Method for detecting Marfan and syndrome-like related mutant genes thereof based on high-throughput sequencing technology

文档序号：872159 发布日期：2021-03-19 浏览：2次中文

阅读说明：本技术 一种基于高通量测序技术检测马凡及其类综合征相关突变基因的方法 (Method for detecting Marfan and syndrome-like related mutant genes thereof based on high-throughput sequencing technology ) 是由段艳宇刘子由于 2020-11-19 设计创作，主要内容包括：本发明公开了一种基于高通量测序技术检测马凡及其类综合征相关突变基因的方法,其步骤包括,(1)样本收集；(2)panel设计；(3)文库构建；(4)上机测序；(5)数据分析注释。本专利发明基于目标区域捕获高通量测序技术流程,通过选取与MF、LDS、SGS和ACC相关的特异有效基因集合使用多重PCR捕获流程,一次性进行高通量捕获测序分析。克服了现有技术需要进行八次核苷酸检测,不仅费时,还耗费患者的费用。本发明与现有技术相比具有针对性强、成本低、流程快等优势。(The invention discloses a method for detecting Marfan and syndrome-like related mutant genes thereof based on a high-throughput sequencing technology, which comprises the steps of (1) collecting samples; (2) designing a panel; (3) constructing a library; (4) sequencing on a computer; (5) and (5) data analysis annotation. The invention is based on a target area capture high-throughput sequencing technical process, and high-throughput capture sequencing analysis is carried out at one time by selecting specific effective gene sets related to MF, LDS, SGS and ACC and using a multiple PCR capture process. Overcomes the defects that the prior art needs to carry out eight times of nucleotide detection, not only wastes time, but also consumes the cost of patients. Compared with the prior art, the method has the advantages of strong pertinence, low cost, fast flow and the like.)

1. A method for detecting Marfan and syndrome-like related mutant genes based on a high-throughput sequencing technology comprises the steps of,

(1) collecting samples:

selecting 8 pathogenic genes related to Marfan and syndromes like the Marfan, namely FBN1, TGFBR1, TGFBR2, SMAD3, TGFB2, TGFB3, SKI and FBN 2;

(2) the panel design:

the kit consists of 7 tubes of reagents,

wherein 5 tubes of reagents are stored at-20 ℃ and comprise 1 tube of IGT-I7 Index (10uM) reagent, 1 tube of IGT-I5 Index (10uM) reagent, 1 tube of Primer pool reagent, 1 tube of IGT-EM808 polymerase mix reagent and 1 tube of Enhancer buffer NB (1N) reagent;

the other 2 tubes of reagents are stored at 4 ℃ and comprise 1 tube of Enhancer buffer M reagent and 1 tube of YF buffer B reagent;

in the designed kit, the Primer pool reagent contains 213 amplicons in total, the sequences of the amplicons are 1 to 213, each amplicon comprises a forward Primer and a reverse Primer, and the sequences of the forward Primer and the reverse Primer are SEQ ID No.1 to SEQ ID No. 426;

(3) library construction:

(3.1) 1 st round of multiplex PCR reaction

Preparing reaction liquid in a PCR tube, a calandria or a PCR plate according to the formula of the following table, and gently blowing and sucking up and down by using a gun to mix uniformly;

operating a PCR instrument, putting the PCR tube into the PCR tube, and carrying out reaction according to the following procedures: the cycle was performed after first 95 ℃ for 210 s: circulating at 98 deg.C for 10s and 60 deg.C for 5min for 18 times, and extending at 72 deg.C for 5 min;

(3.2) magnetic bead purification of pooled products

(3.2.1) adding 27ul of AMPure XP magnetic beads which are balanced at room temperature into 30ul of PCR products processed in the step (3.1), and sucking and uniformly mixing the mixture for 20 times by using a pipette;

(3.2.2) after incubating for 5min at room temperature, placing the PCR tube on a DynaMag-96Side magnetic frame for 3 min;

(3.2.3) completely removing the supernatant, taking down the PCR tube from the magnetic rack, adding 50ul YF buffer B into the tube, and sucking and mixing the mixture by a pipettor for 20 times;

(3.2.4) after incubating for 5min at room temperature, placing the PCR tube on a DynaMag-96Side magnetic frame for 3 min;

(3.2.5) removing the supernatant, placing the PCR tube on a magnetic frame, adding 180ul of 80% ethanol solution with volume percentage concentration into the PCR tube, and standing for 30 s;

