Download conda through the QIIME2 website or miniconda website. After installation, download and install QIIME2

Activate Qiime environment:

conda activate qiime2-amplicon-2023.9

# change out to whatever version has been downloaded

Import data

qiime tools import \

  --type 'SampleData[SequencesWithQuality]' \

  --input-path

/path/to/your/sequencefiles \

  --input-format CasavaOneEightSingleLanePerSampleDirFmt \

  --output-path demux-single-end.qza

Check quality

qiime demux summarize --p-n 10000 --i-data demux-single-end.qza --o-visualization qual_viz

qiime tools view qual_viz.qzv      

Denoise with DADA2

qiime dada2 denoise-single \

--i-demultiplexed-seqs demux-single-end.qza \

--p-trunc-len 200 \  #at what position you should trim your data based on the demux summary above

--o-table table.qza \ #ASV table

--o-representative-sequences rep-sets.qza \

--o-denoising-stats denoising-stats.qza

Filter Sequence Table

qiime feature-table summarize \

--i-table table.qza \

--o-visualization vis_table

qiime tools view vis_table.qzv

Align and Construct Phylogenetic Tree

qiime phylogeny align-to-tree-mafft-fasttree \ # program used for construction

--i-sequences rep-sets.qza \

--o-alignment aligned-rep-seqs.qza \

--o-masked-alignment masked-aligned-rep-seqs.qza \

--o-tree unrooted-tree.qza \

--o-rooted-tree rooted-tree.qza

Compute diversity measures (see QIIME2 for differences in alpha, beta, and emperor plots)

qiime diversity core-metrics-phylogenetic --i-table

table.qza --i-phylogeny rooted-tree.qza --p-sampling-depth 29761

--m-metadata-file /Users/isabellalivingston/Desktop/16S Sequence

Data/GSL_metadata.txt --o-rarefied-table rare_featuretable --o-faith-pd-vector

faithpd_vector --o-observed-features-vector observed_sample_features_vector

--o-shannon-vector shannon_sample_vector --o-evenness-vector

evenness_sample_vector --o-unweighted-unifrac-distance-matrix

unweighted_distance_matrix --o-weighted-unifrac-distance-matrix

weighted_distance_matrix --o-jaccard-distance-matrix jaccard_matrix

--o-bray-curtis-distance-matrix bc_matrix --o-unweighted-unifrac-pcoa-results

unweighted_pcoa --o-weighted-unifrac-pcoa-results weighted_pcoa

--o-jaccard-pcoa-results jaccard_pcoa --o-bray-curtis-pcoa-results bc_pcoa

--o-unweighted-unifrac-emperor unweighted_emperor --o-weighted-unifrac-emperor

weighted_emperor --o-jaccard-emperor jaccard_emperor --o-bray-curtis-emperor

bc_emperor

Test for differences in diversity using metadata

# Alpha Diversity

qiime diversity alpha-group-significance --i-alpha-diversity faithpd_vector.qza

--m-metadata-file /path/to/metadata/txt file

--o-visualization faith-pd-group-significance.qzv

qiime diversity alpha-group-significance --i-alpha-diversity

evenness_sample_vector.qza --m-metadata-file

/Users/isabellalivingston/Desktop/16S\ Sequence\ Data/GSL_metadata.txt

--o-visualization evenness-group-significance.qzv

qiime diversity alpha-group-significance --i-alpha-diversity

shannon_sample_vector.qza --m-metadata-file

/Users/isabellalivingston/Desktop/Metadata\ for\ GSL.tsv --o-visualization

alpha_diversity

# Emperor plots

qiime emperor plot --i-pcoa unweighted_pcoa.qza

--m-metadata-file /Users/isabellalivingston/Desktop/16S\ Sequence\

Data/GSL_metadata.txt --o-visualization unweighted-unifrac-emperor-sex.qzv

qiime emperor plot --i-pcoa bc_pcoa.qza --m-metadata-file

/Users/isabellalivingston/Desktop/16S\ Sequence\ Data/GSL_metadata.txt

--p-custom-axes sex --o-visualization bc-sex-sex.qzv

# Beta diversity

qiime diversity beta-group-significance --i-distance-matrix

unweighted_distance_matrix.qza --m-metadata-file

/Users/isabellalivingston/Desktop/16S\ Sequence\ Data/GSL_metadata.txt

--m-metadata-column sex --o-visualization unweighted-unifrac-sex-significance.qzv

--p-pairwise

Taxonomic Classification

wget http://data.qiime2.org/2018.2/common/gg-13-8-99-515-806-nb-classifier.qza #download classifier of choice

qiime feature-classifier classify-sklearn \

  --i-classifier /path/to/classifier.qza \

  --i-reads rep-sets.qza \

  --o-classification {TAXONOMY}.qza

qiime metadata tabulate \

--m-input-file {TAXONOMY}.qza --o-visualization {TAXONOMY_VIZ}.qzv

qiime taxa barplot \

  --i-table table.qza

\

  --i-taxonomy

{TAXONOMY}.qza \

  --m-metadata-file

/path/to/etadata/txtfile\

  --o-visualization

{TAXA-BAR-PLOTS_VIZ}.qzv

qiime tools view {TAXA-BAR-PLOTS_VIZ}.qzv

Export Data

Qiime tools export \

--input-path taxtable.qza \

--output-path exported-taxa-table

# Should get a confirmation like this: Exported taxtable.qza as BIOMV210DirFmt to directory

exported-taxa-table

# convert from biom to tsv for future analysis

Biom convert -i

/path/to/biom/file -o table.tsv

--to-tsv