Microbial Community Analysis by 16S rRNA Sequencing

The resulting 16S V1–V3 amplicon libraries were sequenced by using a Roche 454 GS FLX+ system and reagents (Roche 454 Life Sciences Corporation, Branford, CT, USA). Genomic DNA from Microbial Mock Community A, Even, Low Concentration (HM-278D v3.1, BEI Resources) obtained as part of the Human Microbiome Project [21 (link)], was diluted 10 times and used as a positive control for PCR and pyrosequencing of 16S amplicons.
The pyrosequencing data analysis pipeline was based on the Quantitative Insights Into Microbial Ecology (QIIME) pipeline version 1.8 [15 (link)]. The 454 read data were subjected to quality processing, chimera filtering, and removal of singleton reads. The taxonomic classification of the quality-processed reads was based on the closed reference clustering of sequences into operational taxonomical units (OTUs), using the UCLUST tool [22 (link)] with a sequence identity level of 97%. The read clusters were further assigned to taxonomies by using the Ribosomal Database Project (RDP) classifier [17 (link)] with a confidence level of 80%. The bacterial community diversity within a sample (α-diversity) was assessed by using the R-package vegan (https://cran.r-project.org/web/packages/vegan). The diversity between samples (β-diversity) was analyzed by using the QIIME implementation of UniFrac [19 (link)] and the weighted UniFrac distance as a measure of β-diversity.