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QIIME_2_workflow.md

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1. Create directory in HOME

mkdir qiime-2-thesis-IDFG
cd qiime-2-thesis-IDFG

2. Create manifest file

See Manifest.txt

Create a text file and populate two columns: sample-id and absolute-filepath.

3. Import into QIIME 2

Used command for single-end reads and demultiplexed.

qiime tools import \
  --type 'SampleData[SequencesWithQuality]' \
  --input-path Manifest.txt \
  --output-path single-end-demux.qza \
  --input-format SingleEndFastqManifestPhred33V2

4. Visualize imported data

mkdir Visualizations

qiime demux summarize \
  --i-data single-end-demux.qza \
  --o-visualization Visualizations/single-end-demux.qzv

qiime tools view Visualizations/single-end-demux.qzv

or open https://view.qiime2.org and drag and drop QZV to visualize in web browser.

5. Quality control with DADA2

After visualizing single-end-demux, chose a quality cutoff of 26, which allowed DADA2 to retain higher quality sequence data compared to lower values for quality cutoff. This cutoff corresponded to a p-trunc-len of 147.

qiime dada2 denoise-single \
  --i-demultiplexed-seqs single-end-demux.qza \
  --p-trunc-len 147 \
  --p-trim-left 0 \
  --o-representative-sequences rep-seqs-dada2.qza \
  --o-table table-dada2.qza \
  --o-denoising-stats stats-dada2.qza

6. Visualize feature table, sequences, and DADA2 statistics

qiime feature-table summarize \
  --i-table table-dada2.qza \
  --m-sample-metadata-file sample-metadata.tsv \
  --o-visualization Visualizations/table-dada2.qzv

qiime feature-table tabulate-seqs \
  --i-data rep-seqs-dada2.qza \
  --o-visualization Visualizations/rep-seqs-dada2.qzv

qiime metadata tabulate \
  --m-input-file stats-dada2.qza \
  --o-visualization Visualizations/stats-dada2.qzv

7. Taxonomy - Train and run a feature classifier using SILVA database

Convert SILVA 16S fasta to QZA

qiime tools import \
  --type 'FeatureData[Sequence]' \
  --input-path silva_138_99_16S.fna \
  --output-path silva-138-99-16S.qza

Extract reference reads based on 515F/926R primers and previous p-trunc-len.

qiime feature-classifier extract-reads \
  --i-sequences silva-138-99-16S.qza \
  --p-f-primer GTGYCAGCMGCCGCGGTAA \
  --p-r-primer CCGYCAATTYMTTTRAGTTT \
  --p-trunc-len 147 \
  --p-min-length 100 \
  --p-max-length 400 \
  --o-reads silva-138-99-515f-926r-ref-seqs.qza

Import taxonomy

qiime tools import \
  --type 'FeatureData[Taxonomy]' \
  --input-format HeaderlessTSVTaxonomyFormat \
  --input-path taxonomy_7_levels.txt \
  --output-path ref-taxonomy.qza

Train the feature classifier

qiime feature-classifier fit-classifier-naive-bayes \
  --i-reference-reads silva-138-99-515f-926r-ref-seqs.qza \
  --i-reference-taxonomy silva-138-99-tax.qza \
  --o-classifier silva-138-99-515f-926r-classifier.qza

Run the feature classifier

qiime feature-classifier classify-sklearn \
  --i-classifier silva-138-99-515f-926r-classifier.qza \
  --i-reads rep-seqs-dada2.qza \
  --o-classification taxonomy.qza

8. Filter out mitochondria, chloroplasts, and Unassigned bacteria

Filter from table

qiime taxa filter-table \
  --i-table table-dada2.qza \
  --i-taxonomy taxonomy.qza \
  --p-exclude mitochondria,chloroplast,Unassigned \
  --o-filtered-table filtered-table.qza

Visualize filtered table

qiime feature-table summarize \
  --i-table filtered-table.qza \
  --m-sample-metadata-file sample-metadata.tsv \
  --o-visualization Visualizations/filtered-table.qzv

