Tumour procurement, DNA extraction, coverage analysis and optimisation of mutation-detection algorithms for human melanoma genomes

Wilmott, James S., Field, Matthew A., Johansson, Peter A., Kakavand, Hojabr, Shang, Ping, De Paoli-Iseppi, Ricardo, Vilain, Ricardo E., Pupo, Gulietta M., Tembe, Varsha, Jakrot, Valerie, Shang, Catherine A., Cebon, Jonathan, Shackleton, Mark, Fitzgerald, Anna, Thompson, John F., Hayward, Nichoas K., Mann, Graham J., and Scolyer, Richard A. (2015) Tumour procurement, DNA extraction, coverage analysis and optimisation of mutation-detection algorithms for human melanoma genomes. Pathology, 47 (7). pp. 683-693.

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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.

Item ID: 43588
Item Type: Article (Research - C1)
ISSN: 1465-3931
Keywords: cancer, coverage, genomics, melanoma, methods, mutation, pathology, sequencing, treatment, whole genome
Funders: Melanoma Institute Australia, Bioplatforms Australia, NSW Ministry of Health, Cancer Council NSW (CC), National Health and Medical Research Council of Australia (NHMRC), Australian Research Council (ARC)
Projects and Grants: CC NSW RG12/13, NHMRC Project grant 402761, NHMRC Project grant 633004, CC NSW Translational Research Program Grant 05TPG1-01, CC NSW Translational Research Program Grant 10TPG1-02, ARC Discovery Grant DP1301004
Date Deposited: 20 Apr 2016 03:39
FoR Codes: 06 BIOLOGICAL SCIENCES > 0601 Biochemistry and Cell Biology > 060102 Bioinformatics @ 25%
06 BIOLOGICAL SCIENCES > 0604 Genetics > 060408 Genomics @ 25%
11 MEDICAL AND HEALTH SCIENCES > 1112 Oncology and Carcinogenesis > 111203 Cancer Genetics @ 50%
SEO Codes: 92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920102 Cancer and Related Disorders @ 100%
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