Variant discovery using next-generation sequencing and its future role in pharmacogenetics

2020 ◽  
Vol 21 (7) ◽  
pp. 471-486
Author(s):  
Laura E Russell ◽  
Ute I Schwarz
Keyword(s):  
Next Generation Sequencing ◽  
Protein Function ◽  
Large Scale ◽  
Clinical Care ◽  
Next Generation ◽  
Clinical Implementation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Variant Discovery ◽  
Generation Sequencing

Next-generation sequencing (NGS) has enabled the discovery of a multitude of novel and mostly rare variants in pharmacogenes that may alter a patient’s therapeutic response to drugs. In addition to single nucleotide variants, structural variation affecting the number of copies of whole genes or parts of genes can be detected. While current guidelines concerning clinical implementation mostly act upon well-documented, common single nucleotide variants to guide dosing or drug selection, in silico and large-scale functional assessment of rare variant effects on protein function are at the forefront of pharmacogenetic research to facilitate their clinical integration. Here, we discuss the role of NGS in variant discovery, paving the way for more comprehensive genotype-guided pharmacotherapy that can translate to improved clinical care.

scholarly journals A Model Study of In Silico Proficiency Testing for Clinical Next-Generation Sequencing

2016 ◽  
Vol 140 (10) ◽  
pp. 1085-1091 ◽  
Author(s):  
Eric J. Duncavage ◽  
Haley J. Abel ◽  
Jason D. Merker ◽  
John B. Bodner ◽  
Qin Zhao ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Proficiency Testing ◽  
In Silico ◽  
Absolute Difference ◽  
Ion Torrent ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Clinical Laboratories ◽  
Generation Sequencing

Context.—Most current proficiency testing challenges for next-generation sequencing assays are methods-based proficiency testing surveys that use DNA from characterized reference samples to test both the wet-bench and bioinformatics/dry-bench aspects of the tests. Methods-based proficiency testing surveys are limited by the number and types of mutations that either are naturally present or can be introduced into a single DNA sample. Objective.—To address these limitations by exploring a model of in silico proficiency testing in which sequence data from a single well-characterized specimen are manipulated electronically. Design.—DNA from the College of American Pathologists reference genome was enriched using the Illumina TruSeq and Life Technologies AmpliSeq panels and sequenced on the MiSeq and Ion Torrent platforms, respectively. The resulting data were mutagenized in silico and 26 variants, including single-nucleotide variants, deletions, and dinucleotide substitutions, were added at variant allele fractions (VAFs) from 10% to 50%. Participating clinical laboratories downloaded these files and analyzed them using their clinical bioinformatics pipelines. Results.—Laboratories using the AmpliSeq/Ion Torrent and/or the TruSeq/MiSeq participated in the 2 surveys. On average, laboratories identified 24.6 of 26 variants (95%) overall and 21.4 of 22 variants (97%) with VAFs greater than 15%. No false-positive calls were reported. The most frequently missed variants were single-nucleotide variants with VAFs less than 15%. Across both challenges, reported VAF concordance was excellent, with less than 1% median absolute difference between the simulated VAF and mean reported VAF. Conclusions.—The results indicate that in silico proficiency testing is a feasible approach for methods-based proficiency testing, and demonstrate that the sensitivity and specificity of current next-generation sequencing bioinformatics across clinical laboratories are high.

scholarly journals Prostate cancer heterogeneity assessment with multi-regional sampling and alignment-free methods

2020 ◽  
Vol 2 (3) ◽  
Author(s):  
Ross G Murphy ◽  
Aideen C Roddy ◽  
Shambhavi Srivastava ◽  
Esther Baena ◽  
David J Waugh ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Cancer Heterogeneity ◽  
Alignment Free ◽  
Treatment Indications ◽  
Patient Heterogeneity ◽  
Genomic Locations ◽  
Generation Sequencing

Abstract Combining alignment-free methods for phylogenetic analysis with multi-regional sampling using next-generation sequencing can provide an assessment of intra-patient tumour heterogeneity. From multi-regional sampling divergent branching, we validated two different lesions within a patient’s prostate. Where multi-regional sampling has not been used, a single sample from one of these areas could misguide as to which drugs or therapies would best benefit this patient, due to the fact these tumours appear to be genetically different. This application has the power to render, in a fraction of the time used by other approaches, intra-patient heterogeneity and decipher aberrant biomarkers. Another alignment-free method for calling single-nucleotide variants from raw next-generation sequencing samples has determined possible variants and genomic locations that may be able to characterize the differences between the two main branching patterns. Alignment-free approaches have been applied to relevant clinical multi-regional samples and may be considered as a valuable option for comparing and determining heterogeneity to help deliver personalized medicine through more robust efforts in identifying targetable pathways and therapeutic strategies. Our study highlights the application these tools could have on patient-aligned treatment indications.

scholarly journals Systematic Evaluation of Sanger Validation of Next-Generation Sequencing Variants

