Computational protocol: Non-pylori Helicobacters (NHPHs) Induce Shifts in Gastric Microbiota in Helicobacter pylori-Infected Patients

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Protocol publication

[…] Paired-end reads were split and truncated based on the unique barcoded-primer sequence of every sample. Paired-end reads of each sample were merged using FLASH version 1.2.7 (). After merging, the generated raw tags were filtered to obtain high-quality clean tags according to QIIME version 1.7.0 (). Chimera sequences were removed using the UCHIME algorithm () to yield clean tags for further analysis.Sequence analysis was performed using UPARSE pipeline version 7.0.1001 (), and sequences with ≥97% similarity were assigned to the same operational taxonomic units (OTUs). Representative sequences for each OTU were screened for further annotation. RDP Classifier (Version 2.2) was used to annotate taxonomic information for each representative sequence based on the Greengenes 13_5 Database (). OTU abundance information was normalized using a standard sequence number corresponding to the sample with the fewest sequences. Subsequent analyses of α and β diversity were performed based on this output-normalized data. [...] A non-parametric t-test adjusted for age and sex based on Monte Carlo permutations was performed to compare the α diversity indices between groups in R statistical software. A PERMANOVA test was used to determine the influences of age, sex, and infection status on β diversity (Weighted UniFrac distance and Bray–Curtis distance). Testing for significantly different phyla and genera between the two groups was performed using Metastats software. The number of permutations used to calculate the P-value (significance threshold = 0.05, false discovery rate threshold = 0.2) was set to 1,000. To explore the functional profiles of different groups, we predicted the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways of the gastric community using the online protocol of PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (). An OTU table based on 16S data was selected for PICRUSt from the Greengenes reference data (version 13.5). The nearest sequenced taxon index (NSTI) was used to assess the accuracy of the predictions for gene function and KEGG pathway enrichment in the gastric bacterial community. Welch’s t-test was employed to analyze the significant differences in KEGG pathways between groups. […]

Pipeline specifications

Software tools QIIME, UCHIME, UPARSE, RDP Classifier, Metastats, PICRUSt
Databases KEGG Greengenes
Applications Phylogenetics, Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Helicobacter pylori, Homo sapiens, Mycoplasma haemofelis
Diseases Bacterial Infections, Haemophilus Infections, Stomach Diseases