title: 16S rRNA from 16 hours of industrial fermentation of 2G  ethanol - Experiment 3
design: Taxonomic analysis to identify  bacterias was performed only for the industrial fermentation (IN). For this,  DNA extraction was performed at the four sampling points as described  previously. The primer pair used, targeting the variable regions 3 and 4 of the  16S rRNA gene, was Bakt_341F (CCTACGGGNGGCWGCAG) and Bakt_805R  (GACTACHVGGGTATCTAATCC) (Herlemann et al., 2011). Primer sequences within the  16S paired read sequences were trimmed out with Cutadapt software (version  1.16) (Martin, 2011). Only the where both the forward primer and the reverse  primer could be identified in the R1 and R2 reads, respectively, were  retained. Insertions, deletions or error rates greater than 20% were not  allowed for primer identification. Amplicon sequence variants (ASVs) were  obtained using DADA2 (version 1.8.0) (Callahan et al., 2016). First, quality  profiles were inspected to truncate the R1 and R2 reads to 228bp and 215bp,  respectively. Reads with more than two expected errors or containing  ambiguous bases were filtered out. Finally, R1 and R2 reads were  independently denoised using the pseudo-pooling approach and the pairs were  merged to obtain ASV sequences. Taxonomic assignment of the V3-V4 16S ASVs  was performed with IDTAXA (Murali et al., 2018) using a classifier trained  with the GTDB (release 83) (Parks et al., 2018). The confidence threshold was  set to 50 and only ASVs with relative abundance greater than 0.01% in at  least two samples were used for subsequent analysis. Correspondence analysis  with the count data was carried out with the phyloseq package (McMurdie and  Holmes, 2013).