Validation and Implementation of a Custom Next-Generation Sequencing Clinical Assay for Hematologic Malignancies.

TitleValidation and Implementation of a Custom Next-Generation Sequencing Clinical Assay for Hematologic Malignancies.
Publication TypeJournal Article
Year of Publication2016
AuthorsKluk MJ, R Lindsley C, Aster JC, Lindeman NI, Szeto D, Hall D, Kuo FC
JournalJ Mol Diagn
Date Published2016 07
KeywordsAlleles, Chromosome Aberrations, DNA Copy Number Variations, Female, fms-Like Tyrosine Kinase 3, Gene Duplication, Gene Frequency, Genetic Predisposition to Disease, Genomics, Hematologic Neoplasms, High-Throughput Nucleotide Sequencing, Humans, Karyotype, Male, Reproducibility of Results, Sensitivity and Specificity, Tandem Repeat Sequences

Targeted next-generation sequencing panels to identify genetic alterations in cancers are increasingly becoming an integral part of clinical practice. We report here the design, validation, and implementation of a comprehensive 95-gene next-generation sequencing panel targeted for hematologic malignancies that we named rapid heme panel. Rapid heme panel is amplicon based and covers hotspot regions of oncogenes and most of the coding regions of tumor suppressor genes. It is composed of 1330 amplicons and covers 175 kb of genomic sequence in total. Rapid heme panel's average coverage is 1500× with <5% of the amplicons with <50× coverage, and it reproducibly detects single nucleotide variants and small insertions/deletions at allele frequencies of ≥5%. Comparison with a capture-based next-generation sequencing assay showed that there is >95% concordance among a wide array of variants across a range of allele frequencies. Read count analyses that used rapid heme panel showed high concordance with karyotypic results when tumor content was >30%. The average turnaround time was 7 days over a 6-month span with an average volume of ≥40 specimens per week and a low sample fail rate (<1%), demonstrating its suitability for clinical application.

Alternate JournalJ Mol Diagn
PubMed ID27339098
PubMed Central IDPMC5707186
Grant ListK08 CA204734 / CA / NCI NIH HHS / United States
P01 CA066996 / CA / NCI NIH HHS / United States