detection-of-rare-mutations-in-ctdna-using-next-generation-sequencing

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

Geneplus-Beijing Institute

An outbreak caused by Shiga-toxin–producing Escherichia coli O104:H4 occurred in Germany in May and June of 2011, with more than 3000 persons infected. Here, we report a cluster of cases associated with a single family and describe an open-source genomic analysis of an isolate from one member of the family. This analysis involved the use of rapid, bench-top DNA sequencing technology, open-source data release, and prompt crowd-sourced analyses. In less than a week, these studies revealed that the outbreak strain belonged to an enteroaggregative E. coli lineage that had acquired genes for Shiga toxin 2 and for antibiotic resistance.

Open-source genomic analysis of Shiga-toxin-producing E. coli O104:H4.

Restriction Enzyme-based Reduced Representation Library (RRL) method represents a relatively feasible and flexible strategy used for Single Nucleotide Polymorphism (SNP) identification in different species. It has remarkable advantage of reducing the complexity of the genome by orders of magnitude. However, comprehensive evaluation for actual efficacy of SNP identification by this method is still unavailable.

Comprehensive evaluation of SNP identification with the Restriction Enzyme-based Reduced Representation Library (RRL) method.

Accurate genotyping is important for genetic testing. Sanger sequencing based typing is the gold standard for genotyping, but it has been underused, due to its high cost and low throughput. In contrast, short-read sequencing provides inexpensive and high throughput sequencing, holding great promise for reaching the goal of cost-effective and high-throughput genotyping. However, the short read length and the paucity of appropriate genotyping methods, pose a major challenge. Here we present RCHSBT - Reliable, Cost-effective and High-throughput Sequence Based Typing pipeline - which takes short sequence reads as input, but uses a unique variant calling, haploid sequence assembling algorithm, can accurately genotype with greater effective length per amplicon than even Sanger sequencing reads. The RCHSBT method was tested for the human MHC loci HLA-A, HLA-B, HLA-C, HLA-DQB1 and HLA-DRB1, upon 96 samples using Illumina PE 150 reads. Amplicons as long as 950 bp were readily genotyped, achieving 100% typing concordance between RCHSBT-called genotypes and genotypes previously called by Sanger sequence. Genotyping throughput was increased over ten times, and cost was reduced over five times, for RCHSBT as compared with Sanger sequence genotyping. We thus demonstrate RCHSBT to be a genotyping method comparable to Sanger sequencing based typing in quality, while being more cost-effective, and higher throughput. This article is protected by copyright. All rights reserved.

A Short-Read Multiplex Sequencing Method for Reliable, Cost-Effective and High-Throughput Genotyping in Large-Scale Studies.

Next-generation sequencing (NGS) is commonly used in a clinical setting for diagnostic and prognostic testing of genetic mutations to select optimal targeted therapies. Herein, we describe the development of a custom NGS assay for detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) in a panel of 51 genes related to breast cancer. We designed and implemented a validation strategy in accordance with principles and guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing work group using artificial, cell-free DNA (cfDNA) with mutant fragments prepared in a simple, rapid, and cost-effective manner. For SNV detection, our test had 96.30% sensitivity at mutant allele frequency ≥0.5% with high specificity (99.9997%) and accuracy (99.9996%). For CNV detection, the approach had 95.83% sensitivity for copy numbers at 1.25× (25.6% extra copies) with high specificity (99.77%) and accuracy (99.76%). In addition, our NGS-based assay demonstrated high intrarun and interrun reproducibility, high consistency compared to digital PCR, and a low cross-contamination rate. An overall assessment using cfDNA and plasma cfDNA samples demonstrated our custom NGS assay yields a reliable and robust detection sensitivity with a mutant allele frequency as low as 0.5% for SNVs and copy number of 1.25× for CNVs.

Technical Validation of a Next-Generation Sequencing Assay for Detecting Clinically Relevant Levels of Breast Cancer-Related Single-Nucleotide Variants and Copy Number Variants Using Simulated Cell-Free DNA.

Meiru Zhao

Geneplus-Beijing Institute

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing