Computational protocol: Correlated production and consumption of chloromethane in the Arabidopsis thaliana phyllosphere

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Protocol publication

[…] For amplicons of the 16S rRNA gene, obtained sequences were screened, failed sequence reads, low quality sequence ends, tags and primers were removed, and non-bacterial ribosome sequences, chimeras detected using black box chimera check software B2C2, and short reads (<250 bp) were discarded as described previously–. Operational taxonomic units (OTUs) were assigned at 5% dissimilarity, commonly used to represent the genus level, validated using taxonomic distance methods, and data reduction analysis performed as described previously,.For cmuA amplicon analysis, Mothur was used to extract flow grams from sff files. Flow grams were de-noised and translated to DNA sequences, and reads with errors in the barcode or primer region, ambiguous bases, or homopolymer runs > 6 bp were removed. Quality filtering and sizing of reads (between 400 and 450 bp), conversion to fasta format, read dereplication, abundance sorting and removal of singletons was done with USEARCH. Chimeras were filtered out using UCHIME within USEARCH. Iterative clustering of OTUs was carried out with UPARSE within USEARCH to a cutoff of 85%. The most abundant sequence within each OTU was chosen as representative of the OTU, and taxonomic affiliation was performed by Blast comparisons to the Genbank database. […]

Pipeline specifications

Software tools mothur, USEARCH, UCHIME, UPARSE
Application 16S rRNA-seq analysis
Organisms Arabidopsis thaliana, Escherichia coli, Bacteria
Chemicals Carbon, Methyl Chloride, Ozone