Genomic characterization of Francisella tularensis and other diverse Francisella species from complex samples

Abstract

Francisella tularensis, the bacterium that causes the zoonosis tularemia, and its genetic near neighbor species, can be difficult or impossible to cultivate from complex samples. Thus, there is a lack of genomic information for these species that has, among other things, limited the development of robust detection assays for F. tularensis that are both specific and sensitive. The objective of this study was to develop and validate approaches to capture, enrich, sequence, and analyze Francisella DNA present in DNA extracts generated from complex samples. RNA capture probes were designed based upon the known pan genome of F. tularensis and other diverse species in the family Francisellaceae. Probes that targeted genomic regions also present in non-Francisellaceae species were excluded, and probes specific to particular Francisella species or phylogenetic clades were identified. The capture-enrichment system was then applied to diverse, complex DNA extracts containing low-level Francisella DNA, including human clinical tularemia samples, environmental samples (i.e., animal tissue and air filters), and whole ticks/tick cell lines, which was followed by sequencing of the enriched samples. Analysis of the resulting data facilitated rigorous and unambiguous confirmation of the detection of F. tularensis or other Francisella species in complex samples, identification of mixtures of different Francisella species in the same sample, analysis of gene content (e.g., known virulence and antimicrobial resistance loci), and high-resolution whole genome-based genotyping. The benefits of this capture-enrichment system include: even very low target DNA can be amplified; it is culture-independent, reducing exposure for research and/or clinical personnel and allowing genomic information to be obtained from samples that do not yield isolates; and the resulting comprehensive data not only provide robust means to confirm the presence of a target species in a sample, but also can provide data useful for source attribution, which is important from a genomic epidemiology perspective.

DNA Capture and Enrichment: A Culture-Independent Approach for Characterizing the Genomic Diversity of Pathogenic Leptospira Species

“…However, other methods for species identification are also possible; we recently described a workflow for detecting and characterizing mixtures of Francisella spp. in enriched samples using species-specific probes [ 29 ]. Because our Leptospira DNA capture and enrichment probe design was based upon the pan-genome of pathogenic Leptospira , probes were included in this array that were specific to each species and, thus, this approach could also be applied to this DNA capture and enrichment system.…”

“…The general design process is explained in detail elsewhere where we describe a similar enrichment system for Francisella spp. [ 29 ], but in brief, the process consisted of (1) bioinformatically “slicing” the coding sequences into 120 nucleotide (nt) fragments and designing complimentary RNA probes with 2x tiling (probes overlap by 60 nts) to maximize coverage; (2) removing probes that were only conserved in a single genome because these sequences may represent contamination; and (3) removing probes that capture highly conserved regions (e.g., rRNA genes) as well as those that show homology with non-target bacteria to minimize hybridization and capture of unwanted sequences. Regions with low GC content are difficult to hybridize [ 29 ]; to compensate for this difficulty, additional probes for these regions were added to the design.…”

“…Certain samples were pooled prior to enrichment whereas others were not ( Table 1 : Pooling), and a slow hybridization method was implemented to prevent the dissociation of probes from AT-rich regions, with ~1000–2000 ng of each library hybridized at 65 °C for 16–24 h. Libraries were then subjected to one or two rounds of DNA capture and enrichment. All these methods are described in more detail elsewhere [ 29 ].…”

“…Reads generated from enriched complex samples were aligned against appropriate reference genomes ( Table 2 ) with minimap2 v2.22 and Samtools as described above and elsewhere [ 29 ].…”

“…To address this need and supplement and improve collective public Leptospira genomic resources, we describe here the design and validation of a pan-pathogenic Leptospira DNA capture and enrichment system that can be used to obtain genomic information from unculturable leptospirosis clinical and animal samples, including frozen and archived samples and those that have been collected after treatment with antibiotics [ 29 ].…”