Abstract
Next generation sequencing (NGS) is routinely used in clinical genetic testing. Quality management of NGS testing is essential to ensure performance is consistently and rigorously evaluated. Three primary metrics are used in NGS quality evaluation: depth of coverage, base quality and mapping quality. To provide consistency and transparency in the utilisation of these metrics we present the Quality Sequencing Minimum (QSM). The QSM defines the minimum quality requirement a laboratory has selected for depth of coverage (C), base quality (B) and mapping quality (M) and can be applied per base, exon, gene or other genomic region, as appropriate. The QSM format is CX_BY(P Y)_MZ(P Z). X is the parameter threshold for C, Y the parameter threshold for B, P Y the percentage of reads that must reach Y, Z the parameter threshold for M, P Z the percentage of reads that must reach Z. The data underlying the QSM is in the BAM file, so a QSM can be easily and automatically calculated in any NGS pipeline. We used the QSM to optimise cancer predisposition gene testing using the TruSight Cancer Panel (TSCP). We set the QSM as C50_B10(85)_M20(95). Test regions falling below the QSM were automatically flagged for review, with 100/1471 test regions QSM-flagged in multiple individuals. Supplementing these regions with 132 additional probes improved performance in 85/100. We also used the QSM to optimise testing of genes with pseudogenes such as PTEN and PMS2. In TSCP data from 960 individuals the median number of regions that passed QSM per sample was 1429 (97%). Importantly, the QSM can be used at an individual report level to provide succinct, comprehensive quality assurance information about individual test performance. We believe many laboratories would find the QSM useful. Furthermore, widespread adoption of the QSM would facilitate consistent, transparent reporting of genetic test performance by different laboratories.
Highlights
Generation sequencing (NGS) is routinely used to investigate if genomic variation has caused, or has the potential to cause human disease in clinical and research settings[1]
The TGLclinical analytical pipeline includes CASAVA v.1.8.2 to demultiplex and create FASTQs per sample from the raw base call (BCL) files and Stampy v.1.0.2013 with BWA v.0.7.5a pre-mapping[14] to map sequence reads to the human reference genome (GRCh37)
We have developed the Quality Sequencing Minimum (QSM), to achieve this
Summary
Generation sequencing (NGS) is routinely used to investigate if genomic variation has caused, or has the potential to cause human disease in clinical and research settings[1]. Such genetic tests must robustly be able to detect pathogenic variants (positive tests) and to exclude the presence of pathogenic variants (negative tests). One of the recommendations is for clear information about test performance and limitations to be provided on the test report, as healthcare professionals need this for clinical decision making[3,4,5] This is well accepted to be best practice there are no specific guidelines for how it can be achieved, and most test reports provide limited or no information
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