Computational protocol: Administration of Lactobacillus salivarius LI01 or Pediococcus pentosaceus LI05 prevents CCl4 induced liver cirrhosis by protecting the intestinal barrier in rats

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Protocol publication

[…] DNA from the isolated caecal contents was used as a template for the amplification of the 16 S rRNA V3 and V4 hypervariable region using the fusion dual barcoded PCR primers F319 (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806 R (5′-GGACTACHVGGGTWTCTA AT-3′). Duplicate PCR products were pooled in equimolar concentrations and then purified using the Qiagen PCR purification kit (Qiagen, Hilden, Germany). Sequencing was performed on an Illumina MiSeq Instrument (Illumina Inc., San Diego, CA) using the 300 bp paired-end protocol.Overlapping paired-end reads from the original DNA fragments were merged using FLASH (version 1.2.10). Quality control was conducted using FastQC (version 0.10.1). The assembled sequences were clustered using the CD-hit-est-based clustering method. PyNAST software (http:/qiime.org/pynast/) was used to analyse and calculate the numbers of sequences and operational taxonomic units (OTUs) for each sample. Next, the sequences were grouped into various OTUs using Felsenstein-corrected similarity matrices; the sequences within an OTU shared at least 97% similarity. The Ribosomal Database Project (RDP) classifier, which is available from the RDP website (http://rdp.cme.msu. edu/classifier/), was used to classify the 16 S rDNA into distinct taxonomic categories by aligning the sequences to a curated database of taxonomically annotated sequences. All 16 S rDNA sequences were mapped to the RDP database using BLASTN to determine the taxonomic assignments. Sequences sharing greater than 97% identity were used to associate a group of OTUs to specific species, whereas those with less than 97% identity were considered novel reads. The microbial diversity in individual conjunctival samples was estimated using rarefaction analysis, the Shannon diversity index (SDI), and the Chao 1 index. Principal coordinate analysis (PCoA) was performed for each sample using OTUs and the ade4 package within the R program (R version 2.15, http://www.R-project.org). [...] For results of biochemical assays and histology score, the Kolmogrov-Smironv test was used to check for normality. Statistical differences between groups were estimated by Kruskal-waills test followed by the Mann-Whitney U test. The frequency of bacterial translocation was compared using the chi-squared test. Analysis of Similarities (ANOSIM) was used to test for clustering of microbial communities using weighted and unweighted UniFrac distance matrices. Wilcoxon rank sum test combined with the Benjamini-Hochberg method was applied to compare bacteria taxa. Correlations between variables were computed using the Spearman rank correlation. The values were presented as mean ± SEM if normally distributed; otherwise the values were presented as median (25th and 75th). P < 0.05 was considered significant. The data were analysed using SPSS version 20.0 for Windows (SPSS Inc., Chicago, IL, USA). […]

Pipeline specifications

Software tools FastQC, CD-HIT, PyNAST, QIIME, RDP Classifier, BLASTN, UniFrac, SPSS
Applications Miscellaneous, 16S rRNA-seq analysis
Organisms Lactobacillus salivarius, Pediococcus pentosaceus, Rattus norvegicus, Lactobacillus rhamnosus GG, Clostridium butyricum, Bacillus licheniformis, Bacteria
Diseases Escherichia coli Infections, Liver Cirrhosis
Chemicals Carbon Tetrachloride