Data Processing:
qiime2 was used to process the amplicon sequence data.

Demultiplex

qiime tools import --type ‘SampleData[PairedEndSequencesWithQuality]’ --input-path <manifest file name> --output-path <name.qza> --input-format PairedEndFastqManifestPhred33V2

qiime tools import --type ‘SampleData[SequencesWithQuality]’ --input-path <manifest file name> --output-path <name.qza> --input-format SingleEndFastqManifestPhred33V2

To evaluate the sequences qiime demux summarize --i-data name.qza --o-visualization name.qzv

Denoise, dereplicate, quality filter & remove chimeras

qiime dada2 denoise-single --i-demultiplexed-seqs <.qza file from above> --p-trim-left 10 --p-trunc-len 290 --p-chimera-method pooled --o-table <name.qza> --o-representative-sequences <name.qza> --o-denoising-stats <name.qza>

Remove singletons

qiime feature-table filter-features --i-table <name of table generated in prior step> --p-min-frequency 2 --o-filtered-table <new table name .qza>

qiime feature-table filter-seqs --i-data <name of rep seq file created in prior step> --i-table <name of filtered table> --o-filtered-data <new repseq file .qza>

Cluster sequences based upon identity

qiime vsearch cluster-features-de-novo --i-table <name.qza> (filtered table) --i-sequences <name.qza> (filtered rep-seqs file) --p-perc-identity 1.0 --o-clustered-table <name.qza> (this is a biom table that has abundances of each cluster) --o-clustered-sequences <name.qza> (this is a new rep-set of sequences - your ASVs)

Assign taxonomy

qiime feature-classifier classify-consensus-vsearch --i-query <name.qza> (this is the rep-set of sequences just created by cluster) --i-reference-reads pr2_4.12.0_18S.qza (this is the PR2 reference set of sequences) --i-reference-taxonomy pr2_4.12.0_18S_tax.qza (this is the corresponding PR2 taxonomy) --p-maxaccepts 1 --o-classification <name.qza> (this is your rep-set sequences and their taxonomy)

BCO-DMO Processing:
- added columns for the latitude and longitude of the sampling locations;
- converted the date field to YYYY-MM-DD format.