title: A method for selectively enriching microbial DNA from contaminating vertebrate host DNA.
abstract: DNA samples derived from vertebrate skin, bodily cavities and body fluids contain both host and microbial DNA; the latter often present as a minor component. Consequently, DNA sequencing of a microbiome sample frequently yields reads originating from the microbe(s) of interest, but with a vast excess of host genome-derived reads. In this study, we used a methyl-CpG binding domain (MBD) to separate methylated host DNA from microbial DNA based on differences in CpG methylation density. MBD fused to the Fc region of a human antibody (MBD-Fc) binds strongly to protein A paramagnetic beads, forming an effective one-step enrichment complex that was used to remove human or fish host DNA from bacterial and protistan DNA for subsequent sequencing and analysis. We report enrichment of DNA samples from human saliva, human blood, a mock malaria-infected blood sample and a black molly fish. When reads were mapped to reference genomes, sequence reads aligning to host genomes decreased 50-fold, while bacterial and Plasmodium DNA sequences reads increased 8-11.5-fold. The Shannon-Wiener diversity index was calculated for 149 bacterial species in saliva before and after enrichment. Unenriched saliva had an index of 4.72, while the enriched sample had an index of 4.80. The similarity of these indices demonstrates that bacterial species diversity and relative phylotype abundance remain conserved in enriched samples. Enrichment using the MBD-Fc method holds promise for targeted microbiome sequence analysis across a broad range of sample types.
authors.affiliation: New England Biolabs Inc., Ipswich, Massachusetts, United States of America.
authors.forename: George R
authors.initials: GR
authors.lastname: Feehery
authors.forename: Erbay
authors.initials: E
authors.lastname: Yigit
authors.forename: Samuel O
authors.initials: SO
authors.lastname: Oyola
authors.forename: Bradley W
authors.initials: BW
authors.lastname: Langhorst
authors.forename: Victor T
authors.initials: VT
authors.lastname: Schmidt
authors.forename: Fiona J
authors.initials: FJ
authors.lastname: Stewart
authors.forename: Eileen T
authors.initials: ET
authors.lastname: Dimalanta
authors.forename: Linda A
authors.initials: LA
authors.lastname: Amaral-Zettler
authors.forename: Theodore
authors.initials: T
authors.lastname: Davis
authors.forename: Michael A
authors.initials: MA
authors.lastname: Quail
authors.forename: Sriharsa
authors.initials: S
authors.lastname: Pradhan
mesh_terms: D000818:Animals; D018899:CpG Islands; D004247:DNA / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D059018:DNA Contamination; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000302:isolation & purification* / Q000378:metabolism; D016054:DNA, Protozoan / Q000302:isolation & purification / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D012463:Saliva / Q000737:chemistry / Q000382:microbiology; D014714:Vertebrates
chemical_list: D004269:DNA, Bacterial; D016054:DNA, Protozoan; D004268:DNA-Binding Proteins; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; D004247:DNA