Harnessing Advances in Long Read Sequencing Technology Towards Improved Clinical Diagnostics

Whether surveying the human genome for pathologically relevant complex structural variation or investigating various microbes related to infectious disease, long read length sequencing is rapidly changing the face of clinical diagnostics, enabling previously unachievable genetic information.  Recent advances in single molecule real time (SMRT) sequencing have enabled faster, more comprehensive clinical diagnostics assays.  Here, we focus specifically on those methods related to uncovering genetic structural variation and finishing of clinically relevant microbial genomes. Despite the progressive advances in next-generation sequencing technologies over the past decade, a variety of hard to sequence repetitive human genetic loci remain ambiguous, including many exonic regions with expected biological function.  We present data resolving numerous structural variants from whole-genome sequencing data achieved using the RSII platform as well as methods for targeting repetitive genetic regions using long-range, high fidelity PCR in tandem with SMRT sequencing.   These results demonstrate the potential for diagnostics that better assess genetic variation in pathologically relevant, repeat rich coding regions and that explore the relationship of structurally complex, non-coding domains using long reads.  Further, these results reinforce the pressing need to derive more comprehensive and accurate human genome references to expand their clinical utility beyond that achieved with traditional, shorter read length NGS technologies.

Similarly, genetic diversity amongst infectious disease isolates spans a multitude of unique pathological structural and epigenetic variants that identify factors related to virulence, origin, and resistance.  Addressing the need to finish microbial genomes to better identify these factors, we present a rapid method to isolate genomic DNA and generate read lengths with N50 values as high as 12,000 base pairs using SMRT sequencing.  Through a series of studies, a demonstration of how readlengths as long as 60,000 base pairs have enabled the ability to assemble complex structural domains, identify structural insertion elements, phase accessory genomes such as plasmids, and how these reads are used to better understand microbial genetics and transmission will be discussed.  One specific example from a solid organ transplantation case will detail the comparison of Staphylococcus aureus isolates in donor and recipient and how microbial finishing can be used to further improve our understanding of the epidemiology of bacterial transmission and the risk of adverse patient outcomes when a compromised organ is considered.  Additionally, better characterization of community isolates leads to improved hospital surveillance, reducing the number of hospital-acquired cases and minimizing the risk of outbreak.