Targeted genome reconstruction strategy for endosymbionts in eukaryotic meta-genomes
The availability of low-cost high-throughput next generation sequencing technologies presents an opportunity for in-silico discovery of endosymbionts. We describe a method for mining a whole genome shotgun metagenome from an insect vector of the disease, citrus greening, to identify members of the endosymbiont community followed by reconstruction and validation of a high-quality draft microbial genome.
The Asian citrus psyllid (D. citri Kuwayama or ACP) is host to 7+ bacterial endosymbionts and is the insect vector of Ca. liberibacter asiaticus, causal agent of citrus greening, a disease that has cost the Florida citrus industry $3.63 billion since 2006. Citrus greening is a complex patho-system which involves interactions between the psyllid vector, the citrus hosts, and Ca. liberibacter asiaticus. Within the psyllids are a living fauna of microbes whose identity and functions are unknown.
DNA from D. citri was sequenced to 108X coverage to produce paired-end and mate-pair Illumina read libraries. The sequences were mined for wolbachia (wACP) reads using 4 sequenced Wolbachia genomes as bait. Putative wACP reads were then assembled using Velvet and MIRA3 assemblers. The resulting wACP contigs were annotated using the RAST and compared to the closest sequenced wolbachia from an insect genome, Wolbachia endosymbiont of Culex quinquefasciatus (wPip). MIRA3 was able to reconstruct a majority of the wPip CDS regions and was therefore, selected for scaffolding using large insert mate-pair libraries. The wACP scaffolds were further improved using Abacas and Mauve contig mover with wPip as reference genome to orient and order the contigs.
In order to determine the presence of the core Wolbachia proteins in our wACP scaffold, we compared them to core Wolbachia proteins identified by OrthoMCL. 1164/1213 wACP proteins had matches of which 669 were to core proteins. This number compares favorably to the number of core proteins (670) found in sequenced Wolbachias.