A Bioinformatics Pipeline for Whole Exome Sequencing: Overview of the Processing and Steps from Raw Data to Downstream Analysis

AbstractRecent advances in next generation sequencing (NGS) technologies have given an impetus to find causality for rare genetic disorders. Since 2005 and aftermath of the human genome project, efforts have been made to understand the rare variants of genetic disorders. Benchmarking the bioinformatics pipeline for whole exome sequencing (WES) has always been a challenge. In this protocol, we discuss detailed steps from quality check to analysis of the variants using a WES pipeline comparing them with reposited public NGS data and survey different techniques, algorithms and software tools used during each step. We observed that variant calling performed on exome and whole genome datasets have different metrics generated when compared to variant callers, GATK and VarScan with different parameters. Furthermore, we found that VarScan with strict parameters could recover 80-85% of high quality GATK SNPs with decreased sensitivity from NGS data. We believe our protocol in the form of pipeline can be used by researchers interested in performing WES analysis for genetic diseases and by large any clinical phenotypes.

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Pilot study of EVIDENCE: High diagnostic yield and clinical utility of whole exome sequencing using an automated interpretation system for patients with suspected genetic disorders

10.1101/628438 ◽

2019 ◽

Cited By ~ 2

Author(s):

Go Hun Seo ◽

Taeho Kim ◽

Jung-young Park ◽

Jungsul Lee ◽

Sehwan Kim ◽

...

Keyword(s):

Family Member ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Diagnostic Yield ◽

Genetic Disorders ◽

Genetic Diseases ◽

Whole Exome ◽

Interpretation System ◽

Automated Interpretation ◽

Age Range

AbstractPurposeEVIDENCE, an automated interpretation system, has been developed to facilitate the entire process of whole exome sequencing (WES) analyses. This study investigated the diagnostic yield of EVIDENCE in patients suspected genetic disorders.MethodsDNA from 330 probands (age range, 0–68 years) with suspected genetic disorders were subjected to WES. Candidate variants were identified by EVIDENCE and confirmed by testing family members and/or clinical reassessments.ResultsThe average number of overlapping organ categories per patient was 4.5 ± 5.0. EVIDENCE reported a total 244 variants in 215 (65.1%) of the 330 probands. After clinical reassessment and/or family member testing, 196 variants were identified in 171 probands (51.8%), including 115 novel variants. These variants were confirmed as being responsible for 146 genetic disorders. One hundred-seven (54.6%) of the 196 variants were categorized as pathogenic or likely pathogenic before, and 146 (74.6%) after, clinical assessment and/or family member testing. Factors associated with a variant being confirmed as causative include rules, such as PVS1, PS1, PM1, PM5, and PP5, and similar symptom scores between that variant and a patient’s phenotype.ConclusionThis new, automated variant interpretation system facilitated the diagnosis of various genetic diseases with a 51% improvement in diagnostic yield.

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Pathogenic variants identified using whole-exome sequencing in Chinese patients with primary ciliary dyskinesia

10.21203/rs.3.rs-156279/v2 ◽

2021 ◽

Author(s):

Yutian Ye ◽

Qijun Huang ◽

Lipeng Chen ◽

Chunxian Liang ◽

Kaixue Zhuang ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Primary Ciliary Dyskinesia ◽

Rare Variants ◽

Genetic Disorders ◽

Autosomal Recessive Disorder ◽

Bioinformatic Analysis ◽

Chinese Patients ◽

Whole Exome ◽

Ciliary Dyskinesia

Abstract Background Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder. The genetic factors contributing to PCD pathogenesis remain elusive for approximately 20–35% of patients with complex and abnormal clinical phenotypes. Our study aimed to identify causative variants of sporadic PCD genes using whole-exome sequencing (WES). Result All patients were diagnosed with PCD based on clinical phenotype or transmission electron microscopy (TEM) images of cilia. WES and bioinformatic analysis were then conducted for patients with PCD. Identified candidate variants were validated by Sanger sequencing. Pathogenicity of candidate variants was then evaluated using in silico software and the American College of Medical Genetics and Genomics (ACMG) database. In total, 15 rare variants were identified in five patients with PCD. Five new variants of CCDC40, DNAH1, DNAAF3, and DNAI1 were considered causative variants and included one splicing and three homozygous variants. Conclusion Our study demonstrated that patients with PCD carry rare causative variants of multiple genes. Our findings indicated that not only known causative genes but also other functional genes should be considered for heterogeneous genetic disorders.

