Detection of Genetic Mutations Using Minor Variant Finder Software. (21st September 2018)

• Record Type: Journal Article
• Authors: Kikuchi, Arizumi; Sawamura, Takahiro; Nonaka, Kenichi
• Is Part Of: American journal of clinical pathology. Volume 150(2018)Supplement 1
• Page Start: S132
• Page End: S132
• Publication Date: 2018-09-21
• Subjects: Diagnosis, Laboratory -- Periodicals; Pathology -- Periodicals; 616.07
• DOI: 10.1093/ajcp/aqy102.316
• Languages: English
• ISSNs: 0002-9173

Abstract

Abstract: Objectives: Capillary sequencer is frequently used to detect variants in the next-generation sequences, but detecting a somatic mutation from formalin-fixed, paraffin-embedded tissue (FFPET) is a challenging task. We evaluated the efficiency of the Minor Variant Finder software (Thermo) developed for the detection of variant in the capillary sequencing and validated the calculation function for "Variant Percent" in this software. Methods: We used three cell lines (MIA PaCa2; A549 with KRAS gene mutated at exon 2, codon 12; and K562) and samples with variant percentages of 2.5%, 5%, 10%, 25%, 50%, and 100% prepared using each genomic DNA. Furthermore, we analyzed the genomic DNA from FFPET from colon cancer patients (n = 44) in KRAS gene exon 2, codons 12 and 13. We performed the capillary electrophoresis in the 3130 genetic analyzer (Thermo) and compared the result with that obtained from the DNA sequencing analysis software (Thermo). Results: The 5% variant cell clearly exhibited a variant signal. We obtained the following variant levels (mean ± standard deviation) in MIA PaCa2 and A549 at each percentage: 2.5% (2.7 ± 0.1, 2.1 ± 0.1), 5% (6.0 ± 0.1, 5.0 ± 0.1), 10% (11.8 ± 0.1, 9.2 ± 0.1), 25% (29.8 ± 0.6, 23.6 ± 0.6), 50% (54.1 ± 0.6, 46.0 ± 0.5), and 100% (94.4 ± 0.8, 94.2 ± 1.4). In FFPET, the percentage of mutation was 31.4%. These results agree with the results obtained using DNA sequencing software for all the samples, and we could obtain clear signals. Conclusion: "Variant Percent" function could not quantitate exact variant percentage; however, it obtained an approximate distinction allowing objective determination rather than only visual. We propose that the Minor Variant Finder software is useful for detection of somatic mutations, especially the low-level mutations, exclusively using FFPET.