Genomic testing based on chromosome microarray (CMA) and Next Generation Sequencing (NGS) revolutionized clinical genetics. That said, microarray, targeted panel, exome and generic whole genome sequencing (WGS) share some of the same shortcomings. Targeted sequencing is sensitive to inappropriate test selection, poor coverage in the intron /exon junctions and often fails to detect copy number variants (CNV) that affect only a few or a single exon. For WGS the main shortcoming is that structural variants in the 50bp-10,000bp range are either completely ignored during analysis or are not reported because of the difficulty to confirm these findings with an orthogonal method. Copy number neutral structural variants such as inversions and reciprocal translocations can also escape detection and complex structural rearrangements are often difficult or impossible to disentangle without the use of an orthogonal technology such as optical genome mapping (OGM). Short repeat expansions (e.g. FragileX syndrome) and long repeat contractions (FSHD) cannot be properly evaluated with short read WGS either.

OGM is a novel technology that can be best described as high-resolution cytogenetics. Depending on the genomic region, its ability to detect structural variants is down to the 5-10 thousand base pair range. Since it is based on microscopic evaluation of individual, very high molecular weight DNA molecules, copy number neutral structural variants and large repeat expansions and contractions can be directly visualized. Combined usage of WGS and OGM not only makes detection of these variants easier, but also provides a method to provide orthogonal confirmation of the findings.

Variants detected by WGS and OGM often classify only as variants of unknown significance since many have not been previously reported. Transcriptome analysis allows both the evaluation of the expression levels of the affected genes and alterations in their promoter usage and splicing. It can provide the required functional characterization allowing classification.

We propose that WGS should always be performed together with OGM and the functional significance of the findings should be verified using transcriptome sequencing.

Learning Objectives:

1. Discuss the reason why microarray, targeted panel , whole exome and even generic whole genome testing might not result in a genetic diagnosis

2. Discuss how to increase sensitivity of genomic testing by incorporating optical genome mapping in the whole genome analysis pipeline

3. Discuss the utility of transcriptome sequencing in the interpretation of variants detected using Whole Genome Sequencing and Optical Genome Mapping.