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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. Dept. of Energy Joint Genome Institute, Walnut Creek, CA (United States); National Research Council Canada, Montreal, QC (Canada)
  2. Dept. of Energy Joint Genome Institute, Walnut Creek, CA (United States)
  3. Illumina, Inc.,San Francisco, CA (United States)
  4. Univ. of North Carolina, Chapel Hill, NC (United States). Carolina Center for Genome Sciences
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) (SC-23)
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 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: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: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 = ,
volume = 6,
place = {United States},
year = {2015},
month = {8}
}

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Cited by: 62 works
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Works referenced in this record:

PCR-Induced Sequence Artifacts and Bias: Insights from Comparison of Two 16S rRNA Clone Libraries Constructed from the Same Sample
journal, December 2005


Review and re-analysis of domain-specific 16S primers
journal, December 2003


Summarizing and correcting the GC content bias in high-throughput sequencing
journal, February 2012

  • Benjamini, Yuval; Speed, Terence P.
  • Nucleic Acids Research, Vol. 40, Issue 10
  • DOI: 10.1093/nar/gks001

Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota
journal, August 2012

  • Bulgarelli, Davide; Rott, Matthias; Schlaeppi, Klaus
  • Nature, Vol. 488, Issue 7409
  • DOI: 10.1038/nature11336

QIIME allows analysis of high-throughput community sequencing data
journal, April 2010

  • Caporaso, J. Gregory; Kuczynski, Justin; Stombaugh, Jesse
  • Nature Methods, Vol. 7, Issue 5
  • DOI: 10.1038/nmeth.f.303

Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms
journal, March 2012

  • Caporaso, J. Gregory; Lauber, Christian L.; Walters, William A.
  • The ISME Journal, Vol. 6, Issue 8
  • DOI: 10.1038/ismej.2012.8

Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample
journal, June 2010

  • Caporaso, J. G.; Lauber, C. L.; Walters, W. A.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue Supplement_1
  • DOI: 10.1073/pnas.1000080107

Effects of GC Bias in Next-Generation-Sequencing Data on De Novo Genome Assembly
journal, April 2013


Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions
journal, September 2010

  • Claesson, Marcus J.; Wang, Qiong; O'Sullivan, Orla
  • Nucleic Acids Research, Vol. 38, Issue 22
  • DOI: 10.1093/nar/gkq873

Illumina-based analysis of microbial community diversity
journal, June 2011


Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB
journal, July 2006

  • DeSantis, T. Z.; Hugenholtz, P.; Larsen, N.
  • Applied and Environmental Microbiology, Vol. 72, Issue 7, p. 5069-5072
  • DOI: 10.1128/AEM.03006-05

MUSCLE: multiple sequence alignment with high accuracy and high throughput
journal, March 2004

  • Edgar, R. C.
  • Nucleic Acids Research, Vol. 32, Issue 5, p. 1792-1797
  • DOI: 10.1093/nar/gkh340

Search and clustering orders of magnitude faster than BLAST
journal, August 2010


UCHIME improves sensitivity and speed of chimera detection
journal, June 2011


Experimental factors affecting PCR-based estimates of microbial species richness and evenness
journal, January 2010

  • Engelbrektson, Anna; Kunin, Victor; Wrighton, Kelly C.
  • The ISME Journal, Vol. 4, Issue 5
  • DOI: 10.1038/ismej.2009.153

The Long-Term Stability of the Human Gut Microbiota
journal, July 2013

  • Faith, Jeremiah J.; Guruge, Janaki L.; Charbonneau, Mark
  • Science, Vol. 341, Issue 6141
  • DOI: 10.1126/science.1237439

Critical Evaluation of Two Primers Commonly Used for Amplification of Bacterial 16S rRNA Genes
journal, February 2008

  • Frank, J. A.; Reich, C. I.; Sharma, S.
  • Applied and Environmental Microbiology, Vol. 74, Issue 8
  • DOI: 10.1128/AEM.02272-07

Meeting Report: The Terabase Metagenomics Workshop and the Vision of an Earth Microbiome Project
journal, January 2010

  • Gilbert, Jack A.; Meyer, Folker; Antonopoulos, Dion
  • Standards in Genomic Sciences, Vol. 3, Issue 3
  • DOI: 10.4056/sigs.1433550

