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:

Tremblay, Julien ^[1]; Singh, Kanwar ^[2]; Fern, Alison ^[2]; Kirton, Edward S. ^[2]; He, Shaomei ^[2]; Woyke, Tanja ^[2]; Lee, Janey ^[2]; Chen, Feng ^[3]; Dangl, Jeffery L. ^[4]; Tringe, Susannah G. ^[2]

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+ Show Author Affiliations

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:

2015-08-04

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