(3.2.6) removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180ul of ethanol solution with the volume percentage concentration of 80%, standing for 30s, and completely removing the supernatant;

(3.2.7) standing at room temperature for 3min to completely volatilize residual ethanol;

(3.2.8) removing the PCR tube from the magnetic frame, adding 24. mu.l of nucleic-free water, pipetting to gently pipette the resuspended beads to avoid air bubbles, and standing at room temperature for 2 min;

(3.2.9) putting the PCR tube on the magnetic frame again, and standing for 3 min;

(3.2.10) sucking 13.5. mu.l of supernatant by a pipette, transferring the supernatant into a new 200. mu.l PCR tube, wherein the supernatant transferred into the tube is the multiplex PCR product;

(3.3) round 2 adaptor sequence PCR reaction

Preparing reaction liquid in a PCR tube, a calandria or a PCR plate according to the formula shown in the table below, and gently blowing, sucking and mixing the reaction liquid up and down by using a gun, wherein the PCR product mixture is the purified multiplex PCR product obtained in the step (3.2.10);

operating the PCR instrument, putting the PCR tube, the calandria or the PCR plate, and carrying out reaction according to the following procedures: the cycle was performed after first 95 ℃ for 210 s: circulating at 98 deg.C for 20s, 68 deg.C for 1min, and 72 deg.C for 30s for 9 times, and extending at 72 deg.C for 5 min;

(3.4) round 2 magnetic bead purification

(3.4.1) taking 30ul of the PCR reaction system treated in the step (3.3), adding 27ul of AMPure XP magnetic beads balanced at room temperature, and sucking and uniformly mixing the mixture for 20 times by using a pipette;

(3.4.2) after incubating at room temperature for 5min, placing the PCR tube on a DynaMag-96Side magnetic frame for 3 min;

(3.4.3) completely removing the supernatant, taking down the PCR tube from the magnetic rack, adding 50ul YF buffer B into the tube, and sucking and mixing the mixture by a pipettor for 20 times;

(3.4.4) after incubating for 5min at room temperature, the PCR tube was placed on a DynaMag-96Side magnetic frame for 3 min;

(3.4.5) removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180ul of 80% ethanol solution in volume percentage into the PCR tube, and standing for 30 s;

(3.4.7) standing at room temperature for 3min to completely volatilize residual ethanol;

(3.4.8) removing the PCR tube from the magnetic frame, adding 24. mu.l of nucleic-free water or 1 XTE buffer (pH8.0), gently pipetting and mixing for 20 times, resuspending the magnetic beads to avoid air bubbles, and standing at room temperature for 2 min;

(3.4.9) putting the PCR tube on the magnetic frame again, and standing for 3 min;

(3.4.10) sucking 20. mu.l of supernatant by a pipette, transferring the supernatant into a new 200. mu.l PCR tube, wherein the supernatant transferred into the tube is the prepared multiplex PCR library;

(3.5) library quantification

The multiplex PCR library obtained in 2ul step (3.4.10) was taken and used3.0fluorometer (qubit dsDNA HS Assay kit) to perform library concentration determination and record library concentration; the concentration range of a normal library formed by saliva gDNA or blood gDNA is 5-40 ng/ul;

(4) sequencing on machine

Sequencing the multiple PCR library obtained in the step (3.5) by using Hiseq Xten PE150 to obtain a gene sequence table, wherein the computer-loading data volume is 150 Mb;

(5) data analysis annotation: after the sequencing data is downloaded, performing bioinformatics analysis on the downloaded data, and detecting variation on genes;

(5.1) the quality control of the off-line data Fastq format: for each pair of original Fastq sequence raw reads generated by each sample library and lane, removing a sequencing joint and sequence reads containing a large number of 'N' bases by using software cutAdpt to obtain sequence Clean reads meeting quality control, and counting the basic information of the Clean reads by using a software fastqc statistical sequence, wherein the basic information comprises the number of sequences and bases, GC content and distribution, sequencing error rate and distribution and base quality distribution;

(5.2) Alignment: using software bwa, aligning the sequence Clean reads with a human reference genome GRCh38 to obtain an alignment information file BAM of the sequence Clean reads on the reference genome;

(5.3) BAM preprocessing of the comparison information file: merging comparison information file BAM files generated by different libraries and Lane of the same sample together by using picard and GATK software, sequencing according to genome coordinates, verifying the comparison information file BAM files, removing a repetitive sequence generated in the PCR process, correcting the quality value of a base group, and obtaining a preprocessed comparison file Clean BAM; counting and comparing the file Clean BAM by using the picard software to obtain quality control information; the quality control information comprises coverage, depth, capture efficiency and uniformity of a target region capture chip, the number of comparable sequences and bases, and the length distribution of insert fragments;