2016 ◽  
Vol 62 (4) ◽  
pp. 647-654 ◽  
Author(s):  
Tyler F Beck ◽  
James C Mullikin ◽  
Leslie G Biesecker ◽  
Keyword(s):  
Next Generation Sequencing ◽  
Sanger Sequencing ◽  
Large Scale ◽  
Best Practice ◽  
Clinical Care ◽  
Systematic Evaluation ◽  
Next Generation ◽  
Current Standard ◽  
Ngs Data ◽  
Generation Sequencing

Abstract BACKGROUND Next-generation sequencing (NGS) data are used for both clinical care and clinical research. DNA sequence variants identified using NGS are often returned to patients/participants as part of clinical or research protocols. The current standard of care is to validate NGS variants using Sanger sequencing, which is costly and time-consuming. METHODS We performed a large-scale, systematic evaluation of Sanger-based validation of NGS variants using data from the ClinSeq® project. We first used NGS data from 19 genes in 5 participants, comparing them to high-throughput Sanger sequencing results on the same samples, and found no discrepancies among 234 NGS variants. We then compared NGS variants in 5 genes from 684 participants against data from Sanger sequencing. RESULTS Of over 5800 NGS-derived variants, 19 were not validated by Sanger data. Using newly designed sequencing primers, Sanger sequencing confirmed 17 of the NGS variants, and the remaining 2 variants had low quality scores from exome sequencing. Overall, we measured a validation rate of 99.965% for NGS variants using Sanger sequencing, which was higher than many existing medical tests that do not necessitate orthogonal validation. CONCLUSIONS A single round of Sanger sequencing is more likely to incorrectly refute a true-positive variant from NGS than to correctly identify a false-positive variant from NGS. Validation of NGS-derived variants using Sanger sequencing has limited utility, and best practice standards should not include routine orthogonal Sanger validation of NGS variants.

scholarly journals SNVMix: predicting single nucleotide variants from next-generation sequencing of tumors

2010 ◽  
Vol 26 (6) ◽  
pp. 730-736 ◽  
Author(s):  
Rodrigo Goya ◽  
Mark G.F. Sun ◽  
Ryan D. Morin ◽  
Gillian Leung ◽  
Gavin Ha ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Generation Sequencing

scholarly journals SNVHMM: predicting single nucleotide variants from next generation sequencing

2013 ◽  
Vol 14 (1) ◽  
pp. 225 ◽  
Author(s):  
Jiawen Bian ◽  
Chenglin Liu ◽  
Hongyan Wang ◽  
Jing Xing ◽  
Priyanka Kachroo ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Generation Sequencing

scholarly journals From next-generation sequencing alignments to accurate comparison and validation of single-nucleotide variants: the pibase software

2012 ◽  
Vol 41 (1) ◽  
pp. e16-e16 ◽  
Author(s):  
Michael Forster ◽  
Peter Forster ◽  
Abdou Elsharawy ◽  
Georg Hemmrich ◽  
Benjamin Kreck ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Next Generation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Accurate Comparison ◽  
Generation Sequencing

scholarly journals Validation of OncoPanel: A Targeted Next-Generation Sequencing Assay for the Detection of Somatic Variants in Cancer

2017 ◽  
Vol 141 (6) ◽  
pp. 751-758 ◽  
Author(s):  
Elizabeth P. Garcia ◽  
Alissa Minkovsky ◽  
Yonghui Jia ◽  
Matthew D. Ducar ◽  
Priyanka Shivdasani ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Copy Number ◽  
Cancer Biology ◽  
Next Generation ◽  
Analytical Validation ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Insertions And Deletions ◽  
Targeted Next Generation Sequencing ◽  
Generation Sequencing

Context.— The analysis of somatic mutations across multiple genes in cancer specimens may be used to aid clinical decision making. The analytical validation of targeted next-generation sequencing panels is important to assess accuracy and limitations. Objective.— To report the development and validation of OncoPanel, a custom targeted next-generation sequencing assay for cancer. Design.— OncoPanel was designed for the detection of single-nucleotide variants, insertions and deletions, copy number alterations, and structural variants across 282 genes with evidence as drivers of cancer biology. We implemented a validation strategy using formalin-fixed, paraffin-embedded, fresh or frozen samples compared with results obtained by clinically validated orthogonal technologies. Results.— OncoPanel achieved 98% sensitivity and 100% specificity for the detection of single-nucleotide variants, and 84% sensitivity and 100% specificity for the detection of insertions and deletions compared with single-gene assays and mass spectrometry–based genotyping. Copy number detection achieved 86% sensitivity and 98% specificity compared with array comparative genomic hybridization. The sensitivity of structural variant detection was 74% compared with karyotype, fluorescence in situ hybridization, and polymerase chain reaction. Sensitivity was affected by inconsistency in the detection of FLT3 and NPM1 alterations and IGH rearrangements due to design limitations. Limit of detection studies demonstrated 98.4% concordance across triplicate runs for variants with allele fraction greater than 0.1 and at least 50× coverage. Conclusions.— The analytical validation of OncoPanel demonstrates the ability of targeted next-generation sequencing to detect multiple types of genetic alterations across a panel of genes implicated in cancer biology.

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