Clinical Validation of KRAS, BRAF, and EGFR Mutation Detection Using Next-Generation Sequencing

Abstract

Objectives To validate next-generation sequencing (NGS) technology for clinical diagnosis and to determine appropriate read depth. Methods We validated the KRAS, BRAF, and EGFR genes within the Ion AmpliSeq Cancer Hotspot Panel using the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA). Results We developed a statistical model to determine the read depth needed for a given percent tumor cellularity and number of functional genomes. Bottlenecking can result from too few input genomes. By using 16 formalin-fixed, paraffin-embedded (FFPE) cancer-free specimens and 118 cancer specimens with known mutation status, we validated the six traditional analytic performance characteristics recommended by the Next-Generation Sequencing: Standardization of Clinical Testing Working Group. Baseline noise is consistent with spontaneous and FFPE-induced C:G→T:A deamination mutations. Conclusions Redundant bioinformatic pipelines are essential, since a single analysis pipeline gave false-negative and false-positive results. NGS is sufficiently robust for the clinical detection of gene mutations, with attention to potential artifacts.

Ion Torrent next-generation sequencing for routine identification of clinically relevant mutations in colorectal cancer patients

“…Ion Torrent NGS assays have been retrospectively evaluated on previously characterised positive and negative archival control samples 10 13 14 20. However, in our study, routine clinical samples have been prospectively received by our central laboratory from several local pathology laboratories.…”

Sequence Artifacts in DNA from Formalin-Fixed Tissues: Causes and Strategies for Minimization

“…Sequence artifacts are often present above the intrinsic background level of sequencing variation of MPS, which is operationally considered to be 1% (23 ). It can be difficult to distinguish artifacts from true low-level mutations which are present as the result of low tumor purity or tumor heterogeneity.…”

“…Sequence artifacts can arise from various sources, including damaged templates preexisting in FFPE DNA (3,4 ), oxidative DNA damage during sample preparation (16 ), DNA polymerase error (17 ), pseudogene amplification (18 ), adaptor sequences and adaptor chimeras (19 ), sequencing chemistry (20 ), sequence alignment (21 ), and spontaneous deamination of nucleotides during thermocycling (22,23 ). Understanding these issues is important for accurate detection of actionable mutations and thus for implementation of precision medicine into the clinic.…”

Clinical Validation and Implementation of a Targeted Next-Generation Sequencing Assay to Detect Somatic Variants in Non-Small Cell Lung, Melanoma, and Gastrointestinal Malignancies

“…23 Using this estimate as a benchmark, we required that variant bases be sequenced at !500Â or !1000Â coverage to detect variant frequencies of !10.0% or !5.0%, respectively (50 variant calls in !500 or !1000 total base reads, respectively). We only sequenced tumor DNA composed of at least 10% neoplastic nuclei (light microscopy approximation, see Materials and Methods), mandating a minimum variant fraction detection threshold of approximately 5% for diploid heterozygous mutations.…”