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Updated: Mar 1, 2026
Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
Published on: April 11, 2016
Integration of Technical, Bioinformatic, and Variant Assessment Approaches in the Validation of a Targeted
Mariam Thomas, Mahadeo A Sukhai, Tong Zhang
1From the Laboratory Medicine Program, Advanced Molecular Diagnostics Laboratory, Departments of Pathology and Genetics (Drs Thomas, Sukhai, Garg, Misyura, Stockley, and Kamel-Reid), the Princess Margaret Cancer Centre (Drs Thomas, Sukhai, Garg, Misyura, Pugh, Stockley, and Kamel-Reid and Ms Zhang), and High Performance Computing and Bioinformatics Services, Princess Margaret Genomics Centre (Dr Harbi and Mr Dolatshahi), University Health Network, Toronto, Ontario, Canada; and the Departments of Medical Biophysics (Drs Pugh and Kamel-Reid) and Laboratory Medicine and Pathobiology (Drs Stockley and Kamel-Reid), The University of Toronto, Toronto, Ontario, Canada.
Context:
- Detection of variants in hematologic malignancies is increasingly important because of a growing number of variants impacting diagnosis, prognosis, and treatment response, and as potential therapeutic targets. The use of next-generation sequencing technologies to detect variants in hematologic malignancies in a clinical diagnostic laboratory setting allows for efficient identification of routinely tested markers in multiple genes simultaneously, as well as the identification of novel and rare variants in other clinically relevant genes.
Objective:
- To apply a systematic approach to evaluate and validate a commercially available next-generation sequencing panel (TruSight Myeloid Sequencing Panel, Illumina, San Diego, California) targeting 54 genes. In this manuscript, we focused on the parameters that were used to evaluate assay performance characteristics.
Data Sources:
- Analytical validation was performed using samples containing known variants that had been identified previously. Cases were selected from different disease types, with variants in a range of genes. Panel performance characteristics were assessed and genomic regions requiring additional analysis or wet-bench approaches identified.
Conclusions:
- We validated the performance characteristics of a myeloid next-generation sequencing panel for detection of variants. The TruSight Myeloid Sequencing Panel covers more than 95% of target regions with depth greater than 500×. However, because of unique variant types such as large insertions or deletions or genomic regions of high GC content, variants in CEBPA, FLT3, and CALR required supplementation with non-next-generation sequencing assays or with informatics approaches to address deficiencies in performance. The use of multiple bioinformatics approaches (2 variant callers and informatics scripts) allows for maximizing calling of true positives, while identifying limitations in using either method alone.
