scholarly journals Use of an integrated pan-cancer oncology enrichment NGS assay to measure tumour mutational burden and detect clinically actionable variants

2020 ◽  
Author(s):  
Valerie Pestinger ◽  
Matthew Smith ◽  
Toju Sillo ◽  
John M Findlay ◽  
Jean-Francois Laes ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Tier 2 ◽  
Single Nucleotide Variants ◽  
Genomic Assays ◽  
Mutational Burden ◽  
Clinical Annotation ◽  
And Control ◽  
Actionable Variants

AbstractIntroductionThe identification of tumour mutational burden (TMB) as a biomarker of response to PD-1 immunotherapy has necessitated the development of genomic assays to measure this. We carried out comprehensive molecular profiling of cancers using the Illumina TruSight Oncology panel (TSO500) and compared to whole genome sequencing.MethodsCancer samples derived from formalin fixed material were profiled on the TSO500 panel, sequenced on an Illumina NextSeq 500 instrument and processed through the TSO500 Docker Pipeline. Either FASTQ files (PierianDx) or VCF files (OncoKDM) were processed to understand clinical actionabilityResultsIn total, 108 samples (a mixture of colorectal, lung, oesophageal and control samples) were processed via the DNA panel. There was good correlation between TMB, SNV, indels and CNV as predicted by TSO500 and WGS (R2>0.9) and good reproducibility, with less than 5% variability between repeated controls. For the RNA panel, 13 samples were processed, with all known fusions observed via orthogonal techniques detected. For clinical actionability 72 Tier 1 variants and 297 Tier 2 variants were identified with clinical trials identified for all patients.ConclusionsThe TruSight Oncology 500 assay accurately measures TMB, MSI, single nucleotide variants, indels, copy number/structural variation and gene fusions when compared to whole genome sequencing and orthogonal technologies. Coupled with a clinical annotation pipeline this provides a powerful methodology for identification of clinically actionable variants.

scholarly journals Unexpected CRISPR off-target mutation pattern in vivo are not typically germline-like

2017 ◽  
Author(s):  
Zhiting Wei ◽  
Funan He ◽  
Guohui Chuai ◽  
Hanhui Ma ◽  
Zhixi Su ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Cas9 Nuclease ◽  
Mutation Pattern ◽  
And Control ◽  
Selection Of

To the EditorSchaefer et al.1 (referred to as Study_1) recently presented the provocative conclusion that CRISPR-Cas9 nuclease can induce many unexpected off-target mutations across the genome that arise from the sites with poor homology to the gRNA. As Wilson et al.2 pointed out, however, the selection of a co-housed mouse as the control is insufficient to attribute the observed mutation differences between the CRISPR-treated mice and control mice. Therefore, the causes of these mutations need to be further investigated. In 2015, Iyer et al.3 (referred to as Study_2) used Cas9 and a pair of sgRNAs to mutate the Ar gene in vivo and off-target mutations were investigated by comparison the control mice and the offspring of the modified mice. After analyzing the whole genome sequencing (WGS) of the offspring and the control mice, they claimed that off-target mutations are rare from CRISPR-Cas9 engineering. Notably, their study only focused on indel off-target mutations. We re-analyzed the WGS data of these two studies and detected both single nucleotide variants (SNVs) and indel mutations.

scholarly journals Sarek: A portable workflow for whole-genome sequencing analysis of germline and somatic variants

2018 ◽  
Author(s):  
Maxime Garcia ◽  
Szilveszter Juhos ◽  
Malin Larsson ◽  
Pall I. Olason ◽  
Marcel Martin ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Open Source ◽  
Genome Sequencing ◽  
Development Project ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Insertion And Deletion ◽  
Sample Heterogeneity

