Utilising emerging technologies for the molecular diagnosis of genomic pathology

Over the last century, the advancement of understanding of the human genome has been profound, and with that, the development of diagnostic genomic testing has been expansive. Next Generation Sequencing technology has enabled the sequencing of the entire human genome in less than 24 hours, something that was unthinkable merely twenty years ago. The field continues to evolve and regularly presents new opportunities to expand the screening and diagnostic capability of genomics within the National Health Service.

While the use of NGS is now common practice throughout the NHS, there remain challenges which this technology has yet to overcome. Elements of the genome remain refractory to analysis, hampered by limitations of both the assay design and the analysis methods used to interpret the data that is generated. Here we evaluate novel technologies which have the potential to overcome these obstacles and contribute to the expansion of the genomic testing repertoire.

Single cell genome analysis is an emerging area of diagnostics; however, the sample input requirements for NGS dictate that cells must undergo a whole genome amplification step to achieve a quantity of DNA sufficient for NGS analysis. Here we evaluate the methods available to perform this and explore the potential of adapting an in-house copy number screening method for use with single cells.

Gene panel screening is standard practice within genomic diagnostics. There are, however, many genes where variant detection and classification is impeded by the presence of highly homologous pseudogene regions. Accurate distinction between gene and pseudogene is not possible with current hybrid capture preparation techniques. As a result, we assess new methods of library preparation that allow short-read sequencing data from single molecules to be linked, enabling contiguous analysis of multi-kilobase regions of the genome.

A further limitation of gene panel screening is that typically reagents are designed to capture only the exons and adjacent canonical splice sites of each target gene. While short read whole genome sequencing is now becoming more commonplace, allowing examination of deeper intronic variation, the technology does not allow the phasing of recessive variants which is sometimes necessary to allow classification of the mutation, meaning testing needs to be expanded beyond the proband to other family members. Here we utilise long read whole genome sequencing for patients where a single recessive variant has been detected by standard of care, to identify the second mutant disease-causing allele.

With the work presented here we outline the difficulties associated with implementing novel genomic technologies, ranging from minor optimisations to those larger obstacles which can go on to prevent long-term implementation of the assay. Despite these challenges, the results achieved demonstrate that although whole genome sequencing in the NHS is becoming more standardised, there remains utility in exploring alternative methodologies which can push the boundaries of variant detection and yield diagnoses for NHS patients that are not currently possible by standard of care.