(5.4) mutation detection: using GATK software to detect small fragment variations on a gene, including Single Nucleotide Polymorphisms (SNPs) and insertions, deletions (indels); and (3) filtering the detected variation, wherein the SNP filtering conditions are as follows: QD <3.37| | FS >31.397| | SOR >10.419| | MQ <20.0| | MQRankSum < -12.49| | ReadPosRankSum < -3.721, and the Indel filtering conditions are as follows: QD <5.2| | FS >52.254| | SOR >9.044| | ReadPosRankSum < -5.504;

annotating the variations that meet the filtering conditions, and noting the corresponding genes, transcripts, variation types, functions, and frequencies in normal populations;

and (5) counting variation results to obtain variation quantity, length distribution, various type variation quantity and the proportion of the variation quantity appearing in the population.

2. The method for detecting Marfan and syndrome-related mutant genes thereof as claimed in claim 1, wherein the concentration of gDNA to be detected is diluted to the same concentration before step (3.1), and then transferred to PCR8 tube.

3. The method for detecting Marfan and syndrome-related mutant genes thereof as claimed in claim 1 or 2, wherein there is further step (3.6) of library quality detection after step (3.5),

taking 1ul of the multiple PCR library obtained in the step (3.4), and measuring the length and purity of the library fragment by using a Qsep100 full-automatic nucleic acid protein analysis system, wherein the target fragment distribution interval of the normal library is between 300bp and 420 bp;

performing library detection on the multiplex PCR library obtained in the step (3.4) by using align 2100 and QPCR;

the result of the multiple PCR library Aligent 2100 fragment obtained in the step (3.4) is qualified when the result is between 250bp and 350bp, and the result of qpcr is qualified when the result is more than 10 nM;

and (4) sequencing the qualified multiplex PCR library in the step (4) to obtain a gene sequence table.

4. The method for detecting Marfan and syndrome-associated mutant genes thereof as claimed in claim 1 or 2, further comprising the step (6) of interpreting the results: explaining the relationship between variant SNPs and variant indels and diseases;

variants were classified into 5 classes by using software REO-HIT according to ACMG/AMP guidelines by software REO-HIT: pathogenic, suspected pathogenic, unknown meaning, suspected benign, benign.

5. The method for detecting Marfan and syndrome-related mutant genes thereof as claimed in claim 4, further comprising the step (7) of providing a report, verifying the sequencing results of the above-detected diseases, suspected diseases, unknown meanings and suspected benign one by one through Sanger's sequencing, and providing the report.

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a high-throughput sequencing technology-based detection method for Marfan and syndrome-like gene mutation thereof.

Background

Marfan syndrome (MFS) is a condition that is primarily manifested as involvement of the skeletal, ocular, and cardiovascular systems, and is prone to aortic dissection and/or aortic rupture. The morbidity of MFS is 0.065-0.2 per mill. In addition, there are other syndromes with phenotypes similar to MFS but with lower prevalence, such as Loeys-Dietz syndrome (LDS), Shprintzen-Goldberg syndrome (SGS) and Bills syndrome (ACC). MF, LDS, SGS and ACC are unigenetic connective tissue diseases with similar clinical phenotypes and large heterogeneity. MFS is predominantly autosomal dominant inheritance, with FBN1 gene encoding fibrillar protein 1 as its major causative gene. The related mutations of the FBN1 gene reported at present exceed 1800, and the proportion of FBN1 gene mutation detected by MFS patients meeting the clinical diagnosis standard is 70-93%. LDS lacks recognized clinical diagnosis standards at present, and the detection of pathogenic gene mutation is the most powerful basis for accurate diagnosis and typing; the pathogenic genes include TGFBR1, TGFBR2, SMAD3, TGFB2 and TGFB 3. SKI gene mutation can be detected in about 90% of patients with Shprintzen-Goldberg syndrome. The pathogenic gene of ACC (also called congenital contracture spider finger) patient is FBN 2. In 2019, the first edition of rare disease diagnosis and treatment guide and the single-gene hereditary cardiovascular disease gene diagnosis guide in China both mention that the differential diagnosis of Marfan and syndromes depends on the identification of pathogenic genes, and the determination of pathogenic sites is the basis of individualized treatment and disease inheritance prevention. There are many methods of gene detection currently used in clinical practice, such as Sanger sequencing, genotyping, high throughput sequencing, Array CGH. Sanger sequencing, as a widely applied technology, has the advantages of rapidness, accuracy, simplicity and convenience and the like, but has the problems of low flux, high relative cost, low automation degree and the like, and is difficult to meet the requirement of one-time detection of multiple genes and multiple variations. Genotyping has various techniques, most of which have the advantages of rapidness, accuracy and low cost, and is very suitable for large-scale detection of known variation, but can only detect known variation of single nucleotides such as SNP and the like. Array CGH is a gold standard for detecting Copy Number Variation (CNV), but cannot detect SNP and Indel variation at the same time.

