Computational protocol: Single fecal microbiota transplantation failed to change intestinal microbiota and had limited effectiveness against ulcerative colitis in Japanese patients

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Protocol publication

[…] The fecal DNA samples were sequenced using the 454 GS FLX Titanium and FLX+ (Roche, Basel, Switzerland) sequencing system. The detailed protocols were described previously. Briefly, the 16S rRNA gene V1 to V2 region was amplified by PCR by using the primers 27Fmod and 338R containing 454 primer sequences A and B and a unique 10-bp barcode sequence in 27Fmod. The PCR amplicons were sequenced to obtain reads, and the reads with an average quality value <25, mismatches to both universal primers, and possible chimeric reads were removed. Among the high-quality reads, 3,000 reads per sample were randomly selected and grouped into operational taxonomic units (OTUs) by clustering using the UCLUST algorithm with a 96% identity threshold. Taxonomic assignments for each OTU were made by similarity search against the public 16S and National Center for Biotechnology Information (NCBI) genome databases using the GLSEARCH program. For assignment at the phylum, family, genus, and species levels, sequence similarity thresholds of 70%, 90%, 94%, and 96%, respectively, were employed. All of the high-quality 16S V1 to V2 sequences analyzed in this study were deposited into the DNA Data-Bank of Japan (DDBJ)/GenBank/ European Molecular Biology Laboratory (EMBL) database under accession number DRA004886. UniFrac distance and principal coordinate analysis were used to assess the similarity of the microbiota structure of each pair of samples. […]

Pipeline specifications

Software tools UCLUST, UniFrac
Databases DDBJ
Application 16S rRNA-seq analysis
Organisms Homo sapiens
Diseases Colitis, Ulcerative, Communicable Diseases, Kidney Diseases, Inflammatory Bowel Diseases