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

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) 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 samples), pooling libraries prior to capture, and lower captured library concentration were all 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 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. Data summary The authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. Scripts used in this study can be accessed on GitHub at . All sequence data generated during this study have been deposited in the European Nucleotide Archive (ENA) Sequence Read Archive (SRA) under project accession numbers PRJEB46822 ( B. anthracis ) and PRJEB50216 ( M. amphoriforme ). Accession numbers for individual samples, along with metadata, laboratory parameters and sequence quality metrics, are available at the University of Glasgow’s data repository, Enlighten, at . ### Competing Interest Statement AD and BB are employed by Daicel Arbor Biosciences, which sells the myBaits Custom hybridization capture kits utilized in this study. The other authors declare that there are no conflicts of interest.