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Updated: Dec 31, 2025
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
Inter-laboratory proficiency testing scheme for tumour next-generation sequencing in Ontario: a pilot study
1Toronto, ON: Advanced Molecular Diagnostics Laboratory, Princess Margaret Cancer Centre, University Health Network (Spence, Stockley); Bioinformatics and HPC Core, Princess Margaret Cancer Centre, University Health Network (Stickle); Cancer Genomics Program, Princess Margaret Cancer Centre, University Health Network (Yu, Chow, Siu); Division of Medical Oncology and Hematology, Princess Margaret Cancer Centre, University Health Network (Siu, Bedard); Department of Medicine, University of Toronto (Siu, Bedard); Department of Clinical Laboratory Genetics, University Health Network (Stockley); Department of Laboratory Medicine and Pathobiology, University of Toronto (Stockley).
Background:
A pilot inter-laboratory proficiency scheme for 5 Ontario clinical laboratories testing tumour samples for the Ontario-wide Cancer Targeted Nucleic Acid Evaluation (octane) study was undertaken to assess proficiency in the identification and reporting of next-generation sequencing (ngs) test results in solid tumour testing from archival formalin-fixed, paraffin-embedded (ffpe) tissue.
Methods:
One laboratory served as the reference centre and provided samples to 4 participating laboratories. An analyte-based approach was applied: each participating laboratory received 10 ffpe tissue specimens profiled at the reference centre, with tumour site and histology provided. Laboratories performed testing per their standard ngs tumour test protocols. Items returned for assessment included genes and variants that would be typically reported in routine clinical testing and variant call format (vcf) files to allow for assessment of ngs technical quality.
Results:
Two main aspects were assessed:■ Technical quality and accuracy of identification of exonic variants■ Site-specific reporting practicesTechnical assessment included evaluation of exonic variant identification, quality assessment of the vcf files to evaluate base calling, variant allele frequency, and depth of coverage for all exonic variants. Concordance at 100% was observed from all sites in the technical identification of 98 exonic variants across the 10 cases. Variability between laboratories in the choice of variants considered clinically reportable was significant. Of the 38 variants reported as clinically relevant by at least 1 site, only 3 variants were concordantly reported by all participating centres as clinically relevant.
Conclusions:
Although excellent technical concordance for ngs tumour profiling was observed across participating institutions, differences in the reporting of clinically relevant variants were observed, highlighting reporting as a gap where consensus on the part of Ontario laboratories is needed.
