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:
-
- USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States); National Research Council Canada, Montreal, QC (Canada)
- USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
- Illumina, Inc., San Francisco, CA (United States)
- 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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journal, August 2018
- Hod, Keren; Dekel, Roy; Aviv Cohen, Nathaniel
- Neurogastroenterology & Motility, Vol. 30, Issue 12
Exposure to Crude Oil and Chemical Dispersant May Impact Marine Microbial Biofilm Composition and Steel Corrosion
journal, June 2018
- Salerno, Jennifer L.; Little, Brenda; Lee, Jason
- Frontiers in Marine Science, Vol. 5
NG-Tax, a highly accurate and validated pipeline for analysis of 16S rRNA amplicons from complex biomes
journal, January 2016
- Ramiro-Garcia, Javier; Hermes, Gerben D. A.; Giatsis, Christos
- F1000Research, Vol. 5
Analysis of soil microbial communities based on amplicon sequencing of marker genes
journal, May 2017
- Schöler, Anne; Jacquiod, Samuel; Vestergaard, Gisle
- Biology and Fertility of Soils, Vol. 53, Issue 5
NG-Tax, a highly accurate and validated pipeline for analysis of 16S rRNA amplicons from complex biomes
journal, January 2016
- Ramiro-Garcia, Javier; Hermes, Gerben D. A.; Giatsis, Christos
- F1000Research, Vol. 5
Molecular Resources from Transcriptomes in the Brassicaceae Family
journal, August 2017
- Lopez, Lua; Wolf, Eva M.; Pires, J. Chris
- Frontiers in Plant Science, Vol. 8
The role of tissue type, sampling and nucleic acid purification methodology on the inferred composition of Pacific oyster ( Crassostrea gigas ) microbiome
journal, June 2019
- Pathirana, E.; McPherson, A.; Whittington, R.
- Journal of Applied Microbiology, Vol. 127, Issue 2
Full‐length 16S rRNA gene classification of Atlantic salmon bacteria and effects of using different 16S variable regions on community structure analysis
journal, July 2019
- Klemetsen, Terje; Willassen, Nils Peder; Karlsen, Christian René
- MicrobiologyOpen, Vol. 8, Issue 10
Functional Gene Array-Based Ultrasensitive and Quantitative Detection of Microbial Populations in Complex Communities
journal, June 2019
- Shi, Zhou; Yin, Huaqun; Van Nostrand, Joy D.
- mSystems, Vol. 4, Issue 4
A common garden experiment with Porphyra umbilicalis (Rhodophyta) evaluates methods to study spatial differences in the macroalgal microbiome
journal, August 2018
- Quigley, Charlotte T. C.; Morrison, Hilary G.; Mendonça, Inara R.
- Journal of Phycology, Vol. 54, Issue 5
Impact of Different Fecal Processing Methods on Assessments of Bacterial Diversity in the Human Intestine
journal, October 2016
- Hsieh, Yu-Hsin; Peterson, Courtney M.; Raggio, Anne
- Frontiers in Microbiology, Vol. 7
Full‐length 16S rRNA gene classification of Atlantic salmon bacteria and effects of using different 16S variable regions on community structure analysis
journal, July 2019
- Klemetsen, Terje; Willassen, Nils Peder; Karlsen, Christian René
- MicrobiologyOpen, Vol. 8, Issue 10
The role of tissue type, sampling and nucleic acid purification methodology on the inferred composition of Pacific oyster ( Crassostrea gigas ) microbiome
journal, June 2019
- Pathirana, E.; McPherson, A.; Whittington, R.
- Journal of Applied Microbiology, Vol. 127, Issue 2
Molecular diet analysis of Anguilliformes leptocephalus larvae collected in the western North Pacific
journal, November 2019
- Chow, Seinen; Inaba, Nobuharu; Nagai, Satoshi
- PLOS ONE, Vol. 14, Issue 11
Uncovering the hidden marine sponge microbiome by applying a multi-primer approach
journal, April 2019
- Yang, Qi; Franco, Christopher M. M.; Zhang, Wei
- Scientific Reports, Vol. 9, Issue 1
Characterizing the Composition of the Pediatric Gut Microbiome: A Systematic Review
journal, December 2019
- Deering, Kane E.; Devine, Amanda; O’Sullivan, Therese A.
- Nutrients, Vol. 12, Issue 1
The Madness of Microbiome: Attempting To Find Consensus “Best Practice” for 16S Microbiome Studies
journal, February 2018
- Pollock, Jolinda; Glendinning, Laura; Wisedchanwet, Trong
- Applied and Environmental Microbiology, Vol. 84, Issue 7
Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.