16S rRNAseq of thermophilic digester
16S rRNA sequencing was performed by DNASense - https://dnasense.com/mga/ .  Amplicon libraries for the bacteria/archaea 16S rRNA gene variable region 4 (abV4C) were prepared by a custom protocol based on an Illumina protocol [4]. Up to 10 ng of extracted DNA was used as template for PCR amplification of the bacteria/archaea 16S rRNA gene variable region 4 (abV4C) amplicons. Each PCR reaction (25 L) contained (12.5 L) PCRBIO Ultra mix and 400 nM of each forward and reverse tailed primer mix. PCR was done with the following program: Initial denaturation at 95 C for 2 min, 30 cycles of amplification (95 C for 15 s, 55 C for 15 s, 72 C for 50 s) and a final elongation at 72 C for 5 min. Duplicate PCR reactions were performed for each sample and the duplicates were pooled after PCR. The forward and reverse, tailed primers were designed according to [4] and contain primers targeting the bacteria/archaea 16S rRNA gene variable region 4 (abV4C): [515FB] GTGYCAGCMGCCGCGGTAA and [806RB] GGACTACNVGGGTWTCTAAT The primer tails enable attachment of Illumina Nextera adaptors necessary for sequencing in a subsequent PCR. The resulting amplicon libraries were purified using the standard protocol for CleanNGS SPRI beads (CleanNA, NL) with a bead to sample ratio of 4:5. DNA was eluted in 25 L of nuclease free water (Qiagen, Germany). DNA concentration was measured using Qubit dsDNA HS Assay kit (Thermo Fisher Scientific, USA). Gel electrophoresis using Tapestation 2200 and D1000/High sensitivity D1000 screentapes (Agilent, USA) was used to validate product size and purity of a subset of amplicon libraries. Sequencing libraries were prepared from the purified amplicon libraries using a second PCR. Each PCR reaction (25 L) contained PCRBIO HiFi buffer (1x), PCRBIO HiFi Polymerase (1 U/reaction) (PCRBiosystems, UK), adaptor mix (400 nM of each forward and reverse) and up to 10 ng of amplicon library template. PCR was done with the following program: Initial denaturation at 95 C for 2 min, 8 cycles of amplification (95 C for 20 s, 55 C for 30 s, 72 C for 60 s) and a final elongation at 72 C for 5 min. The resulting sequencing libraries were purified using the standard protocol for CleanNGS SPRI beads with a bead to sample ratio of 4:5. DNA was eluted in 25 L of nuclease free water. DNA concentration was measured using Qubit dsDNA HS Assay kit  Gel electrophoresis using Tapestation 2200 and D1000/High sensitivity D1000 screentapes was used to validate product size and purity of a subset of sequencing libraries.