title: 16S-L-10
design: Laboratory phase - DNA samples were taken from each experimental system of the accelerated growth experiment to determine how the microbial population changed under the environmental pressure caused by the addition of the FRD material. These samples were submerged in 250 L LifeGuard Soil Preservation Solution (Qiagen) before DNA extraction, amplification of 16S rRNA genes and sequencing at the Australian Centre for Ecogenomics. Cell lysis was achieved by bead beating, followed by DNA extraction using DNeasy PowerSoil Pro Kit (#47016, Qiagen, Germany). Amplification of the V6-V8 portion of the 16S rDNA gene, using the universal primer set 926f (5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAAACTYAAAKGAATTGRCGG-3) and 1392wR (5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGACGGGCGGTGWGTRC-3) with modifications to contain the Illumina specific adapter sequence. Library preparation was done using workflow methods outlined by Illumina (#15044223) with the exception that the NEBNext Ultra II Q5 Mastermix (New England Biolabs #M0544) was used in standard PCR conditions. Agencourt AMPure XP beads were used to achieve DNA purification (Beckman Coulter, Brea, CA, USA)., which was then indexed using Illumina Nextera XT 834 sample Index Kit A-D (Illumina FC-131-1002, San Diego, CA, USA) in the mastermix to assign unique 8-bp barcodes.  A MiSeq instrument with paired-end sequencing was used to pool Equimolar concentrations of the indexed amplicons (Illumina, San Diego, CA, USA) with a 600-cycle V3 300-bp reads. Total read counts ranged from 25,912 to 102,331, with an average of 70,525.Demultiplexed sequences were processed using methods consistent with Coleman et al. (2021). Cutadapt (v. 3.5) was used to remove the universal primer sequences (Martin, 2011  EMBnet journal). Reads were subsequently duplicated and filtered using QIIME2 (v. 2022.2), with chimeras removed by DADA2 (--p-2 trunc-len-f 250 and --p-trunc-len-r 230) (Callahan et al., 2017). The resulting amplicon sequence variants (ASVs) were taxonomically assigned using the nonredundant SILVA database (v. 138, clustered at 99% identity) (Quast et al., 2012). Amplicon data filtration and analyses were performed in R (v. 4.1.3). The following ASVs were discarded: sequences not classified below the phylum level, sequences classified as chloroplasts or mitochondria, sequences not determined to be either archaeal, bacterial or fungal in origin, and ASVs with an abundance of <0.1%. Heatmaps were produced using ggplot2.