

selecting 1 clinical diagnosis polycystic kidney positive sample, building a library by using a TELL-Seq technology, blocking an index sequence by using a conventional blocker, then capturing a target region by using an optimized capture probe set, sequencing by using an Illumina sequencing platform, analyzing the percentage of reads of the target region in a sequencing result to the total number of sequencing reads, and evaluating the blocking effect of the blocker. The operation flow is as follows:

1) building a sample library according to a TELL _ Seq technology;

2) blocking the index sequence according to a conventional blocker, then capturing the target region of the library in the first step by using an optimized capture probe set, and sequencing by using an Illumina sequencing platform;

3) and (3) after the sequencing result is split and corrected by using the tell-read software, counting the percentage of the reads number of the target area in the total number of sequencing reads by using the existing bamdst software, and evaluating the blocking effect.

Blocker evaluation result: table 4 shows that only 0.70% of reads are captured in the target region after TELL-Seq technology is used, which indicates that the index used by the conventional blocker cannot play an effective blocking role for TELL-Seq, and further optimization is needed.

Table 4 capture efficiency results in the optimization process of the block closed index sequence.

Therefore, the index sequence used by the TELL-Seq technology is long, and the conventional blocker cannot play an effective blocking effect. Therefore, a specific blocker is designed and synthesized aiming at the index sequence, and is subjected to blocking treatment, the specific blocker can be specifically and complementarily combined with the 18bp adaptor sequence, the non-specific combination of the probe and the adaptor is avoided, the specific combination of the probe and the target fragment is ensured, the effective blocking effect is achieved on the long index sequence, and the hybridization capture efficiency of the target fragment is improved.

After a specific blocker blocking index sequence is used, the optimized capture probe set is used for capturing a target region, sequencing is carried out through an Illumina sequencing platform, the percentage of reads of the target region in a sequencing result to the total number of sequencing reads is analyzed, and the blocker blocking effect is evaluated. The operation flow is the same as above.

As can be seen from Table 5, when a specific blocker is used for blocking the index sequence after the TELL-Seq library is built, reads of a target region captured by a probe can reach 36.69%, the optimized blocker can play an effective blocking role on the index used by the TELL-Seq, and compared with the conventional blocker, the effect advantage is very obvious.

TABLE 5 Capture efficiency results after Block seal index sequences specified

Example 3 verification of accuracy of SNP-based and INDEL mutation detection

Selecting 3 clinical samples which are clinically diagnosed as polycystic kidney diseases and contain related gene mutation, constructing a library by using a TELL-Seq technology in the application, blocking an index sequence by using a specific blocker, capturing a target region by using a capture probe set, sequencing by using an Illumina sequencing platform, detecting the PKD1 and PKD2 gene full length and the variation sites of PKHD1, ALG8, ALG9, ANKS6, COL4A3, COL4A4, COL4A5, COL4A6, DNAJB11, DZIP1L, FLCN, GANAB, HNF1B, TSC 5, MYH9, LRP 1, TSC2, the whole coding region of VHL genes and the variable splicing region (20 bp) region of exons to introns in the sequencing result, carrying out primary-generation sequencing, verifying the pathogenic mutation sites, and evaluating the accurate capture rate of the application. The operation flow is as follows:

1) extracting sample DNA according to a conventional DNA extraction step;

2) constructing a library and a probe capture sequencing flow according to the application, performing TELL-Seq library construction, blocker closed index sequence and probe capture on the DNA obtained in the step one, and sequencing by using an Illumina platform;

3) after the sequencing result is split, using EMA v0.6.2 software to compare the sequencing result file with a human genome reference sequence (hg19/GRCh37) to obtain a BAM file, and using a sentienon software driver module to detect SNP and INDEL of a target region;

4) according to databases such as ClinVar, 1000G, HGMD, ClinGen, dbVar, gnomaD and the like, vep v96 is used for annotating the detected SNP and INDEL, analyzing the pathogenicity of the mutation on polycystic kidney diseases, and screening pathogenic sites.

5) The pathogenic mutation sites of the PKD1 gene region are subjected to first-generation verification, and the pathogenic sites of other genes are checked by checking original data through IGV, so that the accuracy of sequencing analysis results by using the method is judged.

The results of the detection of the target region variation in 3 samples are shown in Table 6 and FIGS. 3 to 9: one sample has 1 pathogenic Insert site on the PKD1 gene, the other two samples have 1 and 2 pathogenic SNP sites on the PKD1 gene respectively, and the variation exists really through one-generation sequencing verification; the 3 samples have a pathogenic SNP site in other regions except the PKD1 gene, and the original bam file is checked through IGV, so that the SNP results also exist really, and the method for capturing, sequencing and analyzing the SNP and the INDEL has accurate and reliable detection results.

Table 6 capture probe sets of the present application capture sample target area coverage results.

