# Bioinformatics IV: Analysis of Differential Abundant taxa
###### tags: `MAE` `qiime2`

In qiime2

conda init bash
. ~/.bashrc
conda activate /bioware/qiime2-2020.2.0-conda

### Export the ASV table-no chloroplast, no mitochondria

:::success
  qiime tools export
  --input-path table.qza
  --output-path ASV_table
:::

qiime tools export  --input-path table-filtered-no-mitochondria-no-chloroplast.qza   --output-path ASV-filtered-table-no-mitochondria-no-chloroplast

## Convert the exported table from biom format to tsv
:::success
biom convert
-i ASV_table/feature-table.biom
-o ASV_table/feature-table.tsv
--to-tsv
:::

biom convert -i ASV-filtered-table-no-mitochondria-no-chloroplast/feature-table.biom -o ASV-filtered-table-no-mitochondria-no-chloroplast/DAA_feature-table.tsv --to-tsv

#### leave qiime2

conda deactivate

### Get files in new folder

Modify the DAA_feature-table.tsv
Need to remove the first line and the name of the ASV column. Copy as text. name table_frequencies.txt

select the sample name column and the condition you want to test, without headers. Copy as text, name samples.txt

### In Trinity

https://github.com/trinityrnaseq/trinityrnaseq/wiki/Trinity-Differential-Expression

module load bioware
module load trinity

### quality check samples and replicates.

mkdir 01_compare_replicates
cd 01_compare_replicates

### replicas

:::info
--min_rowSums 10

or
--min_rowSums 200
:::

/bioware/trinityrnaseq/Analysis/DifferentialExpression/PtR --matrix ../table_frequencies.txt --samples ../samples.txt --CPM --log2  --min_rowSums 10 --compare_replicates 

cd ..

### overall comparisons

:::info
--min_rowSums 10

or
--min_rowSums 200
:::

mkdir 02_compare_replicates_across_samples

cd 02_compare_replicates_across_samples

#heatmap
/bioware/trinityrnaseq/Analysis/DifferentialExpression/PtR --matrix ../table_frequencies.txt --samples ../samples.txt --CPM --log2  --min_rowSums 10 --sample_cor_matrix

#PCA
/bioware/trinityrnaseq/Analysis/DifferentialExpression/PtR --matrix ../table_frequencies.txt --samples ../samples.txt --CPM --log2  --min_rowSums 10 --center_rows --prin_comp 3 


cd ..

### Differential expression edgeR
:::info

-P 0.001 -C2

or -P 0.05 -C1
:::

#Differential expression edgeR

/bioware/trinityrnaseq/Analysis/DifferentialExpression/run_DE_analysis.pl --matrix  table_frequencies.txt --method edgeR --samples_file samples.txt   --output 03_differential_expression_EDGE  --dispersion 0.1 --reference_sample 1_ocean


cd 03_differential_expression_EDGE 

/bioware/trinityrnaseq/Analysis/DifferentialExpression/analyze_diff_expr.pl --matrix ../table_frequencies.txt -P 0.05 -C 1 --samples ../samples.txt

#clustering 
/bioware/trinityrnaseq/Analysis/DifferentialExpression/define_clusters_by_cutting_tree.pl -R  diffExpr.P0.05_C1.matrix.RData --Ptree 60

#Differential expression edgeR (same but without reordering)

/bioware/trinityrnaseq/Analysis/DifferentialExpression/analyze_diff_expr.pl --matrix  /users/eperedo/saltmarsh/DAA_PRJNA610907/table_frequencies.txt --method edgeR --samples /users/eperedo/saltmarsh/DAA_PRJNA610907/samples.txt  -P 0.05 -C1  --order_columns_by_samples_file --output ordered_by_file

