qiime 2 분석 균군 데이터 코드

10472 단어 균군 분석
1. installation
Linux 서버가 있는 파트너는 콘다 설치를 추천합니다. 윈도우즈 노트북에서 체험하고 싶은 분들은 Virtualbox 가상기기를 사용하여 설치하고 학습할 수 있습니다.
#install conda
conda update conda  #  conda  
conda install wget  #      

#install QIIME2 with conda
wget https://data.qiime2.org/distro/core/qiime2-2018.2-py35-linux-conda.yml  #        
conda env create -n qiime2-2018.2 --file qiime2-2018.2-py35-linux-conda.yml  #         qiime2，           
# OPTIONAL CLEANUP
rm qiime2-2018.2-py35-linux-conda.yml  #      

#activate qiime2
source activate qiime2-2017.12  #      

#deactivate qiime2
souce deactivate   #

2.Obtaining and importing data      
#singleSingleEndFastqManifestPhred33
qiime tools import \
  --type 'SampleData[SequencesWithQuality]' \
  --input-path se-33-manifest \
  --output-path single-end-demux.qza \
  --source-format SingleEndFastqManifestPhred33

    #PairedEndFastqManifestPhred33
qiime tools import \
  --type 'SampleData[PairedEndSequencesWithQuality]' \
  --input-path pe-33-manifest \
  --output-path paired-end-demux.qza \
  --source-format PairedEndFastqManifestPhred33

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

3. Sequence quality control and feature table construction 
qiime dada2 denoise-single \
  --i-demultiplexed-seqs single-end-demux.qza \
  --p-trim-left 0 \
  --p-trunc-len 165 \
  --o-representative-sequences rep-seqs-dada2.qza \
  --o-table table-dada2.qza \
  --o-denoising-stats stats-dada2.qza

mv rep-seqs-dada2.qza rep-seqs.qza
mv table-dada2.qza table.qza

4. FeatureTable and FeatureData summaries 
qiime feature-table summarize \
  --i-table table.qza \
  --o-visualization table.qzv \
  --m-sample-metadata-file sample-metadata.tsv

#      
qiime feature-table tabulate-seqs \
  --i-data rep-seqs.qza \
  --o-visualization rep-seqs.qzv

#qiime tools view rep-seqs.qzv #

5. Generate a tree for phylogenetic diversity analyses 
#     
qiime alignment mafft \
  --i-sequences rep-seqs.qza \
  --o-alignment aligned-rep-seqs.qza

#     
qiime alignment mask \
  --i-alignment aligned-rep-seqs.qza \
  --o-masked-alignment masked-aligned-rep-seqs.qza

#  
qiime phylogeny fasttree \
  --i-alignment masked-aligned-rep-seqs.qza \
  --o-tree unrooted-tree.qza

#         
qiime phylogeny midpoint-root \
  --i-tree unrooted-tree.qza \
  --o-rooted-tree rooted-tree.qza

6.Alpha and beta diversity analysis 
#     
qiime diversity core-metrics-phylogenetic \
  --i-phylogeny rooted-tree.qza \
  --i-table table.qza \
  --p-sampling-depth 12052 \
  --m-metadata-file sample-metadata.tsv \
  --output-dir core-metrics-results

#              ，         ：

# beta   bray_curtis     bray_curtis_distance_matrix.qza 

# alpha   evenness(   ，       )   evenness_vector.qza

# alpha   faith_pd(         )   faith_pd_vector.qza

# beta   jaccard     jaccard_distance_matrix.qza

# alpha   observed_otus(OTU  )   observed_otus_vector.qza

# alpha      (       )   shannon_vector.qza

# beta   unweighted_unifrac    ，      unweighted_unifrac_distance_matrix.qza

# beta   unweighted_unifrac    ，     weighted_unifrac_distance_matrix.qza

#   faith_pd  Alpha           ，      、    ，      
qiime diversity alpha-group-significance \
  --i-alpha-diversity core-metrics-results/faith_pd_vector.qza \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization core-metrics-results/faith-pd-group-significance.qzv

#  evenness        
qiime diversity alpha-group-significance \
  --i-alpha-diversity core-metrics-results/evenness_vector.qza \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization core-metrics-results/evenness-group-significance.qzv

#      ，   qzv   ，    qiime tools view      http://view.qiime2.org/  
#qiime tools view evenness-group-significance.qzv


# BodySite  ，  unweighted_unifrace            
qiime diversity beta-group-significance \
  --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza \
  --m-metadata-file sample-metadata.tsv \
  --m-metadata-category BodySite \
  --o-visualization core-metrics-results/unweighted-unifrac-body-site-significance.qzv \
  --p-pairwise

#        unweighted-unifrac       
qiime emperor plot \
  --i-pcoa core-metrics-results/unweighted_unifrac_pcoa_results.qza \
  --m-metadata-file sample-metadata.tsv \
  --p-custom-axes DaysSinceExperimentStart \
  --o-visualization core-metrics-results/unweighted-unifrac-emperor-DaysSinceExperimentStart.qzv

#Alpha rarefaction plotting
qiime diversity alpha-rarefaction \
  --i-table table.qza \
  --i-phylogeny rooted-tree.qza \
  --p-max-depth 12052 \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization alpha-rarefaction.qzv