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Pathogenic Variants Identified using Whole-exome Sequencing in Chinese Patients with Primary Ciliary Dyskinesia

10.21203/rs.3.rs-156279/v1 ◽

2021 ◽

Author(s):

Yutian Ye ◽

Qijun Huang ◽

Lipeng Chen ◽

Chunxian Liang ◽

Kaixue Zhuang ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Primary Ciliary Dyskinesia ◽

Rare Variants ◽

Genetic Disorders ◽

Autosomal Recessive Disorder ◽

Bioinformatic Analysis ◽

Chinese Patients ◽

Whole Exome ◽

Ciliary Dyskinesia

Abstract Background： Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder. The genetic factors contributing to PCD pathogenesis remain elusive for approximately 20–35% of patients with complex and abnormal clinical phenotypes. Our study aimed to identify causative variants of sporadic PCD genes using whole-exome sequencing (WES). Result：All patients were diagnosed with PCD based on clinical phenotype or transmission electron microscopy (TEM) images of cilia. WES and bioinformatic analysis were then conducted for patients with PCD. Identified candidate variants were validated by Sanger sequencing. Pathogenicity of candidate variants was then evaluated using in silico software and the American College of Medical Genetics and Genomics (ACMG) database. In total, 15 rare variants were identified in five patients with PCD. Five new variants of CCDC40, DNAH1, DNAAF3, and DNAI1 were considered causative variants and included one splicing and three homozygous variants. Conclusion： Our study demonstrated that patients with PCD carry rare causative variants of multiple genes. Our findings indicated that not only known causative genes but also other functional genes should be considered for heterogeneous genetic disorders.

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Exome sequencing in paediatric patients with movement disorders

Orphanet Journal of Rare Diseases ◽

10.1186/s13023-021-01688-6 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Anna Ka-Yee Kwong ◽

Mandy Ho-Yin Tsang ◽

Jasmine Lee-Fong Fung ◽

Christopher Chun-Yu Mak ◽

Kate Lok-San Chan ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Movement Disorders ◽

Rare Variants ◽

Diagnostic Yield ◽

Neurological Diseases ◽

Genetic Diagnosis ◽

Potential Treatment ◽

Paediatric Patients ◽

Whole Exome

Abstract Background Movement disorders are a group of heterogeneous neurological diseases including hyperkinetic disorders with unwanted excess movements and hypokinetic disorders with reduction in the degree of movements. The objective of our study is to investigate the genetic etiology of a cohort of paediatric patients with movement disorders by whole exome sequencing and to review the potential treatment implications after a genetic diagnosis. Results We studied a cohort of 31 patients who have paediatric-onset movement disorders with unrevealing etiologies. Whole exome sequencing was performed and rare variants were interrogated for pathogenicity. Genetic diagnoses have been confirmed in 10 patients with disease-causing variants in CTNNB1, SPAST, ATP1A3, PURA, SLC2A1, KMT2B, ACTB, GNAO1 and SPG11. 80% (8/10) of patients with genetic diagnosis have potential treatment implications and treatments have been offered to them. One patient with KMT2B dystonia showed clinical improvement with decrease in dystonia after receiving globus pallidus interna deep brain stimulation. Conclusions A diagnostic yield of 32% (10/31) was reported in our cohort and this allows a better prediction of prognosis and contributes to a more effective clinical management. The study highlights the potential of implementing precision medicine in the patients.

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A domestic cat whole exome sequencing resource for trait discovery

Scientific Reports ◽

10.1038/s41598-021-86200-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Alana R. Rodney ◽

Reuben M. Buckley ◽

Robert S. Fulton ◽

Catrina Fronick ◽

Todd Richmond ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Genetic Diseases ◽

Domestic Cat ◽

Whole Genome Sequence ◽

Exome Capture ◽

Single Nucleotide Variants ◽

Whole Exome ◽

Olfactory Receptor Genes ◽

Feline Model

AbstractOver 94 million domestic cats are susceptible to cancers and other common and rare diseases. Whole exome sequencing (WES) is a proven strategy to study these disease-causing variants. Presented is a 35.7 Mb exome capture design based on the annotated Felis_catus_9.0 genome assembly, covering 201,683 regions of the cat genome. Whole exome sequencing was conducted on 41 cats with known and unknown genetic diseases and traits, of which ten cats had matching whole genome sequence (WGS) data available, used to validate WES performance. At 80 × mean exome depth of coverage, 96.4% of on-target base coverage had a sequencing depth > 20-fold, while over 98% of single nucleotide variants (SNVs) identified by WGS were also identified by WES. Platform-specific SNVs were restricted to sex chromosomes and a small number of olfactory receptor genes. Within the 41 cats, we identified 31 previously known causal variants and discovered new gene candidate variants, including novel missense variance for polycystic kidney disease and atrichia in the Peterbald cat. These results show the utility of WES to identify novel gene candidate alleles for diseases and traits for the first time in a feline model.