### additional analysis (Deseq2)

/bioware/trinityrnaseq/Analysis/DifferentialExpression/run_DE_analysis.pl --matrix table_frequencies.txt --method DESeq2 --samples_file samples.txt   --output 03_differential_expression #--reference_sample control

cd 03_differential_expression 

/bioware/trinityrnaseq/Analysis/DifferentialExpression/analyze_diff_expr.pl --matrix /users/eperedo/saltmarsh/DAA_PRJNA610907/table_frequencies.txt -P 0.05 -C1 --samples /users/eperedo/saltmarsh/DAA_PRJNA610907/samples.txt

#clustering 
/bioware/trinityrnaseq/Analysis/DifferentialExpression/define_clusters_by_cutting_tree.pl -R  diffExpr.P0.001_C2.matrix.RData --Ptree 60

#Differential expression DESeq2 (same but without reordering)

/bioware/trinityrnaseq/Analysis/DifferentialExpression/analyze_diff_expr.pl --matrix  /users/eperedo/saltmarsh/DAA_PRJNA610907/table_frequencies.txt --method DESeq2 --samples /users/eperedo/saltmarsh/DAA_PRJNA610907/samples.txt  -P 0.001 -C2  --order_columns_by_samples_file --output ordered_by_file

## frequency table for EDGER-- use the raw


qiime taxa collapse --i-table /users/eperedo/MAE2021/qiime/table-filtered-no-mitochondria-no-chloroplast.qza  --i-taxonomy /users/eperedo/MAE2021/qiime/taxonomy.qza --p-level 7 --o-collapsed-table /users/eperedo/MAE2021/qiime/table-filtered-no-mitochondria-no-chloroplast-l7.qza

qiime tools export  --input-path /users/eperedo/MAE2021/qiime/table-filtered-no-mitochondria-no-chloroplast-l7.qza   --output-path /users/eperedo/MAE2021/qiime/ASV-table-filtered-no-mitochondria-no-chloroplast-l7
biom convert -i /users/eperedo/MAE2021/qiime/ASV-table-filtered-no-mitochondria-no-chloroplast-l7/feature-table.biom -o /users/eperedo/MAE2021/qiime/ASV-table-filtered-no-mitochondria-no-chloroplast-l7/DAA_feature-table-l7.tsv --to-tsv

cp /users/eperedo/MAE2021/qiime/ASV-table-filtered-no-mitochondria-no-chloroplast-l7/DAA_feature-table-l7.tsv table-frequencies.txt


## compare replicates

mkdir 01_compare_replicates
cd 01_compare_replicates

### replicas

/bioware/trinityrnaseq/Analysis/DifferentialExpression/PtR --matrix ../table-frequencies.txt --samples ../samples.txt --CPM --log2 --min_rowSums 10 --compare_replicates --barplot_sum_counts 

cd ..


mkdir 02_compare_replicates_across_samples

cd 02_compare_replicates_across_samples

#heatmap
/bioware/trinityrnaseq/Analysis/DifferentialExpression/PtR --matrix ../table-frequencies.txt --samples ../samples.txt --CPM --log2   --sample_cor_matrix --heatmap_colorscheme 'purple,black,yellow'

#PCA
/bioware/trinityrnaseq/Analysis/DifferentialExpression/PtR --matrix ../table-frequencies.txt --samples ../samples.txt --CPM --log2   --center_rows --prin_comp 3 

cd ..


## compare samples

/bioware/trinityrnaseq/Analysis/DifferentialExpression/run_DE_analysis.pl --matrix  table-frequencies.txt --method edgeR --samples_file samples.txt   --output 03_differential_expression_edgeR   --reference_sample ocean

/bioware/trinityrnaseq/Analysis/DifferentialExpression/run_DE_analysis.pl --matrix  table-frequencies.txt --method DESeq2 --samples_file samples.txt   --output 03_differential_expression_DESeq2  --reference_sample ocean


cd 03_differential_expression_EDGE 

/bioware/trinityrnaseq/Analysis/DifferentialExpression/analyze_diff_expr.pl --matrix /users/eperedo/MAE2021/DAA_analysis/taxonomy_level7/table-frequencies.txt -P 1e-3 -C 2 --samples /users/eperedo/MAE2021/DAA_analysis/taxonomy_level7/samples.txt