Adam Hauge

The robust ability of Enterococcus faecalis to survive outside the host and to spread via oral-fecal transmission and its high degree of intrinsic and acquired antimicrobial resistance all complicate the treatment of hospital-acquired enterococcal infections. The conserved E. faecalis core genome serves as an important genetic scaffold for evolution of this bacterium in the modern health care setting and also provides interesting vaccine and drug targets. We used an innovative pooling/sequencing strategy to map a large collection of arrayed transposon insertions in E. faecalis OG1RF and generated an arrayed library of defined mutants covering approximately 70% of the OG1RF genome. Then, we performed high-throughput transposon sequencing experiments using this library to determine core genomic determinants of bile resistance in OG1RF. This collection is a valuable resource for comprehensive, functional enterococcal genomics using both traditional and high-throughput approaches and enables immediate recovery of mutants of interest.

Clinical laboratories have adopted next generation sequencing (NGS) as a gold standard for the diagnosis of hereditary disorders because of its analytic accuracy, high throughput, and potential for cost-effectiveness. We describe the implementation of a single broad-based NGS sequencing assay to meet the genetic testing needs at the University of Minnesota. A single hybrid capture library preparation was used for each test ordered, data was informatically blinded to clinically-ordered genes, and identified variants were reviewed and classified by genetic counselors and molecular pathologists. We performed 2509 sequencing tests from August 2012 till December 2017. The diagnostic yield has remained steady at 25%, but the number of variants of uncertain significance (VUS) included in a patient report decreased over time with 50% of the patient reports including at least one VUS in 2012 and only 22% of the patient reports reporting a VUS in 2017 (p = .002). Among the various clinical specialties, the diagnostic yield was highest in dermatology (60% diagnostic yield) and ophthalmology (42% diagnostic yield) while the diagnostic yield was lowest in gastrointestinal diseases and pulmonary diseases (10% detection yield in both specialties). Deletion/duplication analysis was also implemented in a subset of panels ordered, with 9% of samples having a diagnostic finding using the deletion/duplication analysis. We have demonstrated the feasibility of this broad-based NGS platform to meet the needs of our academic institution by aggregating a sufficient sample volume from many individually rare tests and providing a flexible ordering for custom, patient-specific panels.

CONTEXT: Although next-generation sequencing (NGS) can revolutionize molecular diagnostics, several hurdles remain in the implementation of this technology in clinical laboratories. OBJECTIVES: To validate and implement an NGS panel for genetic diagnosis of more than 100 inherited diseases, such as neurologic conditions, congenital hearing loss and eye disorders, developmental disorders, nonmalignant diseases treated by hematopoietic cell transplantation, familial cancers, connective tissue disorders, metabolic disorders, disorders of sexual development, and cardiac disorders. The diagnostic gene panels ranged from 1 to 54 genes with most of panels containing 10 genes or fewer. DESIGN: We used a liquid hybridization-based, target-enrichment strategy to enrich 10 067 exons in 568 genes, followed by NGS with a HiSeq 2000 sequencing system (Illumina, San Diego, California). RESULTS: We successfully sequenced 97.6% (9825 of 10 067) of the targeted exons to obtain a minimum coverage of 20× at all bases. We demonstrated 100% concordance in detecting 19 pathogenic single-nucleotide variations and 11 pathogenic insertion-deletion mutations ranging in size from 1 to 18 base pairs across 18 samples that were previously characterized by Sanger sequencing. Using 4 pairs of blinded, duplicate samples, we demonstrated a high degree of concordance (>99%) among the blinded, duplicate pairs. CONCLUSIONS: We have successfully demonstrated the feasibility of using the NGS platform to multiplex genetic tests for several rare diseases and the use of cloud computing for bioinformatics analysis as a relatively low-cost solution for implementing NGS in clinical laboratories.