Computational protocol: Metatranscriptomics reveals overall active bacterial composition in caries lesions

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Protocol publication

[…] To increase accuracy in taxonomic assignment, only reads longer than 400 bp were selected. Chimeric PCR products were filtered out using the software Uchime, () and the reads were end-trimmed and quality filtered following Cabrera-Rubio et al. (). Sequences were separated by the sample-specific 8-bp barcode and assigned to a genus using the Ribosomal Database Project classifier, with an 80% confidence threshold (). Sequences were clustered at 97% nucleotide identity over 90% sequence alignment length, and rarefaction curves were obtained using the RDP pyrosequencing pipeline. An attempt was made to assign the reads to the species taxonomic level. In order to do this, a curated database was constructed with the full-length 16S sequences of all species present in the Ribosomal Database Project that belonged to the genera Streptococcus, Lactobacillus, and Veillonella. A BlastN () was then performed against this database with the reads that had been previously assigned to the above-mentioned genera and that were >500 bp. The top hit from each sequence comparison was selected if the alignment length was >500 bp, and the sequence identity >99%. […]

Pipeline specifications

Software tools UCHIME, RDP Classifier, BLASTN
Application 16S rRNA-seq analysis
Organisms Streptococcus mutans
Diseases Brain Diseases, Dental Caries
Chemicals Titanium