False Positives in Multiplex PCR-Based Next-Generation Sequencing Have Unique Signatures

Abstract

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.

IPMNs with co-occurring invasive cancers: neighbours but not always relatives

“…All samples were analysed by targeted next generation sequencing of a custom panel of pancreatic driver genes using Ion AmpliSeq library preparation on an Ion Torrent Personal Genome Machine, and whole exome sequencing was performed on three selected cases by Personal Genome Diagnostics (Baltimore, Maryland, USA) 26. Mutations were identified using NextGENe software followed by visual inspection to minimise risk of artifactual calls 27. The relatedness of the lesions from each patient was determined by a categorical algorithm as described in the ‘Results’ section.…”

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

“…A number of targeted NGS panels have been developed as research technologies, laboratory developed tests, and commercially available products and custom panels for assessing multiple types of clinical specimens 3,5,[10][11][12][13][14][15] . Reports from multiple studies have demonstrated the value of NGS as a sensitive and specific clinical tool for the detection of genomic alterations 3,[10][11][12]14 . Yet studies have also demonstrated the risk of artifacts which can cause false-positive results from challenging cancer biopsies such as FFPE specimens [2][3][4][5]12,14 .…”

“…Reports from multiple studies have demonstrated the value of NGS as a sensitive and specific clinical tool for the detection of genomic alterations 3,[10][11][12]14 . Yet studies have also demonstrated the risk of artifacts which can cause false-positive results from challenging cancer biopsies such as FFPE specimens [2][3][4][5]12,14 . Additionally, recent publications have highlighted high failure rates for NGS using oncology specimens 16 and false-positive calls with commercial targeted NGS panels that are aggravated by the use of low-input DNA 12 .…”

“…Yet studies have also demonstrated the risk of artifacts which can cause false-positive results from challenging cancer biopsies such as FFPE specimens [2][3][4][5]12,14 . Additionally, recent publications have highlighted high failure rates for NGS using oncology specimens 16 and false-positive calls with commercial targeted NGS panels that are aggravated by the use of low-input DNA 12 . As a result, some laboratories have either modified or added additional checks and balances to commercially available targeted NGS technologies to improve performance, often to ensure accuracy or confirm the findings from the bioinformatic analyses 5,10,11 .…”

“…This was achieved by training a decision-tree algorithm using different functional copies of DNA input across 400 FFPE samples with independent measures of truth, and incorporating this algorithm into the bioinformatic software. As a result, the recommended input of 400 amplifiable copies, typically equivalent to ~5-20 ng of FFPE DNA, compares favorably to other methods [10][11][12] , including hybridization-based enrichment where ~250 ng of FFPE DNA is recommended 17,18 . Although the technique is described for use on a MiSeq platform, it can be modified by using Tag PCR primers with instrument-specific adaptors to enable sequence analysis on other NGS platforms.…”