title: Whole genome amplification induced bias in the detection of KRAS-mutated cell populations during colorectal carcinoma tissue testing.
abstract: Whole genome amplification replicates the entire DNA content of a sample and can thus help to circumvent material limitations when insufficient DNA is available for planned genetic analyses. However, there are conflicting data in the literature whether whole genome amplification introduces bias or reflects precisely the spectrum of starting DNA. We analyzed the origins of discrepancies in KRAS (Kirsten rat sarcoma viral oncogene homolog gene) mutation detection in six of ten samples amplified using the GenomePlex® Tissue Whole Genome Amplification kit 5 (WGA5; Sigma-Aldrich, St. Louis, MO, USA) and KRAS StripAssay® (KRAS SA; ViennaLab Diagnostics, Vienna, Austria). We undertook reextraction, reamplification, retyping, authentication, reanalysis, and reinterpretation to determine whether the discrepancies originated during the preanalytical, analytical, and/or interpretative phase of genotyping. We conclude that a combination of glass slide/sample heterogeneity and biased amplification due to stochastic effects in the early phases of whole genome amplification (WGA) may have adversely affected the results obtained. Our findings are relevant for both forensic genetics testing and massively parallel sequencing using preamplification.
authors: Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University Olomouc, Olomouc, Czech Republic.
authors: Jana
authors: J
authors: Stranska
authors: Sylwia
authors: S
authors: Jancik
authors: Rastislav
authors: R
authors: Slavkovsky
authors: Veronika
authors: V
authors: Holinkova
authors: Miroslava
authors: M
authors: Rabcanova
authors: Petr
authors: P
authors: Vojta
authors: Marian
authors: M
authors: Hajduch
authors: Jiri
authors: J
authors: Drabek
mesh_terms: D015179:Colorectal Neoplasms / Q000235:genetics*; D011905:Genes, ras / Q000235:genetics*; D015894:Genome, Human / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D060005:Genotyping Techniques / Q000379:methods; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D017422:Sequence Analysis, DNA
keywords: Bias; Colon cancer; FFPE sample; KRAS mutation genotyping; Whole genome amplification