Computational protocol: The Human Gastric Microbiome Is Predicated upon Infection with Helicobacter pylori

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Protocol publication

[…] A total of 2,825,234 MiSeq paired-end raw sequence forward and reverse reads were merged using ea-utils v1.1.2 (Aronesty, ) with standard settings, followed by a split library step from the Quantitative Insights Into Microbial Ecology (QIIME, v1.9.1) software (Caporaso et al., ). During this step sequence reads shorter than 200 nucleotides, reads that contain ambiguous bases or reads with an average quality score less than 30 were discarded. Chimera were removed with USEARCH v6.1 method in QIIME against 97% clustered GreenGenes reference 16S rRNA gene database (v13.8). In the second step OTU picking was done with QIIME open reference pipeline performing clustering steps at 97% sequence similarity, the taxonomy assignment with UCLUST algorithm (Edgar et al., ), alignment of reference sequences with pyNAST and generation of phylogenetic tree with FastTree. Finally, the OTU table was reduced by removing all OTUs present in only one sample with less than 10 reads. Downstream statistical analysis was performed in R version 3.3.3 (R Core Team, ) using a custom script. Principal Component Analysis (PCA) was performed using the prcomp function with default parameters. P-values for the group overlap in the PCA were calculated according to Goodpaster (Goodpaster and Kennedy, ) and Worley (Worley et al., ). […]

Pipeline specifications

Software tools ea-utils, QIIME, USEARCH, UCLUST, PyNAST, FastTree
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Homo sapiens, Helicobacter pylori, Bacteria