The clinical phenotypes of MF, LDS, SGS and ACC are highly heterogeneous and have different degrees of coincidence, and diagnosis is often difficult to distinguish depending on clinical symptoms alone. In addition, the disease process is hidden, and early diagnosis is difficult. MFS is predominantly autosomal dominant inheritance, with FBN1 gene encoding fibrillar protein 1 as its major causative gene. The related mutations of the FBN1 gene reported at present exceed 1800, and the proportion of FBN1 gene mutation detected by MFS patients meeting the clinical diagnosis standard is 70-93%. The pathogenic genes of Loeys-Dietz syndrome include TGFBR1, TGFBR2, SMAD3, TGFB2 and TGFB3, the pathogenic gene of SGS is SKI, and the pathogenic gene of ACC is FBN 2. The traditional technology is difficult to examine the variation of the genes at one time, and a new generation of sequencing technology provides a new solution. With the development of sequencing technology, the cost, the cycle and the performance of high-throughput sequencing technology are greatly improved, sequencing of whole exome and whole genome becomes a conventional method discovered by scientific research, all types of variation on genome can be detected simultaneously, but the method is not strong in pertinence, large in data volume and high in cost. Clinical application usually pursues fast, economy, simple and convenient, select a set of effective gene set to carry out high-throughput sequencing in order to reduce cost, become one can be used to detect hereditary aorta disease mutant gene effective method.

Disclosure of Invention

The invention aims to solve the technical problem that an effective and low-cost gene mutation method for detecting MF, LDS, SGS and ACC is absent at present, a multiple PCR capture technology is adopted for the technical problem, a selected specific effective gene set is captured by using an amplification principle, then detection is carried out through a high-throughput sequencing platform, and gene mutation of MF, LDS, SGS and ACC is detected and interpreted by using a biological information analysis process and an ACMG genetic interpretation guide, so as to assist clinical accurate diagnosis and treatment of hereditary aortic disease. The results obtained by the method can be applied to the differential diagnosis of MF, LDS, SGS and ACC, the prevention and intervention of diseases, individualized treatment and prenatal diagnosis.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for detecting Marfan and syndrome-like related mutant genes based on a high-throughput sequencing technology comprises the steps of,

(1) collecting samples:

selecting 8 pathogenic genes related to Marfan and syndromes like the Marfan, namely FBN1, TGFBR1, TGFBR2, SMAD3, TGFB2, TGFB3, SKI and FBN 2;

(2) the panel design:

the kit consists of 7 tubes of reagents,

the other 2 tubes of reagents are stored at 4 ℃ and comprise 1 tube of Enhancer buffer M reagent and 1 tube of YF buffer B reagent;

(3) library construction:

(3.1) 1 st round of multiplex PCR reaction

Preparing reaction liquid in a PCR tube according to the formula shown in the table below, and gently blowing, sucking and mixing the reaction liquid up and down by using a gun;

(3.2) magnetic bead purification of pooled products

(3.2.2) after incubating for 5min at room temperature, placing the PCR tube on a DynaMag-96Side magnetic frame for 3 min;

(3.2.3) completely removing the supernatant, taking down the PCR tube from the magnetic rack, adding 50ul YF buffer B into the tube, and sucking and mixing the mixture by a pipettor for 20 times;

(3.2.4) after incubating for 5min at room temperature, placing the PCR tube on a DynaMag-96Side magnetic frame for 3 min;

(3.2.5) removing the supernatant, placing the PCR tube on a magnetic frame, adding 180ul of 80% ethanol solution with volume percentage concentration into the PCR tube, and standing for 30 s;

(3.2.7) standing at room temperature for 3min to completely volatilize residual ethanol;

(3.2.9) putting the PCR tube on the magnetic frame again, and standing for 3 min;

(3.2.10) sucking 13.5. mu.l of supernatant by a pipette, transferring the supernatant into a new 200. mu.l PCR tube, wherein the supernatant transferred into the tube is the multiplex PCR product;

