Technical in-depth comparison of two massive parallel DNA-sequencing methods for formalin-fixed paraffin-embedded tissue from victims of sudden cardiac death

Emma Adolfsson,Alvida Qvick,Henrik Gréen,Daniel Kling,Cecilia Gunnarsson,Jon Jonasson,Anna Gréen

doi:10.1016/j.fsigen.2021.102522

Abstract

Sudden cardiac death (SCD) is a tragic and traumatic event. SCD is often associated with hereditary genetic disease and in such cases, sequencing of stored formalin fixed paraffin embedded (FFPE) tissue is often crucial in trying to find a causal genetic variant. This study was designed to compare two massive parallel sequencing assays for differences in sensitivity and precision regarding variants related to SCD in FFPE material. From eight cases of SCD where DNA from blood had been sequenced using HaloPlex, corresponding FFPE samples were collected six years later. DNA from FFPE samples were amplified using HaloPlex HS, sequenced on MiSeq, representing the first method, as well as amplified using modified Twist and sequenced on NextSeq, representing the second method. Molecular barcodes were included to distinguish artefacts from true variants. In both approaches, read coverage, uniformity and variant detection were compared using genomic DNA isolated from blood and corresponding FFPE tissue, respectively.In terms of coverage uniformity, Twist performed better than HaloPlex HS for FFPE samples. Despite higher overall coverage, amplicon-based HaloPlex technologies, both for blood and FFPE tissue, suffered from design and/or performance issues resulting in genes lacking complete coverage. Although Twist had considerably lower overall mean coverage, high uniformity resulted in equal or higher fraction of genes covered at ≥ 20X. By comparing variants found in the matched samples in a pre-defined cardiodiagnostic gene panel, HaloPlex HS for FFPE material resulted in high sensitivity, 98.0% (range 96.6–100%), and high precision, 99.9% (range 99.5–100%) for moderately fragmented samples, but suffered from reduced sensitivity (range 74.2–91.1%) in more severely fragmented samples due to lack of coverage. Twist had high sensitivity, 97.8% (range 96.8–98.7%) and high precision, 99.9% (range 99.3–100%) in all analyzed samples, including the severely fragmented samples.

Highlights

Sudden cardiac death (SCD) is an event defined as a sudden and unexpected death occurring within an hour of the onset of symptoms, or occurring in people found dead within 24 h of being asymptomatic, who presumably died due to a cardiac arrhythmia or hemodynamic catas trophe [1]
SCD is a traumatic event for relatives of the victim, and finding a potential underlying genetic cause is very important for the relatives since genetic testing of the family may prevent further deaths [34]
Since the only remaining material may be formalin fixed paraffin embedded (FFPE) samples from autopsy, genetic testing must be compatible with this material

Summary

Introduction

Sudden cardiac death (SCD) is an event defined as a sudden and unexpected death occurring within an hour of the onset of symptoms, or occurring in people found dead within 24 h of being asymptomatic, who presumably died due to a cardiac arrhythmia or hemodynamic catas trophe [1]. The disorders have common features in regard to genetics: they are familial rather than sporadic; they are autosomal dominant diseases, and the vast majority show incomplete penetrance. They show marked genetic heterogeneity with multiple types of variants [8]. Previous studies on the genetics of SCD, using targeted sequencing with gene panels of 70–192 genes associated with cardiac disease, showed that potentially pathogenic variants were found in 13–50% of the cases [3,10,11]. Clinical screening of relatives iden tifies an inherited genetic condition in 22–53% of the families, and targeted genetic DNA screening identifies pathological variants in genes coding for cardiac ion channels in up to 35% [12,13,14]

Methods

Results

Discussion

Conclusion