title: Targeted genome enrichment for efficient purification of endosymbiont DNA from host DNA.
abstract: Wolbachiaendosymbionts 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,Wolbachiainfecting 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 fromWolbachiacomplete 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 containingWolbachiausing BLAT and sequences with significant host homology were removed from the bait pool. Filarial parasiteBrugia malayiDNA was used as a test case, as the complete sequence of bothWolbachiaand 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 theWolbachiagenome was captured. We then used the baits to isolate more than 97.1 % of the genome of a distantly relatedWolbachiastrain from the crustaceanArmadillidium vulgare, demonstrating that the method can be used to enrich target DNA from unculturable microbes over large evolutionary distances.
authors.affiliation: 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.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Geniez
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Foster
authors.forename: Sanjay
authors.initials: S
authors.lastname: Kumar
authors.forename: Bouziane
authors.initials: B
authors.lastname: Moumen
authors.forename: Emily
authors.initials: E
authors.lastname: Leproust
authors.forename: Owen
authors.initials: O
authors.lastname: Hardy
authors.forename: Moraima
authors.initials: M
authors.lastname: Guadalupe
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Thomas
authors.forename: Braden
authors.initials: B
authors.lastname: Boone
authors.forename: Cynthia
authors.initials: C
authors.lastname: Hendrickson
authors.forename: Didier
authors.initials: D
authors.lastname: Bouchon
authors.forename: Pierre
authors.initials: P
authors.lastname: Grève
authors.forename: Barton E
authors.initials: BE
authors.lastname: Slatko
keywords: DNA capture; NextGen sequencing; Obligate endosymbiont; SureSelect™; Target enrichment; Wolbachia