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False positives in multiplex PCR-based next-generation sequencing have unique signatures.
Chad M McCall1, Stacy Mosier1, Michele Thiess1
1Department of Pathology, The Sol Goldman Pancreatic Cancer Research Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
The Journal of Molecular Diagnostics : JMD
|July 15, 2014
Summary
Next-generation sequencing (NGS) can detect false mutations in cancer gene panels. Careful analysis is needed to distinguish true mutations from artifacts caused by multiplex PCR primer mispriming.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Next-generation sequencing (NGS) enables rapid mutational analysis of multiple genes in human cancers.
- Multiplex PCR-based panels are increasingly used for cancer gene profiling.
Purpose of the Study:
- To evaluate the accuracy of a multiplex PCR-based NGS panel for detecting EGFR, KRAS, and BRAF mutations in lung and colorectal adenocarcinomas.
- To investigate the cause of apparent novel mutations identified by automated analysis.
Main Methods:
- Implementation of the Ion AmpliSeq Cancer Hotspot Panel for targeted sequencing.
- Automated and individual read analysis of sequencing data.
- Bioinformatic analysis to identify primer binding sites and assess sequence complementarity.
Main Results:
- Automated analysis identified a novel EGFR G873R substitution mutation in 10% of samples.
- Individual read examination revealed these mutations were artifacts, present in short reads near primer binding sites.
- Sequences surrounding the apparent mutations matched panel primer binding sites in 18 of 19 cases.
Conclusions:
- Apparent mutations in multiplex PCR panels can arise from panel primers binding to non-targeted sequences (mispriming).
- Careful sequence analysis is crucial to avoid false-positive results in cancer mutation detection.
- NGS can be used to optimize multiplex PCR primer design by identifying and eliminating those causing mispriming.
