Dataset Information

Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection.

ABSTRACT: INTRODUCTION:Comprehensive genetic cancer profiling using circulating tumor DNA has enabled the detection of National Comprehensive Cancer Network (NCCN) guideline-recommended somatic alterations from a single, non-invasive blood draw. However, reliably detecting somatic variants at low variant allele fractions (VAFs) remains a challenge for next-generation sequencing (NGS)-based tests. We have developed the single-molecule sequencing (SMSEQ) platform to address these challenges. METHODS:The OncoLBx assay utilizes the SMSEQ platform to optimize cell-free DNA extraction and library preparation with variant type-specific calling algorithms to improve sensitivity and specificity. OncoLBx is a pan-cancer panel for solid tumors targeting 75 genes and five microsatellite sites analyzing five classes of NCCN-recommended somatic variants: single-nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), fusions and microsatellite instability (MSI). Circulating DNA was extracted from plasma, followed by library preparation using SMSEQ. Analytical validation was performed according to recently published American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines and established the limit of detection (LOD), sensitivity, specificity, accuracy and reproducibility using 126 gold-standard reference samples, healthy donor samples verified by whole-exome sequencing by an external College of American Pathologists (CAP) reference lab and cell lines with known variants. Results were analyzed using a locus-specific modeling algorithm. RESULTS:We have demonstrated that OncoLBx detects VAFs of???0.1% for SNVs and indels,???0.5% for fusions,???4.5 copies for CNVs and???2% for MSI, with all variant types having specificity???99.999%. Diagnostic performance of paired samples displays 80% sensitivity and?>?99.999% clinical specificity. Clinical utility and performance were assessed in 416 solid tumor samples. Variants were detected in 79% of samples, for which 87.34% of positive samples had available targeted therapy.

SUBMITTER: Atkins A

PROVIDER: S-EPMC6675782 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection.

Atkins Alexander A Gupta Pratyush P Zhang Bing Melody BM Tsai Wen-Sy WS Lucas Julian J Javey Manana M Vora Anagh A Mei Rui R

Molecular diagnosis & therapy 20190801 4

<h4>Introduction</h4>Comprehensive genetic cancer profiling using circulating tumor DNA has enabled the detection of National Comprehensive Cancer Network (NCCN) guideline-recommended somatic alterations from a single, non-invasive blood draw. However, reliably detecting somatic variants at low variant allele fractions (VAFs) remains a challenge for next-generation sequencing (NGS)-based tests. We have developed the single-molecule sequencing (SMSEQ) platform to address these challenges.<h4>Meth ...[more]

PMID: 31209714

Similar Datasets

Project description:BackgroundOne of the most significant issues surrounding next generation sequencing is the cost and the difficulty assembling short read lengths. Targeted capture enrichment of longer fragments using single molecule sequencing (SMS) is expected to improve both sequence assembly and base-call accuracy but, at present, there are very few examples of successful application of these technologic advances in translational research and clinical testing. We developed a targeted single molecule sequencing (T-SMS) panel for genes implicated in ovarian response to controlled ovarian hyperstimulation (COH) for infertility.ResultsTarget enrichment was carried out using droplet-base multiplex polymerase chain reaction (PCR) technology (RainDance®) designed to yield amplicons averaging 1 kb fragment size from candidate 44 loci (99.8% unique base-pair coverage). The total targeted sequence was 3.18 Mb per sample. SMS was carried out using single molecule, real-time DNA sequencing (SMRT® Pacific Biosciences®), average raw read length = 1178 nucleotides, 5% of the amplicons >6000 nucleotides). After filtering with circular consensus (CCS) reads, the mean read length was 3200 nucleotides (97% CCS accuracy). Primary data analyses, alignment and filtering utilized the Pacific Biosciences® SMRT portal. Secondary analysis was conducted using the Genome Analysis Toolkit for SNP discovery l and wANNOVAR for functional analysis of variants. Filtered functional variants 18 of 19 (94.7%) were further confirmed using conventional Sanger sequencing. CCS reads were able to accurately detect zygosity. Coverage within GC rich regions (i.e.VEGFR; 72% GC rich) was achieved by capturing long genomic DNA (gDNA) fragments and reading into regions that flank the capture regions. As proof of concept, a non-synonymous LHCGR variant captured in two severe OHSS cases, and verified by conventional sequencing.ConclusionsCombining emulsion PCR-generated 1 kb amplicons and SMRT DNA sequencing permitted greater depth of coverage for T-SMS and facilitated easier sequence assembly. To the best of our knowledge, this is the first report combining emulsion PCR and T-SMS for long reads using human DNA samples, and NGS panel designed for biomarker discovery in OHSS.

Dataset Information

Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection.

Publications

Detection of Circulating Tumor DNA with a Single-Molecule Sequencing Analysis Validated for Targeted and Immunotherapy Selection.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets