title: DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure.
abstract: The recent development of methods applying next-generation sequencing to microbial community characterization has led to the proliferation of these studies in a wide variety of sample types. Yet, variation in the physical properties of environmental samples demands that optimal DNA extraction techniques be explored for each new environment. The microbiota associated with many species of insects offer an extraction challenge as they are frequently surrounded by an armored exoskeleton, inhibiting disruption of the tissues within. In this study, we examine the efficacy of several commonly used protocols for extracting bacterial DNA from ants. While bacterial community composition recovered using Illumina 16S rRNA amplicon sequencing was not detectably biased by any method, the quantity of bacterial DNA varied drastically, reducing the number of samples that could be amplified and sequenced. These results indicate that the concentration necessary for dependable sequencing is around 10,000 copies of target DNA per microliter. Exoskeletal pulverization and tissue digestion increased the reliability of extractions, suggesting that these steps should be included in any study of insect-associated microorganisms that relies on obtaining microbial DNA from intact body segments. Although laboratory and analysis techniques should be standardized across diverse sample types as much as possible, minimal modifications such as these will increase the number of environments in which bacterial communities can be successfully studied.
authors.affiliation: Committee on Evolutionary Biology, University of Chicago, Chicago, Illinois; Department of Science and Education, Field Museum of Natural History, Chicago, Illinois.
authors.forename: Benjamin E R
authors.initials: BE
authors.lastname: Rubin
authors.forename: Jon G
authors.initials: JG
authors.lastname: Sanders
authors.forename: Jarrad
authors.initials: J
authors.lastname: Hampton-Marcell
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Owens
authors.forename: Jack A
authors.initials: JA
authors.lastname: Gilbert
authors.forename: Corrie S
authors.initials: CS
authors.lastname: Moreau
mesh_terms: D000818:Animals; D001000:Ants / Q000382:microbiology*; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D044822:Biodiversity; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification*; D059014:High-Throughput Nucleotide Sequencing; D064307:Microbiota; D012336:RNA, Ribosomal, 16S / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
keywords: 16S rRNA; DNA extraction; Earth Microbiome Project; ants; host-associated bacteria; insects; microbiome