Discovery of thermophilic Bacillales using reduced-representation genotyping for identification

Abstract

Background: This study demonstrates the use of reduced-representation genotyping to provide preliminary identifications for thermophilic bacterial isolates. The approach combines restriction enzyme digestion and PCR with next-generation sequencing to provide thousands of short-read sequences from across the bacterial genomes. Isolates were obtained from compost, hot water systems, and artesian bores of the Great Artesian Basin. Genomic DNA was double-digested with two combinations of restriction enzymes followed by PCR amplification, using a commercial provider of DArTseq™, Diversity Arrays Technology Pty Ltd. (Canberra, Australia). The resulting fragments which formed a reduced-representation of approximately 2.3% of the genome were sequenced. The sequence tags obtained were aligned against all available RefSeq bacterial genome assemblies by BLASTn to identify the nearest reference genome. Results: Based on the preliminary identifications, a total of 99 bacterial isolates were identified to species level, from which 8 isolates were selected for whole-genome sequencing to assess the identification results. Novel species and strains were discovered within this set of isolates. The preliminary identifications obtained by reduced-representation genotyping, as well as identifications obtained by BLASTn alignment of the 16S rRNA gene sequence, were compared with those derived from the whole-genome sequence data, using the same RefSeq sequence database for the three methods. Identifications obtained with reduced-representation sequencing agreed with the identifications provided by whole-genome sequencing in 100% of cases. The identifications produced by BLASTn alignment of 16S rRNA gene sequence to the same database differed from those provided by whole-genome sequencing in 37.5% of cases, and produced ambiguous identifications in 50% of cases. Conclusions: Previously, this method has been successfully demonstrated for use in bacterial identification for medical microbiology. This study demonstrates the first successful use of DArTseq™ for preliminary identification of thermophilic bacterial isolates, providing results in complete agreement with those obtained from whole-genome sequencing of the same isolates. The growing database of bacterial genome sequences provides an excellent resource for alignment of reduced-representation sequence data for identification purposes, and as the available sequenced genomes continue to grow, the technique will become more effective.

Phylogenetic study of thermophilic genera Anoxybacillus, Geobacillus, Parageobacillus, and proposal of a new classification Quasigeobacillus gen. nov.

“…The placement of the seven genomes derived from the study of thermophilic bacteria in Australia [50] was determined on the basis of their relative position within the core gene maximum likelihood tree and the ANI and AAI values. These genomes had genome sizes ranging between 2.73 to 4.69 Mb, a minimum GC content of 41.81% and a maximum of 55.27%.…”

“…The genus Parageobacillus was represented in the analysis by the genome assemblies of the seven validly described Parageobacillus species, with one representative assembly per species. Additionally, whole genome sequences of two Anoxybacillus , four Geobacillus and one Parageobacillus strains obtained from a study of thermophiles in Australia [50] were included. This group contained 31 chromosome level assemblies, 33 scaffold genomes and 49 genomes up to the contig level.…”

“…Here we report a phylogenetic analysis and comparative genomic analysis of the closely related thermophilic genera Anoxybacillus, Geobacillus and Parageobacillus , as well as determining the preliminary placement of 7 newly isolated strains of these genera [50]. The study was performed using 113 genome assemblies of Anoxybacillus, Geobacillus and Parageobacillus available on RefSeq database [51].…”