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Whole Exome Sequencing of HIV-1 long-term non-progressors identifies rare variants in genes encoding innate immune sensors and signaling molecules

Scientific Reports ◽

10.1038/s41598-018-33481-0 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 3

Author(s):

Sara Konstantin Nissen ◽

Mette Christiansen ◽

Marie Helleberg ◽

Kathrine Kjær ◽

Sofie Eg Jørgensen ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Rare Variants ◽

Signaling Molecules ◽

Innate Immune ◽

Whole Exome ◽

Genes Encoding ◽

Hiv 1

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Multiple Variant Calling Pipelines in Wheat Whole Exome Sequencing

International Journal of Molecular Sciences ◽

10.3390/ijms221910400 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10400

Author(s):

H. Busra Cagirici ◽

Bala Ani Akpinar ◽

Taner Z. Sen ◽

Hikmet Budak

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Variant Calling ◽

High Sensitivity ◽

Whole Exome ◽

The Impact ◽

Multiple Reference ◽

Reference Genomes ◽

Selection Of ◽

Variant Identification

The highly challenging hexaploid wheat (Triticum aestivum) genome is becoming ever more accessible due to the continued development of multiple reference genomes, a factor which aids in the plight to better understand variation in important traits. Although the process of variant calling is relatively straightforward, selection of the best combination of the computational tools for read alignment and variant calling stages of the analysis and efficient filtering of the false variant calls are not always easy tasks. Previous studies have analyzed the impact of methods on the quality metrics in diploid organisms. Given that variant identification in wheat largely relies on accurate mining of exome data, there is a critical need to better understand how different methods affect the analysis of whole exome sequencing (WES) data in polyploid species. This study aims to address this by performing whole exome sequencing of 48 wheat cultivars and assessing the performance of various variant calling pipelines at their suggested settings. The results show that all the pipelines require filtering to eliminate false-positive calls. The high consensus among the reference SNPs called by the best-performing pipelines suggests that filtering provides accurate and reproducible results. This study also provides detailed comparisons for high sensitivity and precision at individual and population levels for the raw and filtered SNP calls.

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Whole‐Exome Sequencing of a Saudi Epilepsy Cohort Reveals Association Signals in Known and Potentially Novel Loci

10.21203/rs.3.rs-915593/v1 ◽

2021 ◽

Author(s):

Amein Kadhem AlAli ◽

Abdulrahman Al-Enazi ◽

Ahmed Ammar ◽

Mahmoud Hajj ◽

Cyril Cyrus ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Rare Variants ◽

High Rate ◽

Variants Of Unknown Significance ◽

Pathogenic Variants ◽

Whole Exome ◽

Novel Variants ◽

Unknown Significance ◽

Rare Genetic Variants

Abstract Background Epilepsy, a serious chronic neurological condition effecting up to 100 million people globally, has clear genetic underpinnings including common and rare variants. In Saudi Arabia the prevalence of epilepsy is high and caused mainly by perinatal and genetic factors. No whole-exome sequencing (WES) studies have been performed to date in Saudi Arabian Epilepsy cohorts. This offers a unique opportunity for the discovery of rare genetic variants impacting this disease as there is a high rate of consanguinity amongst large tribal pedigrees. Results We performed WES on 144 individuals diagnosed with epilepsy, to interrogate known Epilepsy related genes for known and functional novel variants. We also used an American College of Medical Genetics (ACMG) guideline based variant prioritization approach in an attempt to discover putative causative variants. We identified a 32 potentially causative pathogenic variants across 30 different genes in 44/144 (30%) of these Saudi Epilepsy individuals. We also identified 232 variants of unknown significance (VUS) across 101 different genes in 133/144 (92%) subjects. Strong enrichment of variants of likely pathogenicity were observed in previously described epilepsy-associated loci, and a number of putative pathogenic variants in novel loci are also observed. Conclusion Several putative pathogenic variants known to be epilepsy-related loci were identified for the first time in our population, in addition to several potential new loci have been identified which may be prioritized for further investigation.

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