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Updated: Mar 31, 2026
Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing
Published on: October 18, 2013
Tumour procurement, DNA extraction, coverage analysis and optimisation of mutation-detection algorithms for human
James S Wilmott1, Matthew A Field, Peter A Johansson
11Melanoma Institute Australia, North Sydney, NSW 2Sydney Medical School, The University of Sydney, Camperdown, NSW 3Immunogenomics Laboratory, Australian National University, Canberra, ACT 4Oncogenomics Laboratory, QIMR Berghofer Medical Research Institute, Herston, Brisbane, Qld 5Centre for Cancer Research, The University of Sydney at Westmead Millennium Institute, Westmead, NSW 6Bioplatforms Australia, Macquarie University, North Ryde, NSW 7Ludwig Institute for Cancer Research, Olivia Newton-John Cancer and Wellness Centre, Austin Health, Heidelberg, Vic 8The Cancer Development and Treatment Laboratory, Peter MacCallum Cancer Centre and Sir Peter MacCallum Department of Oncology, The University of Melbourne, Vic 9Departments of Melanoma and Surgical Oncology 10Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred Hospital, Camperdown, NSW, Australia; these authors contributed equally.
Abstract:
Whole genome sequencing (WGS) of cancer patients' tumours offers the most comprehensive method of identifying both novel and known clinically-actionable genomic targets. However, the practicalities of performing WGS on clinical samples are poorly defined.This study was designed to test sample preparation, sequencing specifications and bioinformatic algorithms for their effect on accuracy and cost-efficiency in a large WGS analysis of human melanoma samples.WGS was performed on melanoma cell lines (n = 15) and melanoma fresh frozen tumours (n = 222). The appropriate level of coverage and the optimal mutation detection algorithm for the project pipeline were determined.An incremental increase in sequencing coverage from 36X to 132X in melanoma tissue samples and 30X to 103X for cell lines only resulted in a small increase (1-2%) in the number of mutations detected, and the quality scores of the additional mutations indicated a low probability that the mutations were real. The results suggest that 60X coverage for melanoma tissue and 40X for melanoma cell lines empower the detection of 98-99% of informative single nucleotide variants (SNVs), a sensitivity level at which clinical decision making or landscape research projects can be carried out with a high degree of confidence in the results. Likewise the bioinformatic mutation analysis methodology strongly influenced the number and quality of SNVs detected. Detecting mutations in the blood genomes separate to the tumour genomes generated 41% more SNVs than if the blood and melanoma tissue genomes were analysed simultaneously. Therefore, simultaneous analysis should be employed on matched melanoma tissue and blood genomes to reduce errors in mutation detection.This study provided valuable insights into the accuracy of SNV with WGS at various coverage levels in human clinical cancer specimens. Additionally, we investigated the accuracy of the publicly available mutation detection algorithms to detect cancer specific SNVs which will aid researchers and clinicians in study design and implementation of WGS for the identification of somatic mutations in other cancers.