Filter from sequences

qiime taxa filter-seqs \
  --i-sequences rep-seqs-dada2.qza \
  --i-taxonomy taxonomy.qza \
  --p-exclude mitochondria,chloroplast,Unassigned \
  --o-filtered-table filtered-rep-seqs.qza

Visualize filtered rep seqs

qiime feature-table tabulate-seqs \
  --i-data filtered-rep-seqs.qza \
  --o-visualization Visualizations/filtered-rep-seqs.qzv

9. Visualize taxonomy and make a relative abundance barplot

Visualize taxonomy

qiime metadata tabulate \
  --m-input-file taxonomy.qza \
  --o-visualization Visualizations/taxonomy.qzv

Make a taxa barplot displaying relative abundance (using filtered table)

qiime taxa barplot \
  --i-table filtered-table.qza \
  --i-taxonomy taxonomy.qza \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization Visualizations/taxa-bar-plots.qzv

10. Generate a tree for phylogenetic diversity analysis

qiime phylogeny align-to-tree-mafft-fasttree \
  --i-sequences filtered-rep-seqs.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

11. Make an alpha rarefaction plot

qiime diversity alpha-rarefaction \
  --i-table filtered-table.qza \
  --i-phylogeny rooted-tree.qza \
  --p-max-depth 25000 \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization Visualizations/alpha-rarefaction.qzv

View the rarefaction plot and open filtered-table.qzv > Interactive Sample Detail. Choose a Sampling Depth with two considerations: 1) Captures most of the diversity in the rarefaction plot and 2) how many samples are retained in the analysis.

12. Core metrics for Alpha and Beta diversity

qiime diversity core-metrics-phylogenetic \
  --i-phylogeny rooted-tree.qza \
  --i-table table-dada2.qza \
  --p-sampling-depth 12759 \
  --m-metadata-file sample-metadata.tsv \
  --output-dir core-metrics-results

13. Statistical significance for Alpha diversity

cd core-metrics-results

mkdir Alpha_diversity

Shannon

qiime diversity alpha-group-significance \
  --i-alpha-diversity shannon_vector.qza \
  --m-metadata-file ../sample-metadata.tsv \
  --o-visualization Alpha_diversity/shannon_significance.qzv

Observed OTUs

qiime diversity alpha-group-significance \
  --i-alpha-diversity observed_otus_vector.qza \
  --m-metadata-file ../sample-metadata.tsv \
  --o-visualization Alpha_diversity/observed_otus_significance.qzv

Faith PD

qiime diversity alpha-group-significance \
  --i-alpha-diversity faith_pd_vector.qza \
  --m-metadata-file ../sample-metadata.tsv \
  --o-visualization Alpha_diversity/faith_pd_significance.qzv

Evenness

qiime diversity alpha-group-significance \
  --i-alpha-diversity evenness_vector.qza \
  --m-metadata-file ../sample-metadata.tsv \
  --o-visualization Alpha_diversity/evenness_significance.qzv

14. Correlation analyses for Alpha diversity

Shannon

qiime diversity alpha-correlation \
  --i-alpha-diversity shannon_vector.qza \
  --m-metadata-file ../sample-metadata.tsv \
  --o-visualization shannon_correlation_Spearman.qzv

Observed OTUs

qiime diversity alpha-correlation \
  --i-alpha-diversity observed_otus_vector.qza \
  --m-metadata-file ../sample-metadata.tsv \
  --o-visualization observed_otus_correlation_Spearman.qzv

Faith PD

qiime diversity alpha-correlation \
  --i-alpha-diversity faith_pd_vector.qza \
  --m-metadata-file ../sample-metadata.tsv \
  --o-visualization faith_pd_correlation_Spearman.qzv

Evenness

qiime diversity alpha-correlation \
  --i-alpha-diversity evenness_vector.qza \
  --m-metadata-file ../sample-metadata.tsv \
  --o-visualization evenness_correlation_Spearman.qzv

