Computational protocol: The presence of bacteria within tissue provides insights into the pathogenesis of oral lichen planus

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Protocol publication

[…] Genomic DNA was isolated from the mucosal samples using the Power Soil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA). The amplification and sequencing of 16S rRNA genes were performed at ChunLab, Inc. (Seoul, Korea) according to the previously described method using a 454 GS FLX Titanum Sequencing System (Roche Applied Science, Branford, CT, USA). The pyrosequencing data are available in the SRP database under the accession number SRP049562 and SRP065981.The sequenced data were analyzed by using software provided by ChunLab, Inc. After removing PCR primer sequences, reads containing two or more ambiguous nucleotides and reads shorter than 300 bp were discarded. Chimera sequences detected by the Bellerophone method were also removed. The taxonomic classification of each read was assigned against the EzTaxon database-e (http://eztaxon-e.ezbiocloud.net). The cutoff values used for assigning each read to taxonomic assignment were as follows: species (x ≥ 97%), genus (97 > x ≥ 94%), family (94 > x ≥ 90%), order (90 > x ≥ 85%), class (85 > x ≥ 80%), and phylum (80 > x ≥ 75%), where x denotes similarity. The species richness and diversity index were calculated using the Ribosomal RNA database project’s pyrosequencing pipeline (http://pyro.cme.msu.edu). The cutoff value for assigning a sequence to the same group (phylotype) was equal to or greater than 97% similarity. Random subsampling was conducted to equalize variation in the read counts among the samples. The overall phylogenetic distance between communities was estimated using the weighted Fast UniFrac and was visualized using PCoA. [...] Non-parametric methods, including the Mann-Whitney U-test, Kruskal Wallis test, and Spearman’s rank correlation test were used to analyze the data from ex vivo experiments using tissue sections and bacterial samples. The t-test and ANOVA were used to analyze the data from in vitro experiments using HOK-16B cells and purified leukocytes. The association of bacterial species with OLP risk was determined with a logistic regression analysis. All statistical analyses were performed using SPSS Statistics 22 software (SPSS Inc., Chicago, IL, USA). Significance was set at P < 0.05. […]

Pipeline specifications

Software tools Fast Unifrac, SPSS
Applications Miscellaneous, Phylogenetics, 16S rRNA-seq analysis
Organisms Escherichia coli, Bacteria, Homo sapiens