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

By D. Fadrosh, Bing Ma, Pawel Gajer, Naomi A. Sengamalay, Sandra H Ott, R. Brotman, Jacques Ravel. Microbiome, 2014

1,572 citations recorded by the source corpus.

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. 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. 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.

Subfields: Ecology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/7f8859a57e35c66164678710187d4374822411d8

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Citations in the corpus

Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys (2015, background): “This approach is advantageous over that of popular dual-bar-coding schemes (15, 16), which do not allow the flexibility that single-ended forward bar coding does, due to the necessity of a variety of bar codes for both the forward and reverse primers.”
Microbiome Helper: a Custom and Streamlined Workflow for Microbiome Research (2017, methodology): “An alternative approach that used heterogeneity spacers within the barcode provided an improvement to low sequence diversity (12) but required custom barcodes and more-complicated demultiplexing during bioinformatic analysis.”