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Published on: April 11, 2016
Application of Targeted Next-generation Sequencing, TruSeq Custom Amplicon Assay for Molecular Pathology Diagnostics
Erzsébet Csernák1, János Molnár, Gábor E Tusnády
1*Department of Surgical and Molecular Pathology, National Institute of Oncology †"Momentum" Membrane Protein Bioinformatics Research Group, Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary.
Abstract:
The implementation of targeted therapies revolutionized oncology. As the number of new oncogenic driver mutations, which provide molecular targets for prediction of effective and selective therapies, is increasing, the implementation of fast and reliable methods by molecular pathology labs is very important. Here we report our results with TruSeq Custom Amplicon assay performed on formalin-fixed and paraffin-embedded material. The oligo capture probes targeted the hotspot regions of 10 well-known oncogenes linked to clinical diagnosis and treatment of lung and colorectal adenocarcinomas, melanomas, and gastrointestinal stromal tumors. Fifteen previously genotyped formalin-fixed and paraffin-embedded DNA samples from different tumor types were selected for massively parallel sequencing. A bioinformatics pipeline was developed to identify high-quality variants and remove sequence artifacts. With the exception of 1 sample, which was of lower quality than the others, relevant mutations corresponding to tumor types could be reliable detected by the developed bioinformatical pipeline. This study indicates that the application of TruSeq Custom Amplicon assay is a promising tool in molecular pathology diagnostics, but it is important to standardize sample processing (including fixation, isolation procedure, sample selection based on quality assessment, and rigorous variant calling) to achieve the highest success rate and avoid false results.
Insights
The TruSeq Custom Amplicon assay reliably detects oncogenic mutations in formalin-fixed samples for targeted cancer therapies. Standardizing molecular pathology workflows is crucial for accurate results and avoiding false positives.
Area of Science:
- Oncology
- Molecular Pathology
- Genomics
Background:
- Targeted therapies have transformed cancer treatment.
- Increasing oncogenic driver mutations necessitate rapid, reliable diagnostic methods.
- Molecular pathology labs require efficient tools for mutation detection.
Purpose of the Study:
- To evaluate the TruSeq Custom Amplicon assay for detecting oncogenic mutations.
- To assess the assay's performance on formalin-fixed, paraffin-embedded (FFPE) tumor samples.
- To develop a bioinformatics pipeline for variant analysis in FFPE DNA.
Main Methods:
- Utilized the TruSeq Custom Amplicon assay targeting hotspot regions of 10 oncogenes.
- Employed massively parallel sequencing on 15 previously genotyped FFPE DNA samples.
- Developed a bioinformatics pipeline for variant calling and artifact removal.
Main Results:
- The assay successfully detected relevant mutations in most FFPE samples.
- A bioinformatics pipeline identified high-quality variants and removed sequence artifacts.
- One lower-quality sample yielded less reliable results, highlighting the importance of sample quality.
Conclusions:
- The TruSeq Custom Amplicon assay shows promise for molecular pathology diagnostics.
- Standardization of sample processing, from fixation to variant calling, is essential for maximizing success rates.
- Rigorous quality assessment and variant calling are critical to avoid false results in FFPE diagnostics.
