16S of soil: day 14 after manure
Libraries constructed with primers 515F/926R (V4-V5; 300 bp paired-end). 30-cycle PCR using HotStarTaq Plus Master Mix Kit (QIAGEN) under the following conditions: 94C for 3 minutes, followed by 30 cycles of 94C for 30 seconds, 53C for 40 seconds and 72C for 1 minute, after which a final elongation step at 72C for 5 minutes. The samples achieved good signal without hitting amplification plateau. Guided by expected microbial diversity, 300 bp paired-end reads were generated at a depth of 40,000 reads for manure and soil samples (high-diversity samples), and at 20,000 reads for water samples (low-diversity samples) and negative controls. The QIIME2 workflow (version 2018.11) was used to perform quality control and filtering of sequence data. For each of the five library pools separately, raw sequence data were imported into QIIME2, samples were demultiplexed, and primer sequences were trimmed from the sequence reads using q2-cutadapt.