title: MPT-BioMicro-50y
design: Similarly to potential enzyme activities, the 16S rRNA gene amplicon sequencing was determined for soil control, 0.6%, 1.6%, and 3.2% treatments only (n=3). The analysis was conducted as outlined in Graf et al. (2023). Briefly, soil was frozen at -80C then lyophilised and prokaryotic DNA was extracted using the Zymo Research Quick DNA Fecal/Soil Microbe Miniprep Kit (Zymo Research, Irvine, CA) according to manufacturers instructions. Bacterial 16S rRNA genes were PCR-amplified using modified forward primer F515 (5-GTGBCAGCMGCCGCGGTAA-3) and reverse R806 prokaryotic primer (5-GGACTACHVGGGTWTCTAAT-3) (Fadrosh et al. 2014). PCRs were performed using OneTaq DNA Polymerase (New England Biolabs, Ipswich, MA) with no-template negative controls, as per Graf et al. (2023). Following purification using the QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany), amplicons were sequenced using the Illumina MiSeq (Illumina Inc., San Diego, CA) with a 500-cycle v2 chemistry kit (2  250 bp paired-end reads) at the Centre for Environmental Biotechnology, Bangor, United Kingdom. Raw sequences were read and processed (Fadrosh et al. 2014; Bolyen et al. 2019), then taxonomically classified using Silva (v. 132) as the reference database. Analysis of the most abundant taxonomic groups was performed using in-house R scripts, for groups with a relative abundance 2%. Selection started at genus level and groups were added to the immediate upper taxonomic level when none of the samples of that group reached the 2% threshold