Computational protocol: Propionibacterium acnes overabundance and natural killer group 2 member D system activation in corpus‐dominant lymphocytic gastritis

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Protocol publication

[…] DNA extraction for microbiota analysis is described in Supplementary materials and methods. DNA quality and concentration were determined spectrophotometrically with a NanoDrop ND‐3300 instrument and the PicoGreen assay (Thermo Fisher Scientific, Waltham, MA, USA). Only specimens yielding an absorbance ratio of >0.8 at 260/280 nm and an absorbance ratio of ∼2 at 260/230 nm, respectively, were considered for further analyses. For amplification of the bacterial 16S rRNA gene FLX one‐way fusion primers (Lib‐L kit, Primer A, Primer B; Roche 454 Life Science, Branford, CT, USA) with the template‐specific sequence F27 and R357 (supplementary material, Table S2) targeting the V1–2 region of the 16S rRNA gene were used (amplicon length of 349 bp). Primers were chosen on the basis of their performance, enabling superior community capture and taxonomic resolution , and their good polymerase chain reaction (PCR) performance when applied to FFPE samples. PCR amplification was performed as described previously . Reactions for each sample were performed in triplicate, the quality of amplification products was checked visually on agarose gels, and only specimens resulting in reliable PCR amplification were used further. Amplicons were gel‐purified, pooled, and sequenced with the GS FLX Titanium Sequencing Kit XLR70 (Roche 454 Life Science), as described previously . For microbiota analysis, raw files from 454 FLX pyrosequencing were processed with MOTHUR v.1.31.2 according to the standard 454 SOP of MOTHUR . Sequencing errors were reduced with MOTHUR's implementation of PyroNoise , and the command pre.cluster was used to remove sequences that arose because of pyrosequencing errors. Chimeras were removed with UCHIME , and non‐bacterial contaminants were removed by use of the Ribosomal Database Project (RDP) reference . The high‐quality reads were aligned to the SILVA database , . For operational taxonomic unit (OTU)‐based analyses, the processed fasta files from MOTHUR were introduced into QIIME v.1.7.0 . OTUs were formed by clustering the sequences with uclust , with a similarity score of 97% (OTU 97% identity), and taxonomy was assigned by using the RDP classifier and Greengenes reference OTUs. A de novo OTU picking strategy was employed. The biomarker discover program LEfSe (linear discriminant analysis effect size) was used to determine differentially abundant OTUs . A batch file specifying the parameters used for microbiota analyses is given in Supplementary materials and methods. Differences in alpha‐diversity measures were tested by one‐way anova and a post hoc Bonferroni test. Principal coordinate analysis (PCoA) plots were created on the basis of a weighted‐unifrac distance matrix, and statistical differences between groups were calculated with anosim. The presented values are always mean ± standard error of the mean if not indicated otherwise. […]

Pipeline specifications

Software tools mothur, PyroNoise, UCHIME, QIIME, UCLUST, RDP Classifier, LEfSe, UniFrac
Databases Greengenes
Applications Metagenomic sequencing analysis, 16S rRNA-seq analysis
Organisms Helicobacter pylori, Cutibacterium acnes, Homo sapiens
Diseases Gastritis
Chemicals Fatty Acids