title: Targeted, high-depth, next-generation sequencing of cancer genes in formalin-fixed, paraffin-embedded and fine-needle aspiration tumor specimens.
abstract: Implementation of highly sophisticated technologies, such as next-generation sequencing (NGS), into routine clinical practice requires compatibility with common tumor biopsy types, such as formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration specimens, and validation metrics for platforms, controls, and data analysis pipelines. In this study, a two-step PCR enrichment workflow was used to assess 540 known cancer-relevant variants in 16 oncogenes for high-depth sequencing in tumor samples on either mature (Illumina GAIIx) or emerging (Ion Torrent PGM) NGS platforms. The results revealed that the background noise of variant detection was elevated approximately twofold in FFPE compared with cell line DNA. Bioinformatic algorithms were optimized to accommodate this background. Variant calls from 38 residual clinical colorectal cancer FFPE specimens and 10 thyroid fine-needle aspiration specimens were compared across multiple cancer genes, resulting in an accuracy of 96.1% (95% CI, 96.1% to 99.3%) compared with Sanger sequencing, and 99.6% (95% CI, 97.9% to 99.9%) compared with an alternative method with an analytical sensitivity of 1% mutation detection. A total of 45 of 48 samples were concordant between NGS platforms across all matched regions, with the three discordant calls each represented at <10% of reads. Consequently, NGS of targeted oncogenes in real-life tumor specimens using distinct platforms addresses unmet needs for unbiased and highly sensitive mutation detection and can accelerate both basic and clinical cancer research.
authors: Asuragen, Inc., Austin, Texas, USA.
authors: Andrew G
authors: AG
authors: Hadd
authors: Jeff
authors: J
authors: Houghton
authors: Ashish
authors: A
authors: Choudhary
authors: Sachin
authors: S
authors: Sah
authors: Liangjing
authors: L
authors: Chen
authors: Adam C
authors: AC
authors: Marko
authors: Tiffany
authors: T
authors: Sanford
authors: Kalyan
authors: K
authors: Buddavarapu
authors: Julie
authors: J
authors: Krosting
authors: Lana
authors: L
authors: Garmire
authors: Dennis
authors: D
authors: Wylie
authors: Rupali
authors: R
authors: Shinde
authors: Sylvie
authors: S
authors: Beaudenon
authors: Erik K
authors: EK
authors: Alexander
authors: Elizabeth
authors: E
authors: Mambo
authors: Alex T
authors: AT
authors: Adai
authors: Gary J
authors: GJ
authors: Latham
mesh_terms: D044963:Biopsy, Fine-Needle; D045744:Cell Line, Tumor; D052138:Genes, Neoplasm; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000235:genetics* / Q000473:pathology*; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity