EGER Artifactual Mutations Associated with two DNA Sequencers

Abstract

BioTechniquesVol. 42, No. 1 BioFeedbackOpen AccessEGER artifactual mutations associated with two DNA sequencersJing Jie Yu & Daniel C. FlynnJing Jie Yu*Jing Jie Yu, M.D. Daniel C. Flynn, Ph.D. Mary Babb Randolph Cancer Center West Virginia University Morgantown, WV, USA e-mail: E-mail Address: jyu@hsc.wvu.eduMary Babb Randolph Cancer Center West Virginia University, Morgantown, WV, USASearch for more papers by this author & Daniel C. FlynnMary Babb Randolph Cancer Center West Virginia University, Morgantown, WV, USASearch for more papers by this authorPublished Online:16 May 2018https://doi.org/10.2144/000112381AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail In a recent Letter to the Editor of the New England Journal of Medicine titled Assessing EGFR Mutations (February 2, 2006), it was concluded that artifactual mutations in the epidermal growth factor receptor (EGFR), such as C→G/G→A or A→G/T→C transitions, could occur in the analysis of DNA samples extracted from paraffin-embedded sections or laser-microdissected specimens (1). This observation raised an important question concerning whether all EGER mutations reported are authentic, or could there be other artifacts beside those caused by low copy number of template molecules or small amount DNA template of ancient specimens?Recently, we discovered a type of artifactual mutation associated with the use of our CEQ™ Genetic Analysis system (Beckman Coulter, Fullerton, MA, USA). In an investigation of EGFR mutations in a study of erlotinib (Tarceva®) chemoresponse in non-small cell lung (NSCL) cancer patients, we screened patient samples (snap-frozen tissues) for mutation in the EGFR tyrosine kinase domain (exons 18 through 21). Two point mutations within exon 18 were detected in 13 out of 14 samples for a G to A change, and 9 out of 14 for a G to A change, and 9 out of 14 for a G to T alteration. One was a mutation of a G to an A within a cluster of As. To verify these results, we repeated the experiment using an ABI PRISM® DNA sequencer (Applied Biosystems, Foster City, CA, USA) and obtained wild-type Gs for both sites, which agreed with the consensus sequence of EGFR exon 18 (Figure 1, A and B, left and middle panels).The ABI sequencer is not free of problems, however. We have encountered artifacts associated with both types of DNA sequencing systems used in our lab—the CEQ Genetic Analysis system and the ABI Prism DNA sequencer (Figure 1, A and B, right panels). Unfortunately, it is not easy to detect artifacts produced by the systems. Furthermore, this type of artifact cannot be prevented by increasing numbers of PCR, although optimal purification of samples might help somewhat. We recommend that mutations found in the EGFR and perhaps other genes should be verified by two different systems.Figure 1. DNA sequencing results generated by the CEQ Genetic Analysis system and the ABI Prism DNA sequencer from the same non-small cell lung (NSCL) cancer patient sample.The specimen was snap-frozen at −80°C until DNA extraction. Genomic DNA was isolated using the AllPrep DNA/RNA Mini kit (Qiagen, Valencia, CA, USA) and amplified using PCR conditions reported by Lynch et al. (2). The PCR fragments were purified with the QIAquick® Gel Extraction kit (Qiagen) and sequenced (A) using the CEQ 8000 Genetic Analysis system with the GenomeLab™ Dye Terminator Cycle Sequencing (DTCS) Quick Start kit (Beckman Coulter) and (B) using the ABI Prism 377 DNA Sequencer with the BigDye® Terminator Cycle Sequencing kit (Applied Biosystems). Three artifactual mutations were reported by the two DNA sequencing systems. WT, wild-type.More and more clinicians seek guidance in treatment of NSCL cancer and other cancers using EGFR mutations as a guide. They rely on medical genetics researchers and commercial gene sequencing firms to provide vital mutation information. The ABI sequencer and the CEQ system are the two leading DNA sequencing instruments. If we are to provide clinical guidance, we must have highly reliable DNA sequencing results, which may require verification by two different systems that sequence DNA.