ArticleThe Journal of molecular diagnostics : JMD2014
False positives in multiplex PCR-based next-generation sequencing have unique signatures.
Article in The Journal of molecular diagnostics : JMD, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 29 papers.
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The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
29 citing papers in PubMed, 58 citations in OpenAlex.
- The association of complex genetic background with the prognosis of acute leukemia with ambiguous lineage.Scientific reports · 2021Trial
- Mutations of CREBBP and SOCS1 are independent prognostic factors in diffuse large B cell lymphoma: mutational analysis of the SAKK 38/07 prospective clinical trial cohort.Journal of hematology & oncology · 2017Trial
- Evaluation of two highly-multiplexed custom panels for massively parallel semiconductor sequencing on paraffin DNA.PloS one · 2015Trial
- Deletion detection in SARS-CoV-2 genomes from COVID-19 patients: elimination of false positives.Virus evolution · 2026Article
- International society of liquid biopsy (ISLB) perspective on minimal requirements for ctDNA testing in solid tumors.The journal of liquid biopsy · 2025Review
- A Highly Sensitive XNA-Based RT-qPCR Assay for the Identification of ALK, RET, and ROS1 Fusions in Lung Cancer.Diagnostics (Basel, Switzerland) · 2024Article
- Optimizing Insertion and Deletion Detection Using Next-Generation Sequencing in the Clinical Laboratory.The Journal of molecular diagnostics : JMD · 2022Article
- FFPE-Based NGS Approaches into Clinical Practice: The Limits of Glory from a Pathologist Viewpoint.Journal of personalized medicine · 2022Review
- Correlation ofJournal of thoracic disease · 2021Article
- The feasibility of circulating tumor DNA analysis as a marker of recurrence in triple-negative breast cancer.Oncology letters · 2021Article
- The Role of the Pathologist in the Next-Generation Era of Tumor Molecular Characterization.Diagnostics (Basel, Switzerland) · 2021Review
- Targeted capture-based NGS is superior to multiplex PCR-based NGS for hereditary BRCA1 and BRCA2 gene analysis in FFPE tumor samples.BMC cancer · 2019Article
- Article
- Clonal heterogeneity ofOncotarget · 2018Article
- Detection of antimicrobial resistance genes associated with the International Space Station environmental surfaces.Scientific reports · 2018Article
- Article
- Profiling tumour heterogeneity through circulating tumour DNA in patients with pancreatic cancer.Oncotarget · 2017Article
- Application of a multi-gene next-generation sequencing panel to a non-invasive oesophageal cell-sampling device to diagnose dysplastic Barrett's oesophagus.The journal of pathology. Clinical research · 2017Article
- Single nucleotide variant sequencing errors in whole exome sequencing using the Ion Proton System.Biomedical reports · 2017Article
- Detection of false positive mutations in BRCA gene by next generation sequencing.Familial cancer · 2017Article
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
11 authors at 4 institutions in 1 country.
Funding
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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.