(3.3) round 2 adaptor sequence PCR reaction

Preparing a reaction solution in a PCR tube according to the formula of the following table, and gently blowing, sucking and mixing the reaction solution up and down by using a gun, wherein the PCR product mixture is the purified multiplex PCR product obtained in the step (3.2.10);

operating a PCR instrument, putting the PCR tube into the PCR tube, and carrying out reaction according to the following procedures: the cycle was performed after first 95 ℃ for 210 s: circulating at 98 deg.C for 20s, 68 deg.C for 1min, and 72 deg.C for 30s for 9 times, and extending at 72 deg.C for 5 min;

(3.4) round 2 magnetic bead purification

(3.4.2) after incubating at room temperature for 5min, placing the PCR tube on a DynaMag-96Side magnetic frame for 3 min;

(3.4.3) completely removing the supernatant, taking down the PCR tube from the magnetic rack, adding 50ul YF buffer B into the tube, and sucking and mixing the mixture by a pipettor for 20 times;

(3.4.4) after incubating for 5min at room temperature, the PCR tube was placed on a DynaMag-96Side magnetic frame for 3 min;

(3.4.5) removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180ul of 80% ethanol solution in volume percentage into the PCR tube, and standing for 30 s;

(3.4.7) standing at room temperature for 3min to completely volatilize residual ethanol;

(3.4.9) putting the PCR tube on the magnetic frame again, and standing for 3 min;

(3.5) library quantification

(4) sequencing on machine

Sequencing the multiple PCR library obtained in the step (3.5) by using Hiseq Xten PE150 to obtain a gene sequence table, wherein the computer-loading data volume is 150 Mb;

(5) data analysis annotation: after the sequencing data is downloaded, performing bioinformatics analysis on the downloaded data, and detecting variation on genes;

annotating the variations that meet the filtering conditions, and noting the corresponding genes, transcripts, variation types, functions, and frequencies in normal populations;

and (5) counting variation results to obtain variation quantity, length distribution, various type variation quantity and crowd variation frequency information.

For better technical effect, the concentration of gDNA to be detected is diluted to the same concentration before step (3.1), and then transferred to the PCR8 tube.

In order to obtain better technical effect, step (3.6) of library quality detection is carried out after step (3.5),

performing library detection on the multiplex PCR library obtained in the step (3.4) by using align 2100 and QPCR;

and (4) sequencing the qualified multiplex PCR library in the step (4) to obtain a gene sequence table.

In order to obtain better technical effect, the method also comprises the step (6) of interpretation of the result: explaining the relationship between variant SNPs and variant indels and diseases;

variants were classified into 5 classes by using the software REO-HIT according to the ACMG/AMP guidelines: pathogenic, suspected pathogenic, unknown meaning, suspected benign, benign.

In order to obtain better technical effects, the method also comprises the step (7) of giving a report, verifying the sequencing results of the pathogenicity, suspected pathogenicity, unknown significance and suspected virtuosity one by one through the Sanger method sequencing, and giving the report.

The invention is based on a target area capturing high-throughput sequencing technology process, selects a specific effective gene set related to MF, LDS, SGS and ACC, performs high-throughput capturing sequencing analysis by using a multiple PCR capturing process and the following implementation scheme, and aims to decipher the variation condition of pathogenic genes of the MF, LDS, SGS and ACC by using a gene detection technology. The results of the analysis of multigenic variants were read with reference to published guidelines of the American society for genetics (ACMG), molecular Pathology (AMP) and the Chinese medical society for cardiovascular disease Scoring, the accurate cardiovascular disease group, etc. Compared with the prior art, the method has the advantages of strong pertinence, low cost, fast flow and the like.

Reagent manufacturers: illumina Corp.

Drawings

FIG. 1 is a flowchart illustrating genetic variation analysis and interpretation according to an embodiment of the present invention;

FIG. 2 shows the results of quality control library of Qsep100 full-automatic nucleic acid protein analysis system according to the present invention;

FIG. 3 is a flowchart of a detection technique according to an embodiment of the present invention.

Detailed Description

The invention is further explained in detail below with reference to the drawings and the detailed description.

