16S rRNA Gene Sequencing Workflow

As detailed above, RNA and DNA were extracted in parallel and RNA was subsequently reverse transcribed. DNA and cDNA extracts were used as templates for PCR-based amplification for 16S rRNA gene/transcript V4 amplicon generation using the following primers: 515F-ACACTGACGACATGGTTCTACAGTGCCAGCMGCCGCGGTAA and 806R-TACGGTAGCAGAGACTTGGTCTGGACTACNVGGGTWTCTAAT, and thermocycling program: 94°C for 3 min, followed for 32 × [ 94°C for 45s, 50°C for 60s, 72°C for 90s], 72°C for 10 min, and a 4°C hold. Amplicon sequencing was performed on an Illumina MiSeq platform. Sequence analyses were performed using the DADA2 package [63 (link)] implemented in R. Briefly, forward and reverse reads were trimmed with the filterAndTrim() command using the following parameters: trimLeft = c(20,20), maxEE = c(2,2), phix = TRUE, multithread = TRUE, minLen = 120, followed by error assessments and independent forward and reverse read de-replication. Sequencing errors were removed using the dada() command and error-free forward and reverse reads were merged using the mergePairs() command, specifying overhand trimming and a minimum overlap of 120 base pairs. The resulting amplicon sequence variants (ASVs) were assigned taxonomy by alignment against the SILVA 132 database [64 (link)]. ASV count tables and taxonomy assignments were merged into an S4 object for diversity analysis and summary visualization using vegan in phyloseq [65 (link)].