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

Abstract

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 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) <i>Bacillus anthracis,</i> which causes anthrax in both people and animals and whose culture requires high-level containment facilities; and (2) <i>Mycoplasma amphoriforme</i>, a fastidious emerging human respiratory pathogen. We obtained high-quality genomic data for both species directly from clinical samples, with sufficient coverage (>15×) for confident variant calling over at least 80% of the baited genomes for over two thirds of the samples tested. Higher qPCR cycle threshold (<i>Ct</i>) values (indicative of lower pathogen concentrations in the samples), pooling libraries prior to capture, and lower captured library concentration were all statistically associated with lower capture efficiency. The <i>Ct</i> value had the highest predictive value, explaining 52 % of the variation in capture efficiency. Samples with <i>Ct</i> values ≤30 were over six times more likely to achieve the threshold coverage than those with a <i>Ct</i> > 30. We conclude that target-enrichment sequencing provides a valuable alternative to standard WGS following bacterial culture and creates opportunities for an improved understanding of the epidemiology and evolution of many clinically important pathogens for which culture is challenging.