Combined RNAseq and long-read sequencing for unsolved cases


Long-read whole-genome sequencing (LR-WGS) could identify all possibly pathogenic DNA variants in genetic disorders. However, DNA analysis alone is often insufficient to interpret the effect of DNA variation, and by extension whether it could cause disease. To improve diagnostic yield, we have been using transcriptome analysis in a diagnostic setting in undiagnosed disorders. However, interpretation of the results may remain challenging. As combining RNAseq and LR-WGS could increase our understanding of complex variants, we here used LR-WGS and RNA sequencing (Oxford Nanopore Technologies) in unresolved genetic disorders to investigate the capacity for better genetic characterisation and explore the potential of nanopore technology in improving diagnostic yield. We selected undiagnosed individuals, some awaiting diagnosis for more than 15 years, where previous RNAseq analysis indicated strong clues regarding the genetic cause — such as transcript isoform switching, fusion transcripts, transcriptional read-through, and transposon insertion — but further routine diagnostic DNA analysis proved insufficient to identify the precise causative variant. For example, in an undiagnosed individual with a clinical phenotype matching Treacher Collins syndrome, we identified a switch in TCOF1 transcript isoform from one allele. Further DNA analysis, including optical genome mapping, identified a possible breakpoint in TCOF1, but the underlying event could not fully be resolved. LR-WGS subsequently identified a ~3.5 kb mobile element insertion (SVA) in the TCOF1 intronic sequence and together with the observed effect in the RNAseq data, this provided a genetic diagnosis. In all, our results support the idea that Oxford Nanopore Technologies LR-WGS has the potential to be rapidly implemented for undiagnosed genetic disease and stress the need for complementary analysis, such as RNA sequencing, necessary to interpret the clinical impact of complex DNA variants.


Dr. Tjakko van Ham obtained his PhD in genetics (C. elegans functional genomics) in 2009 at the University Medical Centre (UMC), Groningen, Netherlands. After postdoctoral training at Mass General Hospital, Boston, USA and UMC Groningen in genetic brain disorders using zebrafish as a model, he became a group leader at the Erasmus Medical Centre, Rotterdam, Netherlands in 2014. Currently, he is an associate professor leading a translational research laboratory focusing on implementation of new diagnostic approaches in genetic disorders. A main interest is RNA sequencing in undiagnosed genetic disease, which proved highly effective and complementary to genome analysis to assist in diagnosing diverse genetic disorders.

Authors: Tjakko van Ham