Easy and Effective Method for Extracting and Purifying Wolbachia Genomic DNA

A number of methods for extracting the DNA of maternally inherited obligate intracellular bacteria Wolbachia from an insect host and its subsequent purification have been described in previous scholarship. As Wolbachia is present in the hosts' organisms in rather low quantities, these techniques used to be quite labor-intensive. For this paper, we analyzed them in detail, searched for a possibility to simplify and accelerate the protocol, and proposed an easy and effective method for isolating Wolbachia DNA from Drosophila melanogaster with a purity sufficient for genomic sequencing. Our method involves the centrifugation of homogenized flies or just their ovaries, as the most Wolbachia-enriched tissue, followed by the filtration of homogenate and extraction of DNA using a modified version of the Livak buffer protocol. The proportion of Wolbachia DNA in the total DNA was quantified based on the results of sequencing with the use of the Illumina MiSeq platform and a pipeline of bioinformatic analysis. For the two analyzed D. melanogaster lines infected with two different Wolbachia strains, the proportion was at least 68 and 94%, respectively.

Automated summary

Several methods for extracting Wolbachia DNA from insect hosts have been described, but the low quantity of the bacteria made these techniques labor-intensive. This paper proposes a simplified and accelerated method for isolating high-purity Wolbachia DNA from Drosophila melanogaster, using centrifugation, filtration, and a modified version of the Livak buffer protocol. The proportion of Wolbachia DNA in total DNA was quantified using sequencing and bioinformatic analysis.