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Updated: Oct 10, 2025
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Development and extensive analytical validation of deep amplicon sequencing for detecting KRAS and NRAS mutations in
Rastislav Slavkovský1,2, Jana Stránská1,3, Veronika Věnsková1
1Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic.
Abstract:
The presence of wild-type RAS alleles, as determined by genotyping codons 12, 13, 59, 61, 117, and 146, is a prerequisite for personalized anti-EGFR treatment of metastatic colorectal cancer (mCRC) patients. Here we describe analytical validation of in-house developed massively parallel sequencing technology (MPS) in comparison to the in vitro diagnostics (IVD) certified qPCR method. DNA extracted from FFPE samples from CRC patients (n=703) and reference standards (n=33) were tested for KRAS and NRAS mutations in 6 codons of exons 2, 3, and 4 using deep amplicon sequencing (DAS) on a MiSeq benchtop sequencer (Illumina). Two different amplicon lengths and two different library preparation methods (long-RAS and short-RAS) were tested in order to evaluate their impact on DAS performance. In parallel, identical tumor DNA was tested by the following IVD assays: therascreen KRAS RGQ PCR Kit (Qiagen), cobas® KRAS Mutation Test (Roche Diagnostics), and SNaPshot assay (Thermo Fisher Scientific). Both DAS assays detected all the mutations present in reference standards and external quality control samples, except for the artificially generated KRAS codon 146 mutation. The DAS assays performed sufficient analytical specificity and sensitivity (≥0.95). The use of shorter amplicons prolonged the preparation steps but significantly improved the sequencing success rate of FFPE-derived DNA. RAS mutation frequencies in the Czech CRC patients were similar to previous reports, although rare mutations were also detected. DAS with short amplicons is a good strategy for routine assessment of somatic mutations in low-quality FFPE-derived DNA.
Insights
A new massively parallel sequencing (MPS) method accurately detects RAS mutations in metastatic colorectal cancer (mCRC) FFPE samples. This deep amplicon sequencing (DAS) approach, especially with short amplicons, offers a reliable strategy for routine molecular testing.
Area of Science:
- Molecular Oncology
- Genetics
- Cancer Diagnostics
Background:
- RAS (KRAS/NRAS) genotyping is crucial for anti-EGFR therapy selection in metastatic colorectal cancer (mCRC).
- Validated methods are needed for accurate mutation detection in challenging formalin-fixed paraffin-embedded (FFPE) samples.
Purpose of the Study:
- To analytically validate an in-house developed deep amplicon sequencing (DAS) assay for RAS mutation detection.
- To compare the performance of DAS against established in vitro diagnostics (IVD) methods using FFPE-derived DNA.
Main Methods:
- Genotyping of 6 key RAS codons (12, 13, 59, 61, 117, 146) in 703 CRC FFPE samples using DAS on Illumina MiSeq.
- Evaluation of two amplicon lengths (long-RAS, short-RAS) and two library prep methods.
- Comparison with IVD assays: therascreen KRAS, cobas KRAS, and SNaPshot.
Main Results:
- DAS demonstrated high analytical specificity and sensitivity (≥0.95), detecting all mutations in reference standards except one artificial KRAS codon 146.
- Shorter amplicons improved sequencing success rates for FFPE DNA, despite longer preparation times.
- Observed RAS mutation frequencies in Czech CRC patients aligned with prior studies, with detection of rare mutations.
Conclusions:
- Deep amplicon sequencing with short amplicons is a robust strategy for routine assessment of somatic RAS mutations in low-quality FFPE DNA.
- This validated DAS method provides a reliable alternative for RAS genotyping in mCRC patients.
- The findings support the use of advanced sequencing technologies for personalized cancer treatment decisions.
