Journal
ENVIRONMENTAL MICROBIOLOGY
Volume 18, Issue 5, Pages 1403-1414Publisher
WILEY
DOI: 10.1111/1462-2920.13023
Keywords
-
Categories
Funding
- Gordon and Betty Moore Foundation [GMBF3779]
- NSF Dimensions of Biodiversity [1136818]
- National Science Foundation Graduate Research Fellowships
- Directorate For Geosciences
- Division Of Ocean Sciences [1136818] Funding Source: National Science Foundation
Ask authors/readers for more resources
Microbial community analysis via high-throughput sequencing of amplified 16S rRNA genes is an essential microbiology tool. We found the popular primer pair 515F (515F-C) and 806R greatly underestimated (e.g. SAR11) or overestimated (e.g. Gammaproteobacteria) common marine taxa. We evaluated marine samples and mock communities (containing 11 or 27 marine 16S clones), showing alternative primers 515F-Y (5'-GTGYCAGCMGCCGCGG TAA) and 926R (5'-CCGYCAATTYMTTTRAGTTT) yield more accurate estimates of mock community abundances, produce longer amplicons that can differentiate taxa unresolvable with 515F-C/806R, and amplify eukaryotic 18S rRNA. Mock communities amplified with 515F-Y/926R yielded closer observed community composition versus expected (r(2) = 0.95) compared with 515F-Y/806R (r(2) = 0.5). Unexpectedly, biases with 515F-Y/806R against SAR11 in field samples (similar to 4-10-fold) were stronger than in mock communities (similar to 2-fold). Correcting a mismatch to Thaumarchaea in the 515F-C increased their apparent abundance in field samples, but not as much as using 926R rather than 806R. With plankton samples rich in eukaryotic DNA (> 1 mu m size fraction), 18S sequences averaged similar to 17% of all sequences. A single mismatch can strongly bias amplification, but even perfectly matched primers can exhibit preferential amplification. We show that beyond in silico predictions, testing with mock communities and field samples is important in primer selection.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available