Optimizing Whole Exome Sequencing for enhanced clinical investigations

Whole exome sequencing (WES) is widely used as first-tier analysis in clinical investigation. The increased importance of WES has necessitated the development of a completely optimized workflow, from the laboratory setting to the bioinformatics pipeline. From the laboratory perspective, optimizing the entire workflow becomes fundamental to obtaining more reproducible and accurate data, thus enabling a more precise and robust variant calling of clinically relevant genomic regions. Further, it is important to evaluate the new exome panels released on the market and compare them with the existing ones. To ensure the quality of our WES analysis, we used several quality control parameters. We focused on coverage at different levels of mapped reads, but mostly on genotypability and uniformity. In particular, genotypability is a metric that takes into account the read’s mapping quality and also the coverage for a specific base position (it requires at least 4 reads in support). In this thesis, we focused on Twist Bioscience and Roche exome workflows. Our starting point was the introduction of the long fragment (~300 bp) during the library preparation. Firstly, we assessed the impact of the fragmentation method on the final outcome. Subsequently, we optimized the amplification step to ensure improved coverage uniformity and, consequently, to reduce genotypability variability. Indeed, the reduction in variability allows us to consistently achieve well-covered exomes, maintaining a greater uniformity and consequently yielding a more robust dataset of variants, thereby saving sequencing costs. Finally, we assessed the performance of the new exome enrichments provided by both vendors and compared them with the existing ones. We evaluated both the quality of their enrichment and their utility in clinical investigations. With this aim, our evaluation was focused on Twist Bioscience new exome panels: Twist Exome v.2.0 and Twist Exome v.2.0.1 (Twist Exome 2.0 plus Comprehensive Exome Spike-in). Likewise, we evaluated the new KAPA HyperExome v.2 provided by Roche. The new Twist Exome v.2.0.1 is shown to be the most complete design, obtaining high-quality exome sequencing metrics and maximizing coverage of clinically relevant regions. On the other hand, KAPA HyperExome v.2, which surpasses its previous version in WES performances, enabling the generation of higher-quality data and genotyping a greater number of clinically relevant positions