Computational protocol: Assessment of the incorporation of CNV surveillance into gene panel next generation sequencing testing for inherited retinal diseases

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Protocol publication

[…] Sequencing reads were demultiplexed with CASAVA V.1.8.2 and aligned to the hg19 reference genome using Burrows-Wheeler Aligner short read (V.0.6.2) software. Duplicate reads were removed using SAMtools V.0.1.18 before variant calling was performed. We have described the methodology employed for the detection and clinical analysis of SNVs and indels previously. CNV detection was performed using standard parameters for ExomeDepth V.1.1.6. ExomeDepth was presented with sets of aligned and non-duplicate sequencing reads in a binary sequence alignment/map (BAM) file format that were matched by gender and by the enrichment kit used, and had been generated for unrelated individuals with IRD referred for diagnostic testing (online ). [...] We used three distinct strategies to limit the number of potential false-positive CNV events identified by ExomeDepth (). Events that were analysed in a clinical context were all (1) identified against three independent reference sets using ExomeDepth, (2) identified by at least one other CNV software tool (CoNVex, CoNVaDING or both) and (3) visually inspected using the ExomeDepth graphical package.We first limited our analysis of CNV events to those that had been identified by ExomeDepth in comparison to three mutually exclusive reference sets of samples. For each tested individual we created three randomly selected and non-overlapping groups of 30 individuals matched by their gender and the enrichment kit used and presented these to the ExomeDepth algorithm. The overlap between the three reference sets was calculated using bedtools V.2.25.0 intersect. Second, we performed CNV calling using two other publicly available CNV detection algorithms (CoNVex and CoNVaDING). Both algorithms were presented with aligned and non-duplicate sequencing reads in a BAM file format for large groups of individuals matched by gender and the enrichment kit used (as described in online ), and CNV calling was performed using standard parameters for each of these tools. We compared CNV events identified by CoNVex and CoNVaDING with those that had been identified by ExomeDepth using bedtools V.2.25.0 intersect, and included all events identified by ExomeDepth and at least one other CNV detection tool. We limited our third stage of analysis, visual inspection, to those events that were identified against three reference sets using ExomeDepth and by at least one additional CNV detection tool. Visual inspection included an assessment of the consistency of calculated read ratios across all exons within implicated genes, the extent of variation within the selected reference samples for each exon, the nature of the exon CNV status across the cohort and the continuity of abnormal CNV exons within the implicated gene. [...] CNVs were interpreted alongside SNVs and indels that had been detected through routine gene panel NGS diagnostic techniques, as described previously. For each individual, variants were categorised in accordance with the American College of Medical Genetics and Genomics (ACMG) guidelines, and pathogenic/likely pathogenic variants in a disease-causing state were determined to confirm or provisionally confirm a molecular diagnosis of IRD. CNV frequency estimations were calculated through comparison to 682 WGS data sets for individuals with clinical indications of IRD. Six hundred and five samples were generated using Illumina sequencing chemistry as part of the National Institute for Health Research (NIHR) BioResource Rare Diseases project, and the Manta and Canvas software algorithms were used to detect CNVs. Seventy-seven samples were generated using Complete Genomics sequencing chemistry, with CNVs identified using the Complete Genomics V.2.5 variant calling pipeline. Both of these strategies incorporate an assessment of sequencing read depth, an assessment of the read insert sizes and an assessment of sequencing read composition to identify CNV breakpoints/insertion points. […]

Pipeline specifications

Software tools BaseSpace, BWA, SAMtools, ExomeDepth, CoNVaDING, BEDTools, Manta, Canvas, Variant Calling Pipeline
Organisms Cucumber necrosis virus
Diseases Genetic Diseases, Inborn, Refsum Disease, Infantile, Retinal Dystrophies