Computational protocol: Immunogenicity and protective efficacy of recombinant Clostridium difficile flagellar protein FliC

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Protocol publication

[…] Fresh fecal samples were collected from hamsters one week prior to challenge and genomic DNA was prepared from each hamster using the Qiagen QIAamp DNA Stool Mini kit (Qiagen, Valencia, CA, USA). We amplified the bacterial 16S rRNA V1-V2 hypervariable region from the genomic DNA using the forward primer 8F (AGA GTT TGA TCC TGG CTC AG) fused with the Ion Torrent Adaptor A sequence and 1 of 23 unique 10 base pair (bp) barcodes, and reverse primer 357R (CTG CTG CCT YCC GTA) fused with the Ion Torrent Adaptor P1 from each fecal sample. Polymerase chain reactions (PCR) were performed using Platinum PCR SuperMix (Life Technologies) with the following cycling parameters: 94 °C for 10 min, followed by 30 cycles of 94 °C for 30 s, 53 °C for 30 s, 72 °C for 30 s, and a final elongation step of 72 °C for 10 min. Resulting amplicons were purified on a 2% agarose gel stained with SYBR Safe (Life Technologies) using the MinElute PCR Purification kit (Qiagen). Amplicons were further purified with Ampure beads (Beckman-Coulter, Brea, CA, USA), and molar equivalents were determined for each sample using a Bioanalyzer 2100 HS DNA kit (Agilent Technologies, Santa Clara, CA, USA). Samples were pooled into equimolar proportions and sequenced on 314 chips using an Ion Torrent PGM according to manufacturer's instructions (Life Technologies). Resulting sequence reads were removed from the analysis if they were <180 bp, had any barcode or primer errors, contained any ambiguous characters, or contained any stretch of >8 homopolymers. Sequences were assigned to their respective samples based on a 10-nucleotide barcode sequence, and were analyzed further using the Qiime pipeline. Briefly, representative Operational Taxonomic Units (OTU) from each set were chosen at a minimum sequence identity of 97% using UClust and aligned using PyNast against the Greengenes database. Multiple alignments then were used to create phylogenies using FastTree, and taxonomy was assigned to each OTU using the Ribosomal Database Project (RDP) classifier., Principal coordinates analysis (PCoA) was performed based on Beta Diversity using weighted Unifrac distances. […]

Pipeline specifications

Software tools QIIME, UCLUST, PyNAST, FastTree, RDP Classifier, UniFrac
Databases Greengenes
Applications Phylogenetics, 16S rRNA-seq analysis
Organisms Mus musculus, Clostridioides difficile, Homo sapiens, Clostridioides difficile 630
Diseases Infection, Antiphospholipid Syndrome