Dec 05, 2023
BIT 577 QIIME2 Tutorial
- Isabella Livingston1
- 1North Carolina State University
Protocol Citation: Isabella Livingston 2023. BIT 577 QIIME2 Tutorial. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8pqe9g2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 04, 2023
Last Modified: December 05, 2023
Protocol Integer ID: 91804
Keywords: metagenomics, Nanopore sequencing, WGS
Abstract
Command line scripts for processing and analyzing 16S amplicon data using QIIME2.
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 https://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