Abstract
We present an easy-to-use, open-source Optimised Exome analysis tool, OpEx (http://icr.ac.uk/opex) that accurately detects small-scale variation, including indels, to clinical standards. We evaluated OpEx performance with an experimentally validated dataset (the ICR142 NGS validation series), a large 1000 exome dataset (the ICR1000 UK exome series), and a clinical proband-parent trio dataset. The performance of OpEx for high-quality base substitutions and short indels in both small and large datasets is excellent, with overall sensitivity of 95%, specificity of 97% and low false detection rate (FDR) of 3%. Depending on the individual performance requirements the OpEx output allows one to optimise the inevitable trade-offs between sensitivity and specificity. For example, in the clinical setting one could permit a higher FDR and lower specificity to maximise sensitivity. In contexts where experimental validation is not possible, minimising the FDR and improving specificity may be a preferable trade-off for slightly lower sensitivity. OpEx is simple to install and use; the whole pipeline is run from a single command. OpEx is therefore well suited to the increasing research and clinical laboratories undertaking exome sequencing, particularly those without in-house dedicated bioinformatics expertise.
Highlights
We believe the primary advantage of OpEx is as a fully developed validated pipeline requiring minimal user input and no specialised informatic expertise
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Summary
OpEx includes a fixed implementation of tools for read alignment, variant calling and annotation optimised for individual or multiple exome sequencing analysis, outputting data to clinical standards. Platypus calls base substitutions and indels simultaneously This rapid independent variant calling approach enables analysis to keep pace with the sequencing output, allowing early assessment of potential sample quality issues which are not detectable through the CoverView coverage metrics, e.g. a contaminated sample showing excess heterozygosity. It provides early opportunities to assess and act on variant data, for example allowing early identification of a disease-causing variant in a plausible candidate gene These give OpEx a useful advantage over callers that need to call across all samples together to provide optimal performance and cannot be run until all laboratory work for all exomes is complete. As there were so few examples of this rare variant class with which to evaluate performance, we excluded indels >10 bp in our performance evaluation below, in which ‘all calls’ refers to all base substitutions and indels ≤10 bp
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