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    Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton (2015)
    Inter-Research
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    Publication Date: 2022-05-25
    Description: Author Posting. © Inter-Research, 2015. This article is posted here by permission of Inter-Research for personal use, not for redistribution. The definitive version was published in Aquatic Microbial Ecology 75 (2015): 129-137, doi:10.3354/ame01753.
    Description: High-throughput sequencing of small subunit ribosomal RNA (SSU rRNA) genes from marine environments is a widely applied method used to uncover the composition of microbial communities. We conducted an analysis of surface ocean waters with the commonly employed hypervariable 4 region SSU rRNA gene primers 515F and 806R, and found that bacteria belonging to the SAR11 clade of Alphaproteobacteria, a group typically making up 20 to 40% of the bacterioplankton in this environment, were greatly underrepresented and comprised 〈4% of the total community. Using the SILVA reference database, we found a single nucleotide mismatch to nearly all SAR11 subclades, and revised the 806R primer so that it increased the detection of SAR11 clade sequences in the database from 2.6 to 96.7%. We then compared the performance of the original and revised 806R primers in surface seawater samples, and found that SAR11 comprised 0.3 to 3.9% of sequences with the original primers and 17.5 to 30.5% of the sequences with the revised 806R primer. Furthermore, an investigation of seawater obtained from aquaria revealed that SAR11 sequences acquired with the revised 806R primer were more similar to natural cellular abundances of SAR11 detected using fluorescence in situ hybridization counts. Collectively, these results demonstrate that a minor adjustment to the 806R primer will greatly increase detection of the globally abundant SAR11 clade in marine and lake environments, and enable inclusion of this important bacterial lineage in experimental and environmental-based studies.
    Description: This project was supported by NSF award OCE-1233612 to A.A. with contributions from BIOS Grant in aid award to S.McN. and NSF Oceanic Microbial Observatory OCE-0801991 subcontract to BIOS managed by R.P.
    Keywords: SSU rRNA gene ; 16S ; SAR11 ; Bacteria ; Fluorescence in situ hybridization ; FISH
    Repository Name: Woods Hole Open Access Server
    Type: Article
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    Format: application/pdf
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  • 2
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    Improved bacterial 16S rRNA gene (V4 and V4-5) and fungal internal transcribed spacer marker gene primers for microbial community surveys (2017)
    American Society for Microbiology
    Details
    Publication Date: 2022-05-26
    Description: © The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in mSystems 1 (2015): e00009-15, doi:10.1128/mSystems.00009-15.
    Description: Designing primers for PCR-based taxonomic surveys that amplify a broad range of phylotypes in varied community samples is a difficult challenge, and the comparability of data sets amplified with varied primers requires attention. Here, we examined the performance of modified 16S rRNA gene and internal transcribed spacer (ITS) primers for archaea/bacteria and fungi, respectively, with nonaquatic samples. We moved primer bar codes to the 5′ end, allowing for a range of different 3′ primer pairings, such as the 515f/926r primer pair, which amplifies variable regions 4 and 5 of the 16S rRNA gene. We additionally demonstrated that modifications to the 515f/806r (variable region 4) 16S primer pair, which improves detection of Thaumarchaeota and clade SAR11 in marine samples, do not degrade performance on taxa already amplified effectively by the original primer set. Alterations to the fungal ITS primers did result in differential but overall improved performance compared to the original primers. In both cases, the improved primers should be widely adopted for amplicon studies.
    Description: J.A.F. and A.P. are supported by the Gordon and Betty Moore Foundation (GMBF3779) and NSF grant 1136818. A.P. is supported by an NSF Graduate Fellowship. A.A. is supported by NSF grant OCE-1233612. J.K.J. is supported by the Microbiomes in Transition Initiative LDRD Program at the Pacific Northwest National Laboratory, a multiprogram national laboratory operated by Battelle for the DOE under contract DE-AC06-76RL01830. J.A.G. is supported by the U.S. Department of Energy under contract DE-AC02-06CH11357. J.G.C., J.A.G., and R.K. are supported by the Alfred P. Sloan Foundation. R.K. is supported by the Howard Hughes Medical Institute.
    Keywords: Microbial ecology ; Marker genes ; Primers ; 16S ; ITS
    Repository Name: Woods Hole Open Access Server
    Type: Article
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