7.Training feature classifiers with q2-feature-classifier 
wget \
  -O "85_otus.fasta" \
  "https://data.qiime2.org/2018.11/tutorials/training-feature-classifiers/85_otus.fasta"

wget \
  -O "85_otu_taxonomy.txt" \
  "https://data.qiime2.org/2018.11/tutorials/training-feature-classifiers/85_otu_taxonomy.txt"

qiime tools import \
  --type 'FeatureData[Sequence]' \
  --input-path 85_otus.fasta \
  --output-path 85_otus.qza

qiime tools import \
  --type 'FeatureData[Taxonomy]' \
  --source-format HeaderlessTSVTaxonomyFormat \
  --input-path 85_otu_taxonomy.txt \
  --output-path ref-taxonomy.qza

qiime feature-classifier extract-reads \
  --i-sequences 85_otus.qza \
  --p-f-primer CCAGACTCCTACGGGAGGCAG \
  --p-r-primer CGTATTACCGCGGCTGCTG \
  --o-reads ref-seqs.qza

qiime feature-classifier fit-classifier-naive-bayes \
  --i-reference-reads ref-seqs.qza \
  --i-reference-taxonomy ref-taxonomy.qza \
  --o-classifier classifier.qza

qiime feature-classifier classify-sklearn \
  --i-classifier classifier.qza \
  --i-reads rep-seqs.qza \
  --o-classification taxonomy.qza

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

8. Taxonomic analysis 
#       
qiime taxa barplot \
  --i-table table.qza \
  --i-taxonomy taxonomy.qza \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization taxa-bar-plots.qzv

#Differential abundance testing with ANCOM
    #filter to gut samples
qiime feature-table filter-samples \
  --i-table table.qza \
  --m-metadata-file sample-metadata.tsv \
  --p-where "BodySite='gut'" \
  --o-filtered-table gut-table.qza

    #imputation method
qiime composition add-pseudocount \
  --i-table gut-table.qza \
  --o-composition-table comp-gut-table.qza

   9.Subject 
qiime composition ancom \
  --i-table comp-gut-table.qza \
  --m-metadata-file sample-metadata.tsv \
  --m-metadata-column Subject \
  --o-visualization ancom-Subject.qzv

    #specific taxonomic level
qiime taxa collapse \
  --i-table gut-table.qza \
  --i-taxonomy taxonomy.qza \
  --p-level 6 \
  --o-collapsed-table gut-table-l6.qza

qiime composition add-pseudocount \
  --i-table gut-table-l6.qza \
  --o-composition-table comp-gut-table-l6.qza

qiime composition ancom \
  --i-table comp-gut-table-l6.qza \
  --m-metadata-file sample-metadata.tsv \
  --m-metadata-column Subject \
  --o-visualization l6-ancom-Subject.qzv

#shell ##check shell echo $0 ##Bach shell source tab-qiime 10.Importing phylogenetic trees (unrooted) 
qiime tools import \
  --input-path rep_set.tre \
  --output-path unrooted-tree.qza \
  --type 'Phylogeny[Unrooted]'

qiime phylogeny midpoint-root \
  --i-tree unrooted-tree.qza \
  --o-rooted-tree rooted-tree.qza

11.Importing BIOM v1.0.0   
qiime tools import \
  --input-path otu_table_mc2_w_tax_no_pynast_failures.biom \
  --type 'FeatureTable[Frequency]' \
  --source-format BIOMV100Format \
  --output-path feature-table-1.qza

12.Feature Table summarize   
qiime feature-table summarize \
  --i-table feature-table-1.qza \
  --o-visualization table.qzv \
  --m-sample-metadata-file sample-metadata.tsv

13.Alpha and beta diversity analysis 
qiime diversity core-metrics-phylogenetic \
  --i-phylogeny rooted-tree.qza \
  --i-table feature-table-1.qza \
  --p-sampling-depth 51162 \
  --m-metadata-file sample-metadata.tsv \
  --output-dir core-metrics-results

##alpha-group-significance
qiime diversity alpha-group-significance \
  --i-alpha-diversity core-metrics-results/faith_pd_vector.qza \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization core-metrics-results/faith-pd-group-significance.qzv

qiime diversity alpha-group-significance \
  --i-alpha-diversity core-metrics-results/evenness_vector.qza \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization core-metrics-results/evenness-group-significance.qzv

14.beta-group-significance 
qiime diversity beta-group-significance \
  --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza \
  --m-metadata-file sample-metadata.tsv \
  --m-metadata-column BodySite \
  --o-visualization core-metrics-results/unweighted-unifrac-body-site-significance.qzv \
  --p-pairwise

qiime diversity beta-group-significance \
  --i-distance-matrix core-metrics-results/unweighted_unifrac_distance_matrix.qza \
  --m-metadata-file sample-metadata.tsv \
  --m-metadata-column Subject \
  --o-visualization core-metrics-results/unweighted-unifrac-subject-group-significance.qzv \
  --p-pairwise

##PCoA plot
qiime emperor plot \
  --i-pcoa core-metrics-results/unweighted_unifrac_pcoa_results.qza \
  --m-metadata-file sample-metadata.tsv \
  --p-custom-axes DaysSinceExperimentStart \
  --o-visualization core-metrics-results/unweighted-unifrac-emperor-DaysSinceExperimentStart.qzv

qiime emperor plot \
  --i-pcoa core-metrics-results/bray_curtis_pcoa_results.qza \
  --m-metadata-file sample-metadata.tsv \
  --p-custom-axes DaysSinceExperimentStart \
  --o-visualization core-metrics-results/bray-curtis-emperor-DaysSinceExperimentStart.qzv

#Alpha rarefaction plotting
qiime diversity alpha-rarefaction \
  --i-table feature-table-1.qza \
  --i-phylogeny rooted-tree.qza \
  --p-max-depth 51162 \
  --m-metadata-file sample-metadata.tsv \
  --o-visualization alpha-rarefaction.qzv

#Taxonomic analysis
#Differential abundance testing with ANCOM
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