title: 16S rRNA of mus musculus: MMcposFO
design: V3V4  region  16S  rRNA  amplicons  were  generated  using  degenerate  primers  that  contain  the  Illumina  adapter consensus sequence F: (5TCGTCGGCAGCGTCAGATGTGT ATAAGAGACA-GCCTACGGGNGGCWGCAG-3) and  R:  (5GTCTCGTGGGCTCGGAG  ATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3`)81,82.  A detailed  description  of  the  protocol  has  been  published  online  by  Illumina:  16S  Metagenomic  Sequencing  Library Preparation:  Preparing  16S  Ribosomal  RNA  Gene  Amplicons  for  the  Illumina  MiSeq  System.  Samples  were multiplexed  using  the  Illumina  Nextera  XT  Index  Kit  to  construct  barcoded  libraries.  Subsequent  libraries  were multiplexed  and  sequenced  by  500PE  sequencing  on  the  MiSeq  platform.  FastQC  (Babraham  Bioinformatics, Babraham  Institute,  UK)  was  used  to  determine  the  average  quality  scores  of  each  sample  before  and  after  paired reads. The paired ends in each sample were joined, and all sequences less than 250 bp and/or with a Phred score <33 were discarded. Quality filtering was applied using QIIME 53 (at Phred  Q20). We performed operational taxonomic unit (OTU) clustering and alpha- and beta-diversity analysis using QIIME version 1.7 83. A chimera filter was applied using  USEARCH  8.1.  All  sequences  were  clustered  based  on  97%  similarity  to  reference  sequences. The reads that did not meet the 97% similarity criteria were clustered de novo. Taxonomy levels of representative sequences in the OTU table were assigned at 95% similarity based on the SILVA database. We calculated alpha diversity based on the Shannon  diversity  index.  Analysis  of  beta  diversity  statistics  (analysis  of  similarities,  ANOSIM)  was  performed  to determine if differences between the distributions of microbiota profiles from the five datasets were significant.