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Symbiosis. 2012 Dec;58(1-3):201-207. Epub 2013 Jan 6.

Targeted genome enrichment for efficient purification of endosymbiont DNA from host DNA.

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1
New England Biolabs, Inc., Ipswich, MA 01938 USA ; Écologie et Biologie des Interactions, Équipe Écologie, Évolution, Symbiose, University of Poitiers UMR CNRS 7267, 86022 Poitiers, France.

Abstract

Wolbachia endosymbionts are widespread in arthropods and are generally considered reproductive parasites, inducing various phenotypes including cytoplasmic incompatibility, parthenogenesis, feminization and male killing, which serve to promote their spread through populations. In contrast, Wolbachia infecting filarial nematodes that cause human diseases, including elephantiasis and river blindness, are obligate mutualists. DNA purification methods for efficient genomic sequencing of these unculturable bacteria have proven difficult using a variety of techniques. To efficiently capture endosymbiont DNA for studies that examine the biology of symbiosis, we devised a parallel strategy to an earlier array-based method by creating a set of SureSelect™ (Agilent) 120-mer target enrichment RNA oligonucleotides ("baits") for solution hybrid selection. These were designed from Wolbachia complete and partial genome sequences in GenBank and were tiled across each genomic sequence with 60 bp overlap. Baits were filtered for homology against host genomes containing Wolbachia using BLAT and sequences with significant host homology were removed from the bait pool. Filarial parasite Brugia malayi DNA was used as a test case, as the complete sequence of both Wolbachia and its host are known. DNA eluted from capture was size selected and sequencing samples were prepared using the NEBNext® Sample Preparation Kit. One-third of a 50 nt paired-end sequencing lane on the HiSeq™ 2000 (Illumina) yielded 53 million reads and the entirety of the Wolbachia genome was captured. We then used the baits to isolate more than 97.1 % of the genome of a distantly related Wolbachia strain from the crustacean Armadillidium vulgare, demonstrating that the method can be used to enrich target DNA from unculturable microbes over large evolutionary distances.

KEYWORDS:

DNA capture; NextGen sequencing; Obligate endosymbiont; SureSelect™; Target enrichment; Wolbachia

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