AbstractSummaryWhole-genome sequencing (WGS) is a cornerstone of precision medicine, but portable and reproducible open-source workflows for WGS analyses of germline and somatic variants are lacking. We present Sarek, a modular, comprehensive, and easy-to-install workflow, combining a range of software for the identification and annotation of single-nucleotide variants (SNVs), insertion and deletion variants (indels), structural variants, tumor sample heterogeneity, and karyotyping from germline or paired tumor/normal samples. Sarek is implemented in a bioinformatics workflow language (Nextflow) with Docker and Singularity compatible containers, ensuring easy deployment and full reproducibility at any Linux based compute cluster or cloud computing environment. Sarek supports the human reference genomes GRCh37 and GRCh38, and can readily be used both as a core production workflow at sequencing facilities and as a powerful stand-alone tool for individual research groups.AvailabilitySource code and instructions for local installation are available at GitHub (https://github.com/SciLifeLab/Sarek) under the MIT open-source license, and we invite the research community to contribute additional functionality as a collaborative open-source development project.

scholarly journals Contributions of de novo variants to systemic lupus erythematosus

2020 ◽  
Vol 29 (1) ◽  
pp. 184-193 ◽  
Author(s):  
Jonas Carlsson Almlöf ◽  
Sara Nystedt ◽  
Aikaterini Mechtidou ◽  
Dag Leonard ◽  
Maija-Leena Eloranta ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Lupus Erythematosus ◽  
De Novo ◽  
Whole Genome ◽  
Gene Promoters ◽  
Single Nucleotide Variants ◽  
Systemic Lupus ◽  
Promoter Regions

AbstractBy performing whole-genome sequencing in a Swedish cohort of 71 parent-offspring trios, in which the child in each family is affected by systemic lupus erythematosus (SLE, OMIM 152700), we investigated the contribution of de novo variants to risk of SLE. We found de novo single nucleotide variants (SNVs) to be significantly enriched in gene promoters in SLE patients compared with healthy controls at a level corresponding to 26 de novo promoter SNVs more in each patient than expected. We identified 12 de novo SNVs in promoter regions of genes that have been previously implicated in SLE, or that have functions that could be of relevance to SLE. Furthermore, we detected three missense de novo SNVs, five de novo insertion-deletions, and three de novo structural variants with potential to affect the expression of genes that are relevant for SLE. Based on enrichment analysis, disease-affecting de novo SNVs are expected to occur in one-third of SLE patients. This study shows that de novo variants in promoters commonly contribute to the genetic risk of SLE. The fact that de novo SNVs in SLE were enriched to promoter regions highlights the importance of using whole-genome sequencing for identification of de novo variants.

Whole genome sequencing: A new paradigm in the surveillance and control of human tuberculosis

Tuberculosis ◽  
2015 ◽  
Vol 95 (2) ◽  
pp. 91-94 ◽  
Author(s):  
Seyed E. Hasnain ◽  
Ronan F. O'Toole ◽  
Sonam Grover ◽  
Nasreen Z. Ehtesham
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
New Paradigm ◽  
And Control

scholarly journals The Genetic Diversification of a Single Bluetongue Virus Strain Using an In Vitro Model of Alternating-Host Transmission

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 1038 ◽  
Author(s):  
Jennifer H. Kopanke ◽  
Justin S. Lee ◽  
Mark D. Stenglein ◽  
Christie E. Mayo
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genetic Stability ◽  
Bluetongue Virus ◽  
Genomic Structure ◽  
Field Isolate ◽  
Viral Population ◽  
Whole Genome ◽  
Single Nucleotide Variants

Bluetongue virus (BTV) is an arbovirus that has been associated with dramatic epizootics in both wild and domestic ruminants in recent decades. As a segmented, double-stranded RNA virus, BTV can evolve via several mechanisms due to its genomic structure. However, the effect of BTV’s alternating-host transmission cycle on the virus’s genetic diversification remains poorly understood. Whole genome sequencing approaches offer a platform for investigating the effect of host-alternation across all ten segments of BTV’s genome. To understand the role of alternating hosts in BTV’s genetic diversification, a field isolate was passaged under three different conditions: (i) serial passages in Culicoides sonorensis cells, (ii) serial passages in bovine pulmonary artery endothelial cells, or (iii) alternating passages between insect and bovine cells. Aliquots of virus were sequenced, and single nucleotide variants were identified. Measures of viral population genetics were used to quantify the genetic diversification that occurred. Two consensus variants in segments 5 and 10 occurred in virus from all three conditions. While variants arose across all passages, measures of genetic diversity remained largely similar across cell culture conditions. Despite passage in a relaxed in vitro system, we found that this BTV isolate exhibited genetic stability across passages and conditions. Our findings underscore the valuable role that whole genome sequencing may play in improving understanding of viral evolution and highlight the genetic stability of BTV.