Generalized procrustes analysis
journal, March 1975


Patterns in Wetland Microbial Community Composition and Functional Gene Repertoire Associated with Methane Emissions
journal, May 2015

  • He, Shaomei; Malfatti, Stephanie A.; McFarland, Jack W.
  • mBio, Vol. 6, Issue 3
  • DOI: 10.1128/mBio.00066-15

Comparison of microbial diversity determined with the same variable tag sequence extracted from two different PCR amplicons
journal, January 2013


Polymerase chain reaction primers miss half of rRNA microbial diversity
journal, August 2009

  • Hong, SunHee; Bunge, John; Leslin, Chesley
  • The ISME Journal, Vol. 3, Issue 12
  • DOI: 10.1038/ismej.2009.89

Sequencing Intractable DNA to Close Microbial Genomes
journal, July 2012


Accuracy and quality of massively parallel DNA pyrosequencing
journal, January 2007


Specificity and sensitivity of eubacterial primers utilized for molecular profiling of bacteria within complex microbial ecosystems
journal, September 2007


Evaluation of different partial 16S rRNA gene sequence regions for phylogenetic analysis of microbiomes
journal, January 2011


Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies
journal, August 2012

  • Klindworth, Anna; Pruesse, Elmar; Schweer, Timmy
  • Nucleic Acids Research, Vol. 41, Issue 1
  • DOI: 10.1093/nar/gks808

Development of a Dual-Index Sequencing Strategy and Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina Sequencing Platform
journal, June 2013

  • Kozich, James J.; Westcott, Sarah L.; Baxter, Nielson T.
  • Applied and Environmental Microbiology, Vol. 79, Issue 17
  • DOI: 10.1128/AEM.01043-13

Using QIIME to Analyze 16S rRNA Gene Sequences from Microbial Communities
journal, December 2011

  • Kuczynski, Justin; Stombaugh, Jesse; Walters, William Anton
  • Current Protocols in Bioinformatics, Vol. 36, Issue 1
  • DOI: 10.1002/0471250953.bi1007s36

Target Region Selection Is a Critical Determinant of Community Fingerprints Generated by 16S Pyrosequencing
journal, June 2011


Wrinkles in the rare biosphere: pyrosequencing errors can lead to artificial inflation of diversity estimates
journal, January 2010


Reevaluation and Reduction of a PCR Bias Caused by Reannealing of Templates
journal, December 2004


RNAmmer: consistent and rapid annotation of ribosomal RNA genes
journal, April 2007

  • Lagesen, Karin; Hallin, Peter; Rødland, Einar Andreas
  • Nucleic Acids Research, Vol. 35, Issue 9
  • DOI: 10.1093/nar/gkm160

Performance comparison of benchtop high-throughput sequencing platforms
journal, April 2012

  • Loman, Nicholas J.; Misra, Raju V.; Dallman, Timothy J.
  • Nature Biotechnology, Vol. 30, Issue 5
  • DOI: 10.1038/nbt.2198

UniFrac: a New Phylogenetic Method for Comparing Microbial Communities
journal, December 2005


Defining the core Arabidopsis thaliana root microbiome
journal, August 2012

  • Lundberg, Derek S.; Lebeis, Sarah L.; Paredes, Sur Herrera
  • Nature, Vol. 488, Issue 7409
  • DOI: 10.1038/nature11237

Practical innovations for high-throughput amplicon sequencing
journal, September 2013

  • Lundberg, Derek S.; Yourstone, Scott; Mieczkowski, Piotr
  • Nature Methods, Vol. 10, Issue 10
  • DOI: 10.1038/nmeth.2634

FLASH: fast length adjustment of short reads to improve genome assemblies
journal, September 2011


Genome sequencing in microfabricated high-density picolitre reactors
journal, July 2005

  • Margulies, Marcel; Egholm, Michael; Altman, William E.
  • Nature, Vol. 437, Issue 7057, p. 376-380
  • DOI: 10.1038/nature03959

Deciphering the Rhizosphere Microbiome for Disease-Suppressive Bacteria
journal, May 2011


Analysis, Optimization and Verification of Illumina-Generated 16S rRNA Gene Amplicon Surveys
journal, April 2014


