Computational protocol: The making of a miscreant: tobacco smoke and the creation of pathogen-rich biofilms

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Protocol publication

[…] Total RNA was isolated from the biofilms using the mirVana miRNA isolation kit (Applied Biosystems). Microbial cells were lysed and RNA was extracted by Acid-Phenol: Chloroform and ethanol precipitation and eluted in nuclease-free water. Ribosomal RNA was depleted and mRNA enriched by modified capture hybridization approach. Enriched mRNA served as a template for the polyadenylation reaction and cDNA synthesis. Microbial libraries were clustered on the Illumina HiSeq platform, and 150 bp paired-end sequencing was performed. The Illumina base-calling pipeline was used to process the raw fluorescence images and call sequences. Raw reads with >10% unknown nucleotides or with >50% low quality nucleotides (quality value < 20) were discarded. Microbial transcripts were quality filtered using SolexaQA++, and aligned against the Human Oral Microbiome Database using DIAMOND. Aligned sequences were annotated to the KEGG database using Megan 6. The metagenomic sequence classifier Kraken was used along with our custom tool, kraken-biom, for taxonomic identification. Analysis and visualization of the distribution of operational taxonomic units was performed using QIIME and PhyloToAST. Bioconductor package for R, DESeq2, was used to perform differential expression analysis of the annotated microbial transcripts. [...] All reactions were carried out in triplicate and assays duplicated. Bioconductor package for R, DESeq2, was used to perform differential expression analysis of the annotated microbial transcripts. Phylogenetic trees were visualized using PhyloToAST and iTol. Within and between-group comparisons of cytokines and ROS were made using Repeated Measures ANOVA (Tukey HSD) in a Generalized Estimating Equations framework. All analyses were carried out using JMP (Cary, NC) and graphed using the capabilities of R and VORTEX (http://webapp-kumarlab.rhcloud.com/ ). […]

Pipeline specifications

Software tools MEGAN, Kraken, QIIME, PhyloToAST, DESeq2, iTOL
Databases KEGG HOMD
Applications Phylogenetics, Metatranscriptomic sequencing analysis, 16S rRNA-seq analysis
Organisms Nicotiana tabacum, Streptococcus oralis, Streptococcus sanguinis, Streptococcus mitis, Fusobacterium nucleatum, Porphyromonas gingivalis, Filifactor alocis, Tannerella forsythia