False positives in multiplex PCR-based next-generation sequencing have unique signatures.

Journal Article (Journal Article)

Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancers. Recently, we implemented the multiplex PCR-based Ion AmpliSeq Cancer Hotspot Panel (>200 amplicons in 50 genes) to evaluate EGFR, KRAS, and BRAF in lung and colorectal adenocarcinomas. In 10% of samples, automated analysis identified a novel G873R substitution mutation in EGFR. By examining reads individually, we found this mutation in >5% of reads in 50 of 291 samples and also found similar events in 18 additional amplicons. These apparent mutations are present only in short reads and within 10 bases of either end of the read. We therefore hypothesized that these were from panel primers promiscuously binding to nearly complementary sequences of nontargeted amplicons. Sequences around the mutations matched primer binding sites in the panel in 18 of 19 cases, thus likely corresponding to panel primers. Furthermore, because most primers did not show this effect, we demonstrated that next-generation sequencing may be used to better design multiplex PCR primers through iterative elimination of offending primers to minimize mispriming. Our results indicate the need for careful sequence analysis to avoid false-positive mutations that can arise in multiplex PCR panels. The AmpliSeq Cancer panel is a valuable tool for clinical diagnostics, provided awareness of potential artifacts.

Full Text

Duke Authors

Cited Authors

  • McCall, CM; Mosier, S; Thiess, M; Debeljak, M; Pallavajjala, A; Beierl, K; Deak, KL; Datto, MB; Gocke, CD; Lin, M-T; Eshleman, JR

Published Date

  • September 2014

Published In

Volume / Issue

  • 16 / 5

Start / End Page

  • 541 - 549

PubMed ID

  • 25017478

Pubmed Central ID

  • PMC4188281

Electronic International Standard Serial Number (EISSN)

  • 1943-7811

Digital Object Identifier (DOI)

  • 10.1016/j.jmoldx.2014.06.001


  • eng

Conference Location

  • United States