Article Figures & Data
Figures
- Figure1
Summary of technical validity and clinical utility assessment for cancer NGS. (A) NGS basecalling, wherein a DNA sequence and corresponding confidence score is generated from a nuclear genomic DNA template. (B) The next step, which compares all available data to the reference and each other. Variant calling is then performed (underlined bases in panel B), comparing base calls across many reads; many false positive variant calls (x'ed out bases) can be filtered, while true positives (circled bases) should generate a strong signal. (C) Multiple quality metrics are generated during variant calling, which can be compared to cutoffs established during assay validation (dashed lines). (D) Detailed review of available databases and literature (left side) and comparison to clinical history and tumor pathology (right side) to assess clinical utility. VAF, variant allele frequency; QUAL, variant call quality; COSMIC, Catalogue of Somatic Mutations in Cancer; TKIs, tyrosine kinase inhibitor therapies.
- Figure2
Alignment views of four variants detected by NGS. Each panel depicts a representative set of variant reads for single nucleotide variant (A and B) or insertion/deletion variants (C and D) with either high quality (A and C) or low quality (B and D). The human genome reference sequence is the string of bases along the top of each panel. Aligned basecalls matching the reference are listed as dots (plus strand) or commas (minus strand). High quality variants typically have higher variant allele frequencies (VAF), and variants reads have fewer additional variants. Panel D depicts known false positive locus, likely due to the "homopolymer problem" where certain NGS technologies overcall insertion/deletion variants where the reference sequence has five or more of the same nucleotides in a row (in this case, 8 guanines).
Tables
- Table1
Summary of valuable references and guidelines relevant to clinical NGS oncology testing
Source Title Content summary Reference New York State Board of Health "Next Generation" Sequencing (NGS) guidelines for somatic genetic variant detection Detailed standards for technical validity 11 ACMG Standards and guidelines for the interpretation of sequence Variants Guidelines for clinical validity assessment, particularly for germline/constitutional variants 12 ACMG ACMG clinical laboratory standards for next-generation sequencing Broad summaries of major areas of consideration for clinical validation of all NGS assay 13 CDC Assuring the quality of next-generation sequencing in clinical laboratory practice Detailed recommendations for technical validity assessment/validation of all NGS assays 14 CDC Good laboratory practice for clinical next-generation sequencing informatics pipelines Detailed recommendations for clinical validity assessment for all NGS assays 15 Quest Diagnostics (reference laboratory) Annotation of sequence variants in cancer samples processes and pitfalls for routine assays in the clinical laboratory The framework for a repeatable workflow for clinical validity assessment in use at a high volume testing facility is described 16 ACMG: American College of Medical Genetics and Genomics; CDC: United States Centers for Disease Control and Prevention
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