The power of hybridization capture - Illustrated using an expanded gene panel on 100 post mortem samples, focusing on sudden unexplained death

Abstract

Sudden unexpected death (SUD) is an unexpected event that in many cases are caused by diseases with an underlying genetic background. Forensic molecular autopsy is an approach that has gained wide-spread attention, in part explained by the rapid progress of DNA sequencing techniques. The approach leverages genetic data in combination with medical autopsy findings in post-mortem samples to explore a potential underlying genetic cause of death. Traditional forensic approaches to molecular autopsy focus on a small panel of genes, say <200 genes, with strong association to heart conditions whereas clinical genetics tend to capture entire exomes while subsequently selecting targeted panels bioinformatically. The drop in price and the increased throughput has promoted wider exome sequencing as a viable method to discover genetic variants. We explore a targeted gene panel consisting of 2422 genes, selected based on their broad association to sudden unexplained death. A hybridization capture approach from Twist Bioscience based on double stranded DNA probes was used to target exons of the included genes. We selected and sequenced a total of 98 post-mortem samples from historical forensic autopsy cases where the cause of death could not be unambiguously determined based on medical findings and that had a previous negative molecular autopsy. In the current study, we focus on the performance of the hybridization capture technology on a 2422 gene panel and explore metrics related to sequencing success using a mid-end NextSeq 550 as well as a MiSeq FGx platform. With the latter we demonstrate that our sequence data benefits from 2×300 bp sequencing increasing coverage, in particular, for difficult regions where shadow coverage, i.e. regions outside the probes, are utilized. The results further illustrate a highly uniform capture across the panel of genes (mean fold80=1.5), in turn minimizing excessive sequencing costs to reach sufficient coverage, i.e. 20X. We outline a stepwise procedure to select genes associated with SUD through virtual bioinformatical panels extracting tier of genes with increasing strength of association to SUD. We propose some prioritization strategies to filter variants with highest potential and show that the number of high priority genetic variant requiring manual inspections is few (0–3 for all tiers of genes) when all filters are applied.