15. Beta diversity PERMANOVA comparing infected and uninfected frogs (ChytridResult)

mkdir Beta_diversity

Unweighted UniFrac

qiime diversity beta-group-significance \
  --i-distance-matrix unweighted_unifrac_distance_matrix.qza \
  --m-metadata-file ../sample-metadata.tsv \
  --m-metadata-column ChytridResult \
  --o-visualization Beta_diversity/unweighted_unifrac_significance_Chytrid.qzv \
  --p-pairwise

Weighted UniFrac

qiime diversity beta-group-significance \
  --i-distance-matrix weighted_unifrac_distance_matrix.qza \
  --m-metadata-file ../sample-metadata.tsv \
  --m-metadata-column ChytridResult \
  --o-visualization Beta_diversity/weighted_unifrac_significance_Chytrid.qzv \
  --p-pairwise

16. Beta diversity Mantel test (ZoosporeEquivalents)

Unweighted UniFrac

qiime diversity beta-correlation \
  --i-distance-matrix unweighted_unifrac_distance_matrix.qza \
  --m-metadata-file ../sample-metadata.tsv \
  --m-metadata-column ZoosporeEquivalents \
  --p-intersect-ids \
  --o-metadata-distance-matrix Beta_diversity/unweighted_unifrac_correlation_zoospores.qza \
  --o-mantel-scatter-visualization Beta_diversity/unweighted_unifrac_correlation_zoospores.qzv

Weighted UniFrac

qiime diversity beta-correlation \
  --i-distance-matrix unweighted_unifrac_distance_matrix.qza \
  --m-metadata-file ../sample-metadata.tsv \
  --m-metadata-column ZoosporeEquivalents \
  --p-intersect-ids \
  --o-metadata-distance-matrix Beta_diversity/unweighted_unifrac_correlation_zoospores.qza \
  --o-mantel-scatter-visualization Beta_diversity/unweighted_unifrac_correlation_zoospores.qzv

Repeated above commands with latitude and longitude

17. Correlation analyses with Positive infection status samples only

Run following steps and then repeat steps 13 and 15:

1) Make a metadata file that includes only Positive samples

2) Filter the feature table using that metadata file

3) Rerun step 11 creating a separate core-metrics-results-positives

18. adonis analysis - PERMANOVA with multiple variables (infection, latitude, longitude)

Unweighted UniFrac

qiime diversity adonis \
  --i-distance-matrix unweighted_unifrac_distance_matrix.qza \
  --p-formula "ChytridResult * Latitude * Longitude" \
  --m-metadata-file ../sample-metadata.tsv \
  --o-visualization Beta_diversity/uwunifrac_adonis_chytrid_lat_long.qzv

Weighted UniFrac

qiime diversity adonis \
  --i-distance-matrix weighted_unifrac_distance_matrix.qza \
  --p-formula "ChytridResult * Latitude * Longitude" \
  --m-metadata-file ../sample-metadata.tsv \
  --o-visualization Beta_diversity/wunifrac_adonis_chytrid_lat_long.qzv

19. Plot taxonomic relative abundance by infection status

cd ..
mkdir group_plot

Makes a table grouping samples by infection status

Metadata file had two columns: SampleID (Positive and Negative) and SampleName (Infected and Uninfected)

qiime feature-table group \
  --i-table filtered-table.qza \
  --p-axis sample \
  --m-metadata-file sample-metadata.tsv \
  --m-metadata-column ChytridResult \
  --p-mode sum \
  --o-grouped-table group_plot/grouped-table.qza

Makes taxa barplot from the grouped table

qiime taxa barplot \
  --i-table group_plot/grouped-table.qza \
  --i-taxonomy taxonomy.qza \
  --m-metadata-file group_plot/grouped-metadata.tsv \
  --o-visualization group_plot/grouped-taxa-bar-plots.qzv