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Updated: May 2, 2026
Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
Published on: August 29, 2014
An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform
Douglas W Fadrosh, Bing Ma, Pawel Gajer
1Institute for Genome Sciences, Department of Microbiology and Immunology, University of Maryland School of Medicine, 801 W, Baltimore Street, Baltimore, MD 21201, USA. jravel@som.umaryland.edu.
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.
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