A method for detecting Marfan and syndrome-like related mutant genes based on a high-throughput sequencing technology comprises the steps of,

(1) collecting samples:

selecting 8 pathogenic genes related to Marfan and syndromes like the Marfan, namely FBN1, TGFBR1, TGFBR2, SMAD3, TGFB2, TGFB3, SKI and FBN 2;

(2) the panel design:

the kit consists of 7 tubes of reagents,

the other 2 tubes of reagents are stored at 4 ℃ and comprise 1 tube of Enhancer buffer M reagent and 1 tube of YF buffer B reagent;

performing panel design according to the total 8 target genes screened in the step (1), wherein the designed region is an exon region of each gene, and the two sides of the exon region are allowed to extend by 15bp respectively;

in the designed kit, the Primer pool reagent contains 213 amplicons in total, the sequence of the amplicon is 1-213, each amplicon comprises a forward Primer and a reverse Primer, the sequences of the forward Primer and the reverse Primer are SEQ ID No.1-SEQ ID No.426, and the detailed Primer sequences are shown in appendix 3;

the position information of the IGT-I7 Index is disclosed in appendix 1, "96 IGT-I7 Index position information";

the information of IGT-I5 Index is described in appendix 2;

reagent manufacturers: illumina Corp.;

(3) library construction:

(3.0) diluting the concentrations of all gDNAs to be detected to the same concentration, and transferring the gDNAs to a PCR8 union tube, wherein on one hand, the gDNA concentration is convenient for the gun arrangement operation, and on the other hand, the gDNA concentration difference of the final library is reduced for adding the same initial amount of gDNA, so that the library is convenient to mix; reaction numbering is carried out on the upper part of the PCR tube wall or the tube cover, so that the marks are prevented from disappearing due to high temperature or other reasons, and the cross contamination of samples caused by the misoperation of mixing subsequent products is avoided;

(3.1) 1 st round of multiplex PCR reaction

Preparing reaction liquid in a PCR tube (or a calandria or a PCR plate) according to the formula in the following table, and gently blowing and sucking up and down by using a gun to mix uniformly;

(3.2) magnetic bead purification of pooled products

(3.2.2) after incubating for 5min at room temperature, placing the PCR tube on a DynaMag-96Side magnetic frame for 3 min;

(3.2.3) completely removing the supernatant, taking down the PCR tube from the magnetic rack, adding 50ul YF buffer B into the tube, and sucking and mixing the mixture by a pipettor for 20 times;

(3.2.4) after incubating for 5min at room temperature, placing the PCR tube on a DynaMag-96Side magnetic frame for 3 min;

(3.2.5) removing the supernatant, placing the PCR tube on a magnetic frame, adding 180ul of 80% ethanol solution with volume percentage concentration into the PCR tube, and standing for 30 s;

(3.2.6) removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180ul of 80% ethanol solution with volume percentage concentration into the PCR tube, standing for 30s, completely removing the supernatant, and recommending to remove the residual ethanol solution at the bottom by using a 10 mu l pipette;

(3.2.7) standing at room temperature for 3min to completely volatilize residual ethanol;

(3.2.9) putting the PCR tube on the magnetic frame again, and standing for 3 min;

(3.2.10) sucking 13.5. mu.l of supernatant by a pipette, transferring the supernatant into a new 200. mu.l PCR tube, wherein the supernatant transferred into the tube is the multiplex PCR product;

(3.3) round 2 adaptor sequence PCR reaction

Preparing reaction liquid in a PCR tube (or a calandria or a PCR plate) according to the formula shown in the table below, and gently blowing and sucking up and down by using a gun to mix uniformly, wherein the PCR product mixture is the purified multiplex PCR product obtained in the step (3.2.10);

operating a PCR instrument, putting the PCR tube into the PCR tube, and carrying out reaction according to the following procedures: the cycle was performed after first 95 ℃ for 210 s: circulating at 98 deg.C for 20s, 68 deg.C for 1min, and 72 deg.C for 30s for 9 times, and extending at 72 deg.C for 5 min;

(3.4) round 2 magnetic bead purification

(3.4.2) after incubating at room temperature for 5min, placing the PCR tube on a DynaMag-96Side magnetic frame for 3 min;

(3.4.3) completely removing the supernatant, taking down the PCR tube from the magnetic rack, adding 50ul YF buffer B into the tube, and sucking and mixing the mixture by a pipettor for 20 times;

(3.4.4) after incubating for 5min at room temperature, the PCR tube was placed on a DynaMag-96Side magnetic frame for 3 min;

(3.4.5) removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180ul of 80% ethanol solution in volume percentage into the PCR tube, and standing for 30 s;

(3.4.6) removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180ul of 80% ethanol solution in volume percentage into the PCR tube, standing for 30s, completely removing the supernatant, and recommending to remove the residual ethanol solution at the bottom by using a 10-microliter pipettor;

