Computational protocol: Xiexin Tang improves the symptom of type 2 diabetic rats by modulation of the gut microbiota

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Protocol publication

[…] Qubit®3.0 (Life Invitrogen) was used to quantify the purified PCR products and every twenty-four amplicons whose barcodes were different were mixed equally. Following Illumina’s genomic DNA library preparation procedure, Illumina Pair-End libraries were labeled with different multiplex indexing barcodes for sequencing (2 × 250) on an Illumina MiSeq platform (Shanghai BIOZERON Co., Ltd) in accordance with standard protocols. The raw reads were deposited into the National Center for Biotechnology Information Sequence Read Archive database.Demultiplexing and quality filtering were performed by the Quantitative Insights into Microbial Ecology (QIIME) version 1.17. OTUs were clustered using UPARSE at 97% similarity (version 7.1 http://drive5.com/uparse/) and chimeric sequences were identified and removed using UCHIME. The phylogenetic affiliation of each 16S rRNA gene sequence was defined by Ribosomal Database Project (RDP) Classifier (http://rdp.cme.msu.edu/) against the silva (SSU128) 16S rRNA database with a confidence threshold of 70%.Statistical analysis of the animal experiment was by SPSS 11.5 (SPSS, Inc., Chicago, IL). Obvious differences were accepted at p values of <0.05. The rarefaction analysis based on Mothur v.1.21 was conducted to reveal the diversity by Shannon diversity indices. PCoA was applied to examine dissimilarities in community composition and microbiota abundances were constructed based on the unweighted UniFrac distance metric. Furthermore, LEfSe was performed by non-parametric factorial Kruskal-Wallis (KW) sum-rank test and then we performed the linear discriminant analysis to assess effect size of each differentially abundant taxon or OTU. CCA and Heat-map analysis were conducted to investigate correlations between gut microbiota and diabetes related metabolize indexes. […]

Pipeline specifications

Software tools QIIME, UPARSE, UCHIME, RDP Classifier, mothur, LEfSe
Applications Phylogenetics, Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Rattus norvegicus, Homo sapiens, Bacteria, Escherichia coli, Lachnospiraceae
Chemicals Fatty Acids