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. 2017 Jun 5;7:2774. doi: 10.1038/s41598-017-03139-4

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Two-step generation of 16S amplicons for PacBio sequencing using M13-tagged barcoded primers. The first round of PCR amplifies full-length 16S rRNA fragments using gene-specific primers tailed with universal M13 forward or reverse sequence. In the second step, a unique combination of barcode sequences is added to 16S amplicons from each sample using M13 forward and reverse primers tagged with 16-base PacBio barcodes at their 5′ ends. Barcoded amplicons are subsequently pooled for SMRTbell library construction and multiplexed sequencing.