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analysis.sh
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## output files are in ~/qiime-analysis
## scripts are in ~/Final_Project_Cyano
conda activate qiime2-2022.8
cd ~/qiime-analysis
mkdir fastp_fq
./fastp.sh 150 /tmp/gen711_project_data/cyano/fastqs fastp_fq
mkdir qiime_out
qiime tools import \
--type "SampleData[PairedEndSequencesWithQuality]" \
--input-format CasavaOneEightSingleLanePerSampleDirFmt \
--input-path fastp_fq \
--output-path qiime_out/cyano.qza
qiime cutadapt trim-paired \
--i-demultiplexed-sequences qiime_out/cyano.qza \
--p-cores 4 \
--p-front-f GTGYCAGCMGCCGCGGTAA \
--p-front-r CCGYCAATTYMTTTRAGTTT \
--p-discard-untrimmed \
--p-match-adapter-wildcards \
--verbose \
--o-trimmed-sequences cutadapt_cyano.qza
qiime demux summarize \
--i-data cutadapt_cyano.qza \
--o-visualization demux_cyano.qzv
#Denoising
qiime dada2 denoise-paired \
--i-demultiplexed-seqs cutadapt_cyano.qza \
--p-trunc-len-f 0 \
--p-trunc-len-r 0 \
--p-trim-left-f 0 \
--p-trim-left-r 0 \
--p-n-threads 4 \
--o-denoising-stats denoising-stats.qza \
--o-table feature_table.qza \
--o-representative-sequences rep-seqs.qza
qiime metadata tabulate \
--m-input-file denoising-stats.qza \
--o-visualization denoising-stats.qzv
qiime feature-table tabulate-seqs \
--i-data rep-seqs.qza \
--o-visualization rep-seqs.qzv
# Taxonomy assignment
#classify rep seqs
qiime feature-classifier classify-sklearn \
--i-classifier /tmp/gen711_project_data/cyano/classifier_16S_V4-V5.qza \
--i-reads rep-seqs.qza \
--o-classification cyano_taxo
#
qiime feature-classifier classify-consensus-vsearch \
--i-query rep-seqs.qza \
--i-reference-reads /tmp/gen711_project_data/reference_databases/silva132_99.qza \
--i-reference-taxonomy /tmp/gen711_project_data/reference_databases/majority_taxonomy_all_levels.qza \
--p-maxaccepts 10 \
--p-query-cov 0.80 \
--p-perc-identity 0.9 \
--p-threads 36 \
--o-classification taxonomy.qza
# Barplot
qiime taxa barplot \
--i-table feature_table.qza \
--m-metadata-file /tmp/gen711_project_data/cyano/metadata.tsv \
--i-taxonomy taxonomy.qza \
--o-visualization my-barplot.qzv
# Feature table
qiime feature-table filter-samples \
--i-table feature_table.qza \
--m-metadata-file /tmp/gen711_project_data/cyano/metadata.tsv \
--o-filtered-table new_samples_table.qza
# PCA Plot
qiime phylogeny align-to-tree-mafft-fasttree \
--i-sequences rep-seqs.qza \
--o-alignment algn \
--o-masked-alignment masked-aligned-rep-seqs \
--o-tree unrooted-tree \
--o-rooted-tree rooted-tree \
--p-n-threads 8
#
qiime diversity core-metrics-phylogenetic \
--i-phylogeny rooted-tree.qza \
--i-table feature_table.qza \
--p-sampling-depth 1500 \
--m-metadata-file /tmp/gen711_project_data/cyano/metadata.tsv \
--output-dir core-metrics-phylogenetics
#
qiime feature-table relative-frequency \
--i-table core-metrics-phylogenetics/rarefied_table.qza \
--o-relative-frequency-table core-metrics-phylogenetics/relative_rarefied_table
#
qiime diversity pcoa-biplot \
--i-features core-metrics-phylogenetics/relative_rarefied_table.qza \
--i-pcoa core-metrics-phylogenetics/unweighted_unifrac_pcoa_results.qza \
--o-biplot core-metrics-phylogenetics/unweighted_unifrac_pcoa_biplot
#error
qiime emperor biplot \
--i-biplot core-metrics-phylogenetics/unweighted_unifrac_pcoa_biplot.qza \
--m-sample-metadata-file /tmp/gen711_project_data/cyano/metadata.tsv \
--o-visualization core-metrics-phylogenetics/unweighted_unifrac_pcoa_biplot
###