Inside Job: Targeted Assembly of Obligate Intracellular Bacterial Genomes

Obligate intracellular bacteria (OIB), unlike other bacterial genera, are entirely dependent upon their eukaryotic hosts for survival. Members of this group include species from the Chlamydia, Anaplasma, Rickettsia, and Coxiella genera, many of which are pathogenic to humans. Approximately one billion people are at risk of infection with OIBs, however the mechanisms these bacteria employ to survive within the host cell are not well understood. Reconstituting the genomes of OIBs can elucidate the unique host-pathogen interactions that take place to allow these bacteria to effectively replicate in their host cells. Due to their absolute dependence on their hosts, however, genome sequencing and assembly of OIBs can be complicated by the inability to separate bacterial sequences from host DNA. 

Here, we present the ATCC's obligate intracellular bacterial assembly pipeline. Briefly, short Illumina and long Oxford Nanopore Technologies (ONT) reads are both employed to produce accurate hybrid assemblies. Both sets of reads are first trimmed according to ATCC's quality standards. The trimmed reads then enter an initial classification stage, invoking an extensive and custom-designed host genome database to reduce the amount of confounding sequence due to eukaryotic DNA. The resulting reads then follow the same standard bacterial assembly procedure. The findings presented here display the success of this method, focusing on Chlamydia, Pisirickettsia, and Rickettsia species.