Dataset: T. rotula microbiome global sample

The V3-V4 rDNA region of the 16S rRNA gene was amplified from all samples using a dual index approach in 20 µl reactions containing 1µl genomic DNA, 0.4 U of Taq (Lucigen) polymerase, and a final concentration of 200 μM dNTPs, 2 mM MgCl₂, and 0.5 μM of each primer (16S V3 Forward – CCTACGGGNGGCWSCAG and 16S V4 Reverse – GGACTACNVGGGTWTCTAAT). The PCR was run for 30 seconds at 98 °C, followed by 30-35 cycles at 98 °C for 10 seconds, 55 °C for 30 sec and 72 °C for 30 sec, with a final extension at 72 °C for 2 min. Triplicate 20 µl reactions were pooled and gel-purified for each sample.

Raw paired-end libraries were quality controlled using the ea-utils V.1.04.807 script fastq-mcf to a mate-pair quality mean of 25 with a 10 basepair (bp) sliding window and a minimum length of 150 bp. Forward and reverse reads were merged using the USEARCH V.8.1.1861 script fastq_mergepairs with a minimum overlap length of 10 bp and zero differences in the overlap region followed by clustering using Minimum Entropy Decomposition (MED) V.2.0 with standard settings. Chloroplasts were filtered using the QIIME V.1.9.1 script filter_fasta.py followed by an additional clustering step in MED with a minimum substantive abundance (-M) of 53 sequences required to form a single node. Chimeras were identified and removed using the USEARCH V.11.0.667 script uchime2_ref in sensitive mode against the Silva v128 database. Silva database v128 was used to assign taxonomy to OTUs and libraries were then rarefied using the script multiple_rarefactions_even_depth.py ten times to an even depth of 501 sequences per sample and OTU tables were merged using merge_otu_tables.py in QIIME V.1.9.1.

BCO-DMO Processing:
- flagged N/A as missing data identifier (replaced ;with nd);
- converted date format to YYYY-MM-DD
- renamed fields to conform with BCO-DMO naming conventions;
- removed commas from the Sample_Site column.