scholarly journals Identification of Pathogenic Structural Variants in Rare Disease Patients through Genome Sequencing

2019 ◽  
Author(s):  
James M. Holt ◽  
Camille L. Birch ◽  
Donna M. Brown ◽  
Manavalan Gajapathy ◽  
Nadiya Sosonkina ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Rare Disease ◽  
Genome Sequencing ◽  
Genomic Analysis ◽  
Whole Genome ◽  
Structural Variants ◽  
Single Nucleotide Variants ◽  
Standard Clinical Practice ◽  
Wide Range ◽  
Genetic Features

AbstractPurposeClinical whole genome sequencing is becoming more common for determining the molecular diagnosis of rare disease. However, standard clinical practice often focuses on small variants such as single nucleotide variants and small insertions/deletions. This leaves a wide range of larger “structural variants” that are not commonly analyzed in patients.MethodsWe developed a pipeline for processing structural variants for patients who received whole genome sequencing through the Undiagnosed Diseases Network (UDN). This pipeline called structural variants, stored them in an internal database, and filtered the variants based on internal frequencies and external annotations. The remaining variants were manually inspected and then interesting findings were reported as research variants to clinical sites in the UDN.ResultsOf 477 analyzed UDN cases, 286 cases (≈ 60%) received at least one structural variant as a research finding. The variants in 16 cases (≈ 4%) are considered “Certain” or “Highly likely” molecularly diagnosed and another 4 cases are currently in review. Of those 20 cases, at least 13 were identified originally through our pipeline with one finding leading to identification of a new disease. As part of this paper, we have also released the collection of variant calls identified in our cohort along with heterozygous and homozygous call counts. This data is available at https://github.com/HudsonAlpha/UDN_SV_export.ConclusionStructural variants are key genetic features that should be analyzed during routine clinical genomic analysis. For our UDN patients, structural variants helped solve ≈ 4% of the total number of cases (≈ 13% of all genome sequencing solves), a success rate we expect to improve with better tools and greater understanding of the human genome.

scholarly journals Assessing reproducibility of inherited variants detected with short-read whole genome sequencing

2022 ◽  
Vol 23 (1) ◽  
Author(s):  
Bohu Pan ◽  
Luyao Ren ◽  
Vitor Onuchic ◽  
Meijian Guan ◽  
Rebecca Kusko ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Precision Medicine ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Single Nucleotide Variants ◽  
Short Read ◽  
Sequencing Platforms ◽  
The Impact ◽  
Two Populations

Abstract Background Reproducible detection of inherited variants with whole genome sequencing (WGS) is vital for the implementation of precision medicine and is a complicated process in which each step affects variant call quality. Systematically assessing reproducibility of inherited variants with WGS and impact of each step in the process is needed for understanding and improving quality of inherited variants from WGS. Results To dissect the impact of factors involved in detection of inherited variants with WGS, we sequence triplicates of eight DNA samples representing two populations on three short-read sequencing platforms using three library kits in six labs and call variants with 56 combinations of aligners and callers. We find that bioinformatics pipelines (callers and aligners) have a larger impact on variant reproducibility than WGS platform or library preparation. Single-nucleotide variants (SNVs), particularly outside difficult-to-map regions, are more reproducible than small insertions and deletions (indels), which are least reproducible when > 5 bp. Increasing sequencing coverage improves indel reproducibility but has limited impact on SNVs above 30×. Conclusions Our findings highlight sources of variability in variant detection and the need for improvement of bioinformatics pipelines in the era of precision medicine with WGS.

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