Target-enrichment sequencing yields valuable genomic data for difficult-to culture bacteria of public health importance

Tristan P. W. Dennis1, Barbara K. Mable1, Brian Brunelle2, Alison Devault2, Ryan Carter1 4 , Clare L. Ling3,4, Blandina T. Mmbaga5, Jo E. B. Halliday1, Katarina Oravcova1 , Taya L. Forde1 *
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Genomic data contribute invaluable information to the epidemiological investigation of pathogens of public health importance. However, whole genome sequencing (WGS) of bacteria  typically relies on culture, which represents a major hurdle for generating such data for a wide 38 range of species for which culture is challenging. In this study, we assessed the use of culture free target-enrichment sequencing as a method for generating genomic data for two bacterial  species: 1) Bacillus anthracis, which causes anthrax in both people and animals and whose  culture requires high level containment facilities; and 2) Mycoplasma amphoriforme, a  fastidious emerging human respiratory pathogen. We obtained high quality genomic data for  both species directly from clinical samples, with sufficient coverage (>15X) for confident variant  calling over at least 80% of the baited genomes for over two thirds of the samples tested.  Higher qPCR cycle threshold (Ct) values (indicative of lower pathogen concentrations in the 46 samples), pooling libraries prior to capture, and lower captured library concentration were all 47 statistically associated with lower capture efficiency. The Ct value had the highest predictive  value, explaining 52% of the variation in capture efficiency. Samples with Ct values ≤ 30 were  over 6 times more likely to achieve the threshold coverage than those with a Ct > 30. We 50 conclude that target-enrichment sequencing provides a valuable alternative to standard WGS 51 following bacterial culture and creates opportunities for an improved understanding of the 52 epidemiology and evolution of many clinically important pathogens for which culture is 53 challenging.