(3.4.7) standing at room temperature for 3min to completely volatilize residual ethanol;

(3.4.9) putting the PCR tube on the magnetic frame again, and standing for 3 min;

(3.5) library quantification

(3.6) library quality testing

Taking 1ul of the multiple PCR library obtained in the step (3.4), and measuring the length and purity of the library fragment by using a Qsep100 full-automatic nucleic acid protein analysis system, wherein the target fragment distribution interval of the normal library is between 300bp and 420 bp; the detection result is shown in figure 2;

performing library detection on the multiplex PCR library obtained in the step (3.4) by using align 2100 and QPCR;

(4) sequencing on machine

Sequencing the qualified multiplex PCR library obtained in the step (3.6) by using Hiseq Xten PE150 to obtain a gene sequence table, wherein the computer data amount is 150 Mb;

(5) data analysis annotation: after the sequencing data is downloaded, performing bioinformatics analysis on the downloaded data, and detecting variation on genes;

(5.3) BAM preprocessing of the comparison information file: combining BAM files of comparison information files generated by different libraries and Lane of the same sample together by using picard and GATK software, sequencing according to genome coordinates, verifying the BAM files of the comparison information files, removing a repetitive sequence generated in the PCR process, correcting a base quality value, and obtaining a Clean BAM of the comparison file after pretreatment; counting and comparing the file Clean BAM by using the picard software to obtain quality control information; the quality control information comprises coverage, depth, capture efficiency and uniformity of a target region capture chip, the number of comparable sequences and bases, and the length distribution of insert fragments;

(5.4) mutation detection: detecting small fragment variation on the gene by using GATK software, wherein the small fragment variation comprises Single Nucleotide Polymorphism (SNP) and insertion and deletion indels;

and (3) filtering the detected variation, wherein the SNP filtering conditions are as follows: QD <3.37| | FS >31.397| | SOR >10.419| | MQ <20.0| | MQRankSum < -12.49| | ReadPosRankSum < -3.721, and the Indel filtering conditions are as follows: QD <5.2| | FS >52.254| | SOR >9.044| | ReadPosRankSum < -5.504;

annotating the variations that meet the filtering conditions to obtain variation information, wherein the variation information comprises genes, transcripts, variation types, functions and frequencies in normal population;

counting variation results to obtain variation quantity, length distribution, various type variation quantity and proportion appearing in population;

(6) interpretation of the results: explaining the relationship between variant SNPs and variant indels and diseases;

the variants were classified into 5 classes by using the software REO-HIT according to the ACMG/AMP guidelines (Genet Med.2015 May; 17 (5): 405-: pathogenic, suspected pathogenic, unknown meaning, suspected benign, benign;

the software REO-HIT processing procedure is shown in FIG. 1:

the method comprises the specific steps of carrying out,

loading a database: loading ClinVar, CGD and OMIM databases;

collecting variation information: reading the variation information obtained in the step (5.4) one by one to obtain basic variation information, population variation frequency information, disease variation information, variation function prediction information and variation conservation;

processing interpretation evidence:

i) by database comparison with the collected mutation information, it was sequentially judged whether each mutation satisfied ACMG/AMP guidelines (Genet med.2015 May; 17(5): 405-424.) said pathogenic or benign evidence;

wherein the evidence of pathogenicity includes: strong pathogenic PVS, strong pathogenic PS, medium pathogenic PM and weak pathogenic PP;

benign evidence includes: independent benign BA, strong benign BS, weak benign BP;

ii) combining the evidence of variation, classifying each variation according to the ACMG/AMP combination rules as: pathogenic, suspected pathogenic, unknown meaning, suspected benign, benign;

(7) issue a report

And (3) verifying the sequencing results of the detected diseases, suspected diseases, unknown meanings and suspected benign one by one through Sanger sequencing, and giving a report.

Comparative example

Sanger sequencing is a gold standard for the detection of DNA mutations, but due to its limited sensitivity, it is not possible to simultaneously probe multiple targets in parallel.

If Sanger sequencing is adopted to detect FBN1, TGFBR1, TGFBR2, SMAD3, TGFB2, TGFB3, SKI and FBN2 genes, Sanger sequencing is required to be carried out on each exon respectively, and the cost is high.

The target region capture high-throughput sequencing used in the invention relates to the targeted enrichment of the exons of FBN1, TGFBR1, TGFBR2, SMAD3, TGFB2, TGFB3, SKI and FBN2 genes, and the detection of all the exons of the genes can be realized only once by parallel sequencing of Illumina XTen.

