Aims Targeted next-generation sequencing (NGS) panels, which identify genomic alterations, are the stronghold of molecular oncology laboratories. In spite of technological advances, the quantity and quality of DNA from formalin-fixed paraffin-embedded tissue and paucicellular specimens are barriers to successful sequencing. Here, we describe an NGS assay employing single tube stem-loop inhibition mediated amplification technology that delivers highly accurate results with low input DNA. Rigorous quality metrics, regular monitoring and in-depth validation make the test attractive for clinical laboratories.
Methods The study used a customised NGS panel, targeting 48 genes across several solid tumour types. Validation, in accordance with guidelines from New York State, sequenced patient samples harbouring 136 known variants, including single-nucleotide variants (SNVs) and indels. Specimen types included formalin-fixed paraffin embedded blocks, core biopsies and cytology material. Neoplastic cellularity of the tumours ranged from 10% to 80%.
Results The assay was highly specific and sensitive with excellent accuracy, reproducibility and repeatability/precision. Concordant results for identification of SNVs and indels were obtained from specimens with DNA input of 2–3 ng, tissue with 10% neoplastic cellularity and variant allelic frequencies of 2.5%–3%. Over 99% of the target areas are shown to achieve at least 500X coverage when parsed through two bioinformatics pipelines. With over 2000 clinical specimens analysed, the success of the panel for reporting of results is 95.3%
Conclusions The advanced technology enables accurate identification of clinically relevant variants with uniformity of coverage and an impressive turn-around-time. The overall workflow and cost-effectiveness provide added value.
- Diagnostic Techniques and Procedures
- Pathology, Molecular
Data availability statement
All data relevant to the study are included in the article or uploaded as online supplemental information. Not applicable.
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Handling editor Runjan Chetty.
Contributors SB: conceptualisation, methodology, formal analysis and writing. EC: conceptualisation and investigation. CF: conceptualisation, methodology and project administration. SH and MM: conceptualisation, methodology, writing-review and editing. HF: guarantor, conceptualisation, methodology, analysis, investigation, analysis and writing-original draft.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests HF has received consulting fees from Opentrons Labworks and PIllar Biosciences. SH has received consulting fees from Opentron Labworks and Loxo Oncology and honoraria from Illumina and Medscape.
Provenance and peer review Not commissioned; externally peer reviewed.
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