Sequencing and enrichment of Candidatus Liberibacter asiaticus from its psyllid host using filtration and host DNA removal

A barrier to cost-effective sequence analysis of obligately biotrophic bacterial mutualists or pathogens is host DNA contamination. The pathogen 'Candidatus Liberibacter asiaticus' (CLas), whose growth in a variety of citrus species results in citrus greening disease (huanglongbing), is a prime example of a bacterium whose host-associated lifestyle has presented a significant challenge to generating genome sequences for comparative genomic analyses or antibiotic resistance screening. In addition to citrus, CLas also replicates within and is transmitted by the Asian citrus psyllid, Diaphorina citri, to citrus when the insect feeds. While methods to enrich and sequence CLas from the Asian citrus psyllid exist, they are laborious, costly, and result in biases (e.g., during multiple displacement amplification) that limit the utility of the resulting genomes for downstream use. To overcome these issues, we've developed a simple homogenization, filtration, and DNase-treatment methodology to directly sequence CLas from individual Diaphorina citri adults and have shown that CLas comprises upwards of 60% of the total sequencing reads from these samples. Furthermore, enrichment of other D. citri bacterial endosymbionts was achieved, highlighting the applicability of this approach as a simple yet comprehensive method to simultaneously explore multiple host-associated bacterial populations. While CLas enrichment and sequencing was more effective in lab-reared D. citri individuals (in which CLas reaches high titers), we leveraged this method to sequence DNA from wild-caught D. citri individuals from a Florida citrus grove and were able to sequence and assemble nearly complete citrus mitochondrial and plastid genomes, fungal mitochondrial genomes, and bacterial genomes associated with wild D. citri populations. Hence, results from the application of this method can directly inform agricultural management strategies involving surveillance of insect populations related to food preferences and their microbiomes.