Example 4 accuracy verification based on CNV detection

2 clinical samples which are clinically diagnosed as polycystic kidney diseases and contain related copy number variation are selected, a TELL-Seq technology library in the application is used, after an index sequence is blocked by a specific blocker, a capture probe set is used for capturing a target region, an Illumina sequencing platform is used for sequencing, the PKD1 and PKD2 gene full length, PKHD1, ALG8, ALG9, ANKS6, COL4A3, COL4A4, COL4A5, COL4A6, DNAJB11, DZIP1L, FLCN, GANAB, HNF1B, LRP5, MYH9, TSC1, TSC2, the whole coding region of VHL gene and the CNV of a variable splicing region (exon is extended to intron 20bp) region in the sequencing result are detected, the PKD1 gene region of the 2 samples is used for carrying out the verification of the PKV analysis result, the MLV analysis result is compared with the CNV analysis result, and the application is accurate. The operation flow is as follows:

1) extracting sample DNA according to a conventional DNA extraction step;

3) after the sequencing result is split, comparing the sequencing result file with a human genome reference sequence (hg19/GRCh37) by using EMA v0.6.2 software to obtain a BAM file, and performing CNV (CNV) detection on the compared BAM file by using DECoN software and pancn software to obtain CNV results of 2 samples;

4) and analyzing the CNV of the PKD1 gene region of 2 samples by using MLPA, and comparing and verifying the detection result of the application, thereby judging the accuracy of the CNV result analyzed by using the method for capturing, sequencing and analyzing.

TABLE 7 CNV test results of PKD1 gene by DECoN software

TABLE 8 CNV assay results of the panelcn software for PKD1 gene

Sample	Gene	Exon	RC.norm	medRC.norm	CN
						PKD-1	PKD1	PKD1.exon3.chr16.2169095.2169206	2354	3461	CN1
PKD-1	PKD1	PKD1.intro45.chr16.2169207.2169287	1788	2921	CN1
						PKD-5	PKD1	PKD1.exon3.chr16.2169095.2169206	1955	3250	CN1
PKD-5	PKD1	PKD1.intro45.chr16.2169207.2169287	1419	2742	CN1

Results of CNV detection in 2 samples (see tables 7-8, FIGS. 10-11): using DECoN software analysis, 2 samples all detected deletion CNVs in exon 3 and one intron region adjacent thereto of the PKD1 gene; the same results were obtained in 2 samples analysed using the pancn software; the verification result of MLPA is consistent with the detection result, which shows that the detection result is accurate and reliable when the method is used for capturing, sequencing and analyzing CNV.

The foregoing descriptions of specific exemplary embodiments of the present application have been presented for purposes of illustration and description. It is not intended to limit the application to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the present application and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the present application and various alternatives and modifications thereof. It is intended that the scope of the application be defined by the claims and their equivalents.

Claims

1. A library construction method for polycystic kidney disease gene sequencing is characterized by comprising the following steps:

1) adding index to the sample DNA;

2) sealing the index by using a blocker;

3) adding a capture probe to capture a target area;

4) library elution and PCR amplification;

5) library purification and quantification.

2. The library construction method of claim 1, wherein 1) is based on a TELL-Seq technique for adding an index to the sample DNA.

3. The library construction method of any one of claims 1-2, wherein the blocker in 2) is a blocker sequence capable of specific complementary binding to an 18bp linker sequence.

4. The library construction method according to any one of claims 1 to 3, wherein the capture probe in 3) is a probe against the full length of the genes PKD1, PKD2, and a probe against the full coding regions and variable splicing regions of the genes PKHD1, ALG8, ALG9, ANKS6, COL4A3, COL4A4, COL4A5, COL4A6, DNAJB11, DZIP1L, FLCN, GANAB, HNF1B, LRP5, MYH9, TSC1, TSC2 and VHL.

5. The library construction method of any one of claims 1 to 4, wherein the probe sequence is as shown in SEQ ID No.1 to 227.

6. The library construction method of any one of claims 1 to 5, wherein the method comprises the steps of:

1) adding index to the sample DNA based on the TELL-Seq technology:

3) and (3) adding a capture probe to capture a target area: centrifuging and uniformly mixing the mixed DNA template and the required reagent, and carrying out hybridization reaction by a PCR hybridization program;

5) Library purification and quantification.

7. A capture probe set for constructing a polycystic kidney disease gene sequencing library is characterized in that the probe sequence is shown as SEQ ID NO. 1-227.

8. A product for use in sequencing library construction of polycystic kidney disease genes comprising the capture probe set of claim 7.

9. Use of the capture probe set of claim 7 in the preparation of a kit for the detection of polycystic kidney disease.

10. A polycystic kidney disease detection system is characterized by comprising a library construction module, a sequencing and data analysis module and a variation result annotation and interpretation module;

the library construction module for performing the library construction method of any one of claims 1 to 6;

the sequencing and data analysis module is configured to perform the following:

1) and (4) sequencing by using an Illumina platform sequencer.

4) detecting SNP, CNV and SV of a target region;