Innovating and improving genome diagnostics using targeted long-read sequencing

Abstract

Though short-read sequencing is increasingly becoming the standard method in genome diagnostic workflows, numerous other methods are still required. The detection of repeats, pseudogenes, complex genomic rearrangements, phasing, orientation, and methylation information are difficult — or even impossible — to acquire using just short-read sequencing. This makes diagnostics time consuming, expensive, difficult to maintain, and limited. To overcome this, we aim to introduce nanopore sequencing to streamline and improve our diagnostic workflow. We have designed a neurological disease gene panel for Oxford Nanopore adaptive sampling to simultaneously analyze genes carrying repeat regions, genomic regions where methylation status affects pathogenicity (e.g. FRM1), and genes with pseudogenes (e.g. PMS2). We optimized the laboratory process by altering various technical variables, such as short-read elimination, shearing methodologies, and flow cell loading conditions. Our optimized technical workflow reaches an output of approximately 15 Gb (13–18 Gb, GridION) with a mean read length of 10 kb, resulting in 95% 30x coverage of 455/471 genes of our gene panel. We further validated the Oxford Nanopore adaptive sampling gene panel by retrospectively sequencing 20 patients with a known variant in one of the included genes and determined the coverage needed for accurate variant calling. We used an in-house developed variant interpretation pipeline (https://github.com/molgenis/vip/). Currently, the validation is still ongoing, but first results show that repeat length determination and methylation status are fully concordant with diagnostic test results. Nanopore sequencing seems a relevant technique in a future diagnostic workflow and the possibilities of adaptive sampling are promising.

Biography

Eddy de Boer is a researcher and PhD candidate in the department of genetics at the University Medical Center Groningen where he works in the development and innovation team of the diagnostics section. In this role he designs and develops new methodologies and downstream data analysis to improve genetic diagnostic care in a broad range of projects. Most of his current projects are focused on nanopore long-read sequencing.