The invention has the characteristics that: a gene mutation detection method of MF, LDS, SGS and ACC based on high-throughput sequencing technology and its application are characterized by that it can make gene detection for MF, LDS, SGS and ACC, and at the same time, in the course of experiment a labeling technology can be used to raise accuracy of result detection.

Gene lists As shown in Table 1, the target regions are each gene exon and each region flanked by 15bp extensions.

The detection method comprises the following steps:

(1) the genome DNA to be detected is interrupted, the main band is 300-400bp

(2) Purifying the broken DNA fragment, repairing the tail end, adding a joint and carrying out PCR amplification

(3) The tagged joint is used in the process of capturing and building a library, so that the sequencing noise pollution is reduced, the PCR error is corrected, the accuracy of the result is improved, and particularly, the low-frequency mutation is easy to detect;

(4) hybridizing the amplified product with a target region capture chip, and amplifying and purifying to obtain a target DNA sequencing library

(5) Sequencing with sequencing library to obtain the sequence of the current gene

(6) Bioinformatic analysis using sequencing sequences including sequence quality control, sequence alignment, alignment file pre-processing and detection of SNPs, Indel variations, variation filtering and annotation

(7) The results of the variation are interpreted and classified into 5 categories, pathogenic, suspected pathogenic, unknown meaning, suspected benign, and benign.

In the detection process, the sequencing data volume is required to reach more than 150Mb, the sequencing depth reaches 150X, and the coverage is higher than 99.9%.

The invention relates to a gene mutation detection method of MF, LDS, SGS and ACC based on a high-throughput sequencing technology and application thereof, and can simultaneously capture all coding regions and flanking regions of 8 genes shown in a third part by adopting a multiplex PCR technology. The relationship between the variation and the disease is accurately interpreted, and important basis is provided for the screening, early diagnosis, differential diagnosis, birth guidance and exercise suggestion of MF, LDS, SGS and ACC. Has the advantages of comprehensiveness, low cost and the like.

Appendix 1:

position information for IGT-I7 Index, using BOX1, 96-well plates,

A01

A02

A03

A04

A05

A06

A07

A08

A09

A10

A11

A12

B01

B02

B03

B04

B05

B06

B07

B08

B09

B10

B11

B12

C01

C02

C03

C04

C05

C06

C07

C08

C09

C10

C11

C12

D01

D02

D03

D04

D05

D06

D07

D08

D09

D10

D11

D12

E01

E02

E03

E04

E05

E06

E07

E08

E09

E10

E11

E12

F01

F02

F03

F04

F05

F06

F07

F08

F09

F10

F11

F12

G01

G02

G03

G04

G05

G06

G07

G08

G09

G10

G11

G12

H01

H02

H03

H04

H05

H06

H07

H08

H09

H10

H11

H12

。

appendix 2: IGT-I5 Index sequence information

The IGT-I5 Index end sample separation sequence is related to a sequencing platform:

MiniSeq, NextSeq, HiSeq 3000/4000, and Hiseq X Ten sequencing platforms, please use the sample sequences in the Index column;

MiSeq, HiSeq 2000/2500 and NovaSeq sequencing platforms, please use the subsampling sequence of the Inprimer column;

3. in the case of single-ended sequencing, the data can be split only by column-dividing sequence of IGT-I7 Index.

Name	Indcx sample separation sequence	Inprimcr partial sample sequence
			IGT-15-31#	GTAGAGGA	TCCTCTAC
IGT-15-32#	CCGCCTTA	TAAGGCGG
			IGT-15-33#	ATAGTACG	CGTACTAT
IGT-I5-34#	TTCTGCCT	AGGCAGAA

。

Appendix 3:

the Primer pool reagent contains 213 amplicons with the sequences of 1-213, each amplicon comprises a forward Primer and a reverse Primer, the sequences of the forward Primer and the reverse Primer are SEQ ID No.1-SEQ ID No.426, which are shown in the following table,

SEQUENCE LISTING

<110> heart cerebrovascular prevention and cure education department key laboratory

FIRST AFFILIATED HOSPITAL OF GANNAN MEDICAL University

Paragraph, yanyu

Liu, Zi is composed of

<120> method for detecting Marfan and syndrome-like related mutant genes thereof based on high-throughput sequencing technology

<130> 2020

<160> 426

<170> PATENTIN VERSION 3.5

<210> 1

<211> 32

<212> DNA