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J Basic Microbiol. 2016 Mar;56(3):321-6. doi: 10.1002/jobm.201500420. Epub 2015 Oct 1.

An extended single-index multiplexed 16S rRNA sequencing for microbial community analysis on MiSeq illumina platforms.

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Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada.
Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada.


The primary 16S rRNA sequencing protocol for microbial community analysis using Illumina platforms includes a single-indexing approach that allows pooling of hundreds of samples in each sequencing run. The protocol targets the V4 hypervariable region (HVR) of 16S rRNA using 150 bp paired-end (PE) sequencing. However, the latest improvement in Illumina chemistry has increased the read length up to 600 bp using 300 bp PE sequencing. To take advantage of the longer read length, a dual-indexing approach was previously developed for targeting different HVRs. However, due to simple working protocols, the single-index 150 bp PE approach still continues to be attractive to many researchers. Here, we described an extended single-indexing protocol for 300 bp PE illumina sequencing that targets the V3-V4 HVRs of 16S rRNA. The new primer set led to increased read length and alignment resolution, as well as increased richness and diversity of resulting microbial profile compared to that obtained from150 bp PE protocol for V4 sequencing. The β-diversity profile also differed qualitatively and quantitatively between the two approaches. Both primer sets had high coverage rates and specificity to detect dominant phyla; however, their coverage rate with regards to the rare biosphere varied. Our data further confirms that the choice of primer is the most deterministic factor in sequencing coverage and specificity.


Paired-end sequencing; Sequencing coverage; Single-index; Taxonomic classification; V3-V4 hypervariable regions

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