A Molecular View of Microbial Diversity and the Biosphere
journal, May 1997


A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing
journal, October 2007

  • Parameswaran, Poornima; Jalili, Roxana; Tao, Li
  • Nucleic Acids Research, Vol. 35, Issue 19
  • DOI: 10.1093/nar/gkm760

Diversity and heritability of the maize rhizosphere microbiome under field conditions
journal, April 2013

  • Peiffer, J. A.; Spor, A.; Koren, O.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 16
  • DOI: 10.1073/pnas.1302837110

PCR Biases Distort Bacterial and Archaeal Community Structure in Pyrosequencing Datasets
journal, August 2012


FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments
journal, March 2010


Evaluation of PCR-Generated Chimeras, Mutations, and Heteroduplexes with 16S rRNA Gene-Based Cloning
journal, February 2001


The SILVA ribosomal RNA gene database project: improved data processing and web-based tools
journal, November 2012

  • Quast, Christian; Pruesse, Elmar; Yilmaz, Pelin
  • Nucleic Acids Research, Vol. 41, Issue D1
  • DOI: 10.1093/nar/gks1219

Performance Comparison of Illumina and Ion Torrent Next-Generation Sequencing Platforms for 16S rRNA-Based Bacterial Community Profiling
journal, September 2014

  • Salipante, Stephen J.; Kawashima, Toana; Rosenthal, Christopher
  • Applied and Environmental Microbiology, Vol. 80, Issue 24
  • DOI: 10.1128/AEM.02206-14

Microbial diversity in the deep sea and the underexplored "rare biosphere"
journal, July 2006

  • Sogin, M. L.; Morrison, H. G.; Huber, J. A.
  • Proceedings of the National Academy of Sciences, Vol. 103, Issue 32
  • DOI: 10.1073/pnas.0605127103

A renaissance for the pioneering 16S rRNA gene
journal, October 2008


Naive Bayesian Classifier for Rapid Assignment of rRNA Sequences into the New Bacterial Taxonomy
journal, June 2007

  • Wang, Q.; Garrity, G. M.; Tiedje, J. M.
  • Applied and Environmental Microbiology, Vol. 73, Issue 16
  • DOI: 10.1128/AEM.00062-07

Phylogenetic structure of the prokaryotic domain: The primary kingdoms
journal, November 1977

  • Woese, C. R.; Fox, G. E.
  • Proceedings of the National Academy of Sciences, Vol. 74, Issue 11
  • DOI: 10.1073/pnas.74.11.5088

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    journal, June 2019

    • Pathirana, E.; McPherson, A.; Whittington, R.
    • Journal of Applied Microbiology, Vol. 127, Issue 2
    • DOI: 10.1111/jam.14326

    Deciphering the associations between soil microbial diversity and ecosystem multifunctionality driven by long-term fertilization management
    journal, January 2018


    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
    • DOI: 10.1002/mbo3.898

    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
    • DOI: 10.1007/s00374-017-1205-1

    NG-Tax, a highly accurate and validated pipeline for analysis of 16S rRNA amplicons from complex biomes
    journal, January 2016


    What's Normal? Microbiomes in Human Milk and Infant Feces Are Related to Each Other but Vary Geographically: The INSPIRE Study
    journal, April 2019

    • Lackey, Kimberly A.; Williams, Janet E.; Meehan, Courtney L.
    • Frontiers in Nutrition, Vol. 6
    • DOI: 10.3389/fnut.2019.00045

    NG-Tax, a highly accurate and validated pipeline for analysis of 16S rRNA amplicons from complex biomes
    journal, January 2016


    Effects of Ice-Algal Aggregate Export on the Connectivity of Bacterial Communities in the Central Arctic Ocean
    journal, May 2018

    • Rapp, Josephine Z.; Fernández-Méndez, Mar; Bienhold, Christina
    • Frontiers in Microbiology, Vol. 9
    • DOI: 10.3389/fmicb.2018.01035

    Molecular Resources from Transcriptomes in the Brassicaceae Family
    journal, August 2017


    How conserved are the conserved 16S-rRNA regions?
    journal, January 2017

    • Martinez-Porchas, Marcel; Villalpando-Canchola, Enrique; Ortiz Suarez, Luis Enrique
    • PeerJ, Vol. 5
    • DOI: 10.7717/peerj.3036