Next generation 16s rrna gene amplicon sequencing

The microbial community was analysed using Illumina next-generation 16S rRNA gene amplicon sequencing. Both the luminal suspension and the washed bran residue were analysed for the time points 1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 48 and 72 h (Fig. S1). Biological replicates were only analysed from time points 2, 10 and 48 h.
DNA was extracted and DNA quality verified as described by De Paepe et al. [40 (link)]. Samples were sent out to LGC Genomics (Teddington, Middlesex, UK) for library preparation and sequencing on an Illumina Miseq platform. To assess sequencing quality, a mock community sample, with a composition as listed by De Paepe et al. [40 (link)] was included in triplicate.
The V3–V4 region of the 16S rRNA gene was amplified by PCR using primers derived from Klindworth et al. [46 (link)], with a slight modification to the reverse primer, 785R (5′-GACTACHVGGGTATCTAAKCC-3′), by introducing another degenerated position (K) to make it more universal. The library preparation was performed, as described by De Paepe et al. [40 (link)]. The sequence data have been submitted to the NCBI (National Center for Biotechnology Information) database under accession number SRP127353.

The microbial community at different time points along the fermentation was analyzed by Illumina next-generation 16S rRNA gene amplicon sequencing. Taxonomic analysis were assessed by sequencing the bacterial 16S rRNA gene in the V3-V4 region using the amplification primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) adapted to incorporate the transposon-based Illumina Nextera adapters (Illumina, United States) and a sample barcode sequence allowing multiplexed sequencing (García-López et al., 2020 (link)). High-throughput sequencing was performed at the Institute for Integrative Systems Biology (Université Laval, Québec, Canada) on a MiSeq platform using 2 × 300 bp paired-end sequencing (Illumina, United States). The raw sequence data have been submitted to European Nucleotide Archive (ENA) database with accession number PRJEB52762.