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Open Access Highly Accessed Methodology

An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform

Douglas W Fadrosh1, Bing Ma1, Pawel Gajer1, Naomi Sengamalay1, Sandra Ott1, Rebecca M Brotman2 and Jacques Ravel1*

Author Affiliations

1 Institute for Genome Sciences, Department of Microbiology and Immunology, University of Maryland School of Medicine, 801 W. Baltimore Street, Baltimore, MD 21201, USA

2 Institute for Genome Sciences, Department of Epidemiology and Public Health, University of Maryland School of Medicine, 801 W. Baltimore Street, Baltimore, MD 21201, USA

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Microbiome 2014, 2:6  doi:10.1186/2049-2618-2-6

Published: 24 February 2014

Abstract

Background

To take advantage of affordable high-throughput next-generation sequencing technologies to characterize microbial community composition often requires the development of improved methods to overcome technical limitations inherent to the sequencing platforms. Sequencing low sequence diversity libraries such as 16S rRNA amplicons has been problematic on the Illumina MiSeq platform and often generates sequences of suboptimal quality.

Results

Here we present an improved dual-indexing amplification and sequencing approach to assess the composition of microbial communities from clinical samples using the V3-V4 region of the 16S rRNA gene on the Illumina MiSeq platform. We introduced a 0 to 7 bp “heterogeneity spacer” to the index sequence that allows an equal proportion of samples to be sequenced out of phase.

Conclusions

Our approach yields high quality sequence data from 16S rRNA gene amplicons using both 250 bp and 300 bp paired-end MiSeq protocols and provides a flexible and cost-effective sequencing option.