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Title: Primer and platform effects on 16S rRNA tag sequencing

Abstract

Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as well as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. In conclusion, beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.

Authors:
 [1];  [2];  [2];  [2];  [2];  [2];  [2];  [3];  [4];  [2]
  1. USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); National Research Council Canada, Montreal, QC (Canada)
  2. USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
  3. Illumina, Inc., San Francisco, CA (United States)
  4. Univ. of North Carolina, Chapel Hill, NC (United States). Dept. of Biology and Howard Hughes Medical Inst., Curriculum in Genetics and Molecular Biology, Dept. of Microbiology and Immunology
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1512103
Alternate Identifier(s):
OSTI ID: 1256969
Grant/Contract Number:  
AC02-05CH11231; KP/CH57/1; IOS-0958245
Resource Type:
Accepted Manuscript
Journal Name:
Frontiers in Microbiology
Additional Journal Information:
Journal Volume: 6; Journal Issue: AUG; Journal ID: ISSN 1664-302X
Publisher:
Frontiers Research Foundation
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 16S rRNA gene sequencing; microbial population and community ecology; high throughput sequencing; microbial diversity; community assembly; amplification; sequencing error

Citation Formats

Tremblay, Julien, Singh, Kanwar, Fern, Alison, Kirton, Edward S., He, Shaomei, Woyke, Tanja, Lee, Janey, Chen, Feng, Dangl, Jeffery L., and Tringe, Susannah G. Primer and platform effects on 16S rRNA tag sequencing. United States: N. p., 2015. Web. doi:10.3389/fmicb.2015.00771.
Tremblay, Julien, Singh, Kanwar, Fern, Alison, Kirton, Edward S., He, Shaomei, Woyke, Tanja, Lee, Janey, Chen, Feng, Dangl, Jeffery L., & Tringe, Susannah G. Primer and platform effects on 16S rRNA tag sequencing. United States. doi:https://doi.org/10.3389/fmicb.2015.00771
Tremblay, Julien, Singh, Kanwar, Fern, Alison, Kirton, Edward S., He, Shaomei, Woyke, Tanja, Lee, Janey, Chen, Feng, Dangl, Jeffery L., and Tringe, Susannah G. Tue . "Primer and platform effects on 16S rRNA tag sequencing". United States. doi:https://doi.org/10.3389/fmicb.2015.00771. https://www.osti.gov/servlets/purl/1512103.
@article{osti_1512103,
title = {Primer and platform effects on 16S rRNA tag sequencing},
author = {Tremblay, Julien and Singh, Kanwar and Fern, Alison and Kirton, Edward S. and He, Shaomei and Woyke, Tanja and Lee, Janey and Chen, Feng and Dangl, Jeffery L. and Tringe, Susannah G.},
abstractNote = {Sequencing of 16S rRNA gene tags is a popular method for profiling and comparing microbial communities. The protocols and methods used, however, vary considerably with regard to amplification primers, sequencing primers, sequencing technologies; as well as quality filtering and clustering. How results are affected by these choices, and whether data produced with different protocols can be meaningfully compared, is often unknown. Here we compare results obtained using three different amplification primer sets (targeting V4, V6–V8, and V7–V8) and two sequencing technologies (454 pyrosequencing and Illumina MiSeq) using DNA from a mock community containing a known number of species as well as complex environmental samples whose PCR-independent profiles were estimated using shotgun sequencing. We find that paired-end MiSeq reads produce higher quality data and enabled the use of more aggressive quality control parameters over 454, resulting in a higher retention rate of high quality reads for downstream data analysis. While primer choice considerably influences quantitative abundance estimations, sequencing platform has relatively minor effects when matched primers are used. In conclusion, beta diversity metrics are surprisingly robust to both primer and sequencing platform biases.},
doi = {10.3389/fmicb.2015.00771},
journal = {Frontiers in Microbiology},
number = AUG,
volume = 6,
place = {United States},
year = {2015},
month = {8}
}

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Cited by: 62 works
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FIGURE 1 FIGURE 1: Timeline indicating major breakthroughs in experimental and theoretical work in the field of 16S rRNA gene sequencing.

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    • Pollock, Jolinda; Glendinning, Laura; Wisedchanwet, Trong
    • Applied and Environmental Microbiology, Vol. 84, Issue 7
    • DOI: 10.1128/aem.02627-17