2015
DOI: 10.1002/jobm.201500420
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An extended single‐index multiplexed 16S rRNA sequencing for microbial community analysis on MiSeq illumina platforms

Abstract: 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 previo… Show more

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Cited by 96 publications
(68 citation statements)
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“…A library of V4 region of 16S rRNA was constructed using modified F515 and R806 primers 39 as described previously 40 . PCR reaction for each sample was performed in duplicate and contained 1.0 µl of pre-normalized DNA, 1.0 µl of each forward and reverse primers (10 µM), 12 µl HPLC grade water (Fisher Scientific, Ottawa, ON, Canada) and 10 µl 5 Prime Hot MasterMix (5 Prime, Inc., Gaithersburg, MD, USA).…”
Section: Methodsmentioning
confidence: 99%
“…A library of V4 region of 16S rRNA was constructed using modified F515 and R806 primers 39 as described previously 40 . PCR reaction for each sample was performed in duplicate and contained 1.0 µl of pre-normalized DNA, 1.0 µl of each forward and reverse primers (10 µM), 12 µl HPLC grade water (Fisher Scientific, Ottawa, ON, Canada) and 10 µl 5 Prime Hot MasterMix (5 Prime, Inc., Gaithersburg, MD, USA).…”
Section: Methodsmentioning
confidence: 99%
“…The V4 region of 16S rRNA gene was targeted for PCR amplification using modified F515/R806 primers 34 as described before 35 . The reverse PCR primer was indexed with 12-base Golay barcodes allowing for multiplexing of samples.…”
Section: Methodsmentioning
confidence: 99%
“…The PCR amplification was targeted to amplify the V4 region of the 16S rRNA gene using modified F515/R806 primers (Caporaso et al, 2012) as described previously (Derakhshani et al, 2016). In brief, the reverse PCR primer was indexed with 12-base Golay barcodes, allowing for multiplexing of samples.…”
Section: Methodsmentioning
confidence: 99%