Computational protocol: Comparative analysis of the intestinal flora in type 2 diabetes andnondiabetic mice

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Protocol publication

[…] The forward and reverse paired-end reads with 300 bp were trimmed to 258 bp using fastx_trimmer with options (-f 3 and –l 260) in FASTX-Toolkit 0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/). Bacterial community analyses based on 16S rRNA genes were performed using the QIIME software package (Version 1.9.1) [] and the Greengenes 13_8 dataset [] as reference. The trimmed reads were joined according to the fastq-join parameter [] within the join_paired_ends.py script. The joined reads were quality filtered and merged into one file using the split_librarys_fastq.py script with options (–q 19 and –barcord_type ‘not-barcoded’). The sequences were clustered at 97% sequence identity using USEARCH v6.1.544 [] against the Greengenes dataset. Chimeric sequences were detected using the ChimeraSlayer algorithm [] within the identify_chimeric_seqs.py script. After filtering of the chimeric sequences, reassignment using the assign_taxonomy.py script along with the RDP classifier [] was performed to assign the taxonomy of each representative sequence of OTUs. To calculate beta diversity, the UniFrac [] was used within the beta_diversity_through_plots.py script along with a tree file that was constructed with a set of representative sequences of the OTUs using the make_phylogeny.py script with the default parameter. The alpha diversity was calculated using the alpha_diversity.py script. Both diversities were calculated using 72,000 reads. […]

Pipeline specifications

Software tools FASTX-Toolkit, QIIME, ea-utils, USEARCH, ChimeraSlayer, RDP Classifier, UniFrac
Databases Greengenes
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Mus musculus, Escherichia coli, Firmicutes, Bacteroidetes, Bacteria, Lachnospiraceae