Exome sequencing_Wuhan biological Optical Valley Science and technology

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Exome sequencing

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Exome sequencing

Product Description

Case Interpretation

Whole Exome Sequencing (WES) refers to the use of sequence capture technology to capture and enrich DNA from exome regions for high-throughput sequencing, which can directly identify genetic variants associated with protein function variation. Exome sequencing has been applied to the search for disease-causing and susceptibility genes associated with a variety of complex diseases.

technology roadmap

technical parameter

Sample requirements

Sequencing strategy

Lead time

Sample type: genomic DNA

Total sample concentration: ≥20 ng/μl (Qubit quantification)

Total sample mass: ≥3.0 μg (total of 3 times of library building, excluding consuming of sample assay, Qubit quantification)

Sample purity: OD260/280 =1.7~2.2

Electrophoresis requirements: clear main band, no degradation or mild degradation, no serious RNA or protein contamination. (latest electrophoresis result of the sample delivery date)

Sequencing type: Illumina/MGI PE150.

Sequencing depth: germ cell mutations ≥ 100X (10G).

Somatic mutations ≥150X (15G)

45 days

Product advantages

a. High specificity: Use mainstream kit, have stable capture efficiency, comprehensive exon region coverage and high capture specificity;

b. Cost-effective: comprehensive functional annotation of exon regions, which can effectively identify disease- or trait-related regions and save research costs;

c. High information efficiency: Compared with whole genome sequencing, it has deeper coverage and better data precision, less interference information and easy to analyse.

Analysis of genetic factors of premature ovarian failure by exon sequencing in case 1
Research background:
Premature ovarian failure usually refers to women's amenorrhea before the age of 40, and 1% of women suffer from this disease. The etiology of the disease is complex, such as infection, surgery, autoimmune diseases, environmental factors and genetic factors can lead to premature ovarian failure. At present, at least 10-15% of the diseased individuals are considered to be affected by genetic factors.
Research content:
The depth of sequencing of one patient with premature ovarian failure and one sister without disease (whose parents are close relatives) in the Middle East family (MO1DA) is 50 × The full exon capture sequencing of IV-1 showed that one patient suffered from premature ovarian failure (IV-1) and the other patient did not suffer from this disease (IV-3). The sequencing results showed that there was a large segment of homozygous region on chromosome 7 of IV-1, and six genes located in 7q21.3~22.2 were found to have mutations. Five of the genes were excluded according to the known function or expression domain of the gene, and a frameshift mutation (c.968delC) occurred in one gene - STAG3. Through Sanger sequencing, it was confirmed that the mutation and phenotype (premature ovarian failure) were co-separated. The four sisters with premature ovarian failure had a homozygous mutation of STAG3, while the other members without the disease did not have the mutation. Using STAG3 mutant mice, it was verified that STAG3 mutation was the cause of premature ovarian failure.

Figure 1. A.MO1DA pedigree map; B. Structure of STAG3 gene (red arrow represents mutation site)

Research results:
The study used exon sequencing technology to find for the first time that the mutation of STAG3 gene in meiotic gene can lead to recessive premature ovarian failure in patients with premature ovarian failure in the Middle East family (MO1DA), which has also been confirmed in mouse animal models and patients with premature ovarian failure.

Case 2 Whole Exome Sequencing Helps Explore New Functional Genes of Heart Disease
Research background:
Valvular heart disease can affect any of the heart valves and is often associated with syndrome disorder. Previous studies have shown that valvular heart disease (HVD) has a complex genetic structure. Although the incidence of different types of HVD, such as mitral valve prolapse and mitral aortic valve (BAV), is high, only a few cases have potential single-gene etiology in non-syndromic background.
Research content:
Two consanguineous families were identified, and each family had two affected family members who had developed progressive heart valve disease very early. Full-exome sequencing showed that there were homologous and truncated nonsense alleles of ADAMTS19 gene in all four affected individuals. The expression analysis of lacZ reporter gene and single-cell RNA-seq showed that Adamts19 was a new marker of valve interstitial cells. In the inference of the gene regulatory network of valve interstitial cells, Adamts19 is located in a highly identifiable network driven by the transcription factor of the Wnt signal pathway downstream, i.e., lysophoid enhancer-binding factor 1.
Research results:
Two unrelated consanguineous families with the recessive genetic pattern of early onset HVD without syndrome were analyzed by full exon capture sequencing. Homozygous rare loss of function (LOF) alleles were found in the ADAMTS19 gene of four affected individuals. The recessive linkage analysis of ADAMTS19 locus in the two families showed that there was no significant difference due to the limited number of affected individuals. Although there is no significant linkage, ADAMTS19 represents the only gene with homozygous and rare functional deletion alleles in all affected individuals, making it a candidate gene for HVD observed. Compared with other ADAMTS-associated diseases, no syndrome is the characteristic of identifying patients affected by HVD, indicating that the functional deficiency of ADAMTS19 leads to isolated, non-syndromic and progressive HVD in humans.

reference：

　　1. Caburet S, Arboleda V A, Llano E, et al. Mutant cohesin in premature ovarian failure[J]. New England Journal of Medicine, 2014, 370(10): 943-949.

　　2.F Wünnemann, Ta-Shma A , Preuss C , et al. Loss of ADAMTS19 causes progressive non-syndromic heart valve disease[J]. Nature Genetics, 2020, 52(1).