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DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure.

Rubin BE, Sanders JG, Hampton-Marcell J, Owens SM, Gilbert JA, Moreau CS - Microbiologyopen (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Committee on Evolutionary Biology, University of Chicago, Chicago, Illinois; Department of Science and Education, Field Museum of Natural History, Chicago, Illinois.

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Principal coordinate plots of unweighted UniFrac distances between all sequenced samples (A), Pseudomyrmex flavicornis samples (B), Pseudomyrmex nigrocinctus samples (C), and Cephalotes varians samples (D). Colors correspond to different species in panel A and different colonies in B–D. Adults and larvae are represented as open and filled symbols, respectively.
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fig04: Principal coordinate plots of unweighted UniFrac distances between all sequenced samples (A), Pseudomyrmex flavicornis samples (B), Pseudomyrmex nigrocinctus samples (C), and Cephalotes varians samples (D). Colors correspond to different species in panel A and different colonies in B–D. Adults and larvae are represented as open and filled symbols, respectively.

Mentions: No pattern was apparent between extraction protocols in PCoA plots using unweighted UniFrac distances between samples (Fig. 4), but differences between species, colonies, and life stages were clear. Neither ANOSIM nor PERMANOVA analyses found significant differences between extraction methodologies when all samples were included (P > 0.1; Fig. 4), regardless of the beta diversity metric used. The lack of significant effect held true when only samples within individual species were compared (P > 0.1; Fig. 4). However, Species hosted significantly different communities according to all beta diversity metrics (ANOSIM, PERMANOVA, P = 0.001; Fig. 4). Within species, colonies hosted significantly different communities (P < 0.005; Fig. 4). While extractions from three abdomens and one abdomen were not significantly different for P. nigrocinctus or Cephalotes varians (P > 0.1), there were significant differences between the larval and adult extractions within P. flavicornis (P = 0.001; Fig. 4). The results from all statistical tests of beta diversity differences can be found in Table S4.


DNA extraction protocols cause differences in 16S rRNA amplicon sequencing efficiency but not in community profile composition or structure.

Rubin BE, Sanders JG, Hampton-Marcell J, Owens SM, Gilbert JA, Moreau CS - Microbiologyopen (2014)

Principal coordinate plots of unweighted UniFrac distances between all sequenced samples (A), Pseudomyrmex flavicornis samples (B), Pseudomyrmex nigrocinctus samples (C), and Cephalotes varians samples (D). Colors correspond to different species in panel A and different colonies in B–D. Adults and larvae are represented as open and filled symbols, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263514&req=5

fig04: Principal coordinate plots of unweighted UniFrac distances between all sequenced samples (A), Pseudomyrmex flavicornis samples (B), Pseudomyrmex nigrocinctus samples (C), and Cephalotes varians samples (D). Colors correspond to different species in panel A and different colonies in B–D. Adults and larvae are represented as open and filled symbols, respectively.
Mentions: No pattern was apparent between extraction protocols in PCoA plots using unweighted UniFrac distances between samples (Fig. 4), but differences between species, colonies, and life stages were clear. Neither ANOSIM nor PERMANOVA analyses found significant differences between extraction methodologies when all samples were included (P > 0.1; Fig. 4), regardless of the beta diversity metric used. The lack of significant effect held true when only samples within individual species were compared (P > 0.1; Fig. 4). However, Species hosted significantly different communities according to all beta diversity metrics (ANOSIM, PERMANOVA, P = 0.001; Fig. 4). Within species, colonies hosted significantly different communities (P < 0.005; Fig. 4). While extractions from three abdomens and one abdomen were not significantly different for P. nigrocinctus or Cephalotes varians (P > 0.1), there were significant differences between the larval and adult extractions within P. flavicornis (P = 0.001; Fig. 4). The results from all statistical tests of beta diversity differences can be found in Table S4.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Committee on Evolutionary Biology, University of Chicago, Chicago, Illinois; Department of Science and Education, Field Museum of Natural History, Chicago, Illinois.

Show MeSH