Perform DE analysis (Trinity)
Create count matrix
Locate util/abundance_estimates_to_matrix.pl in the trinityrnaseq-2.2.0 distribution, and run
cd ~/LSLNGS2015/Trinity
PATH_TO_TRINITY/util/abundance_estimates_to_matrix.pl \
--est_method RSEM --out_prefix Trinity_genes \
RSEM_Sp_ds/Sp_ds.genes.results RSEM_Sp_hs/Sp_hs.genes.results \
RSEM_Sp_log/Sp_log.genes.results RSEM_Sp_plat/Sp_plat.genes.results
PATH_TO_TRINITY/util/abundance_estimates_to_matrix.pl \
--est_method RSEM --out_prefix Trinity_trans \
RSEM_Sp_ds/Sp_ds.isoforms.results RSEM_Sp_hs/Sp_hs.isoforms.results \
RSEM_Sp_log/Sp_log.isoforms.results RSEM_Sp_plat/Sp_plat.isoforms.resultsOptions
--est_method RSEM or eXpress
--out_prefixoutput file prefix (default: 'matrix')
--cross_sample_fpkm_norm choose one of the three normalization methods, TMM, UpperQuartile, none (default: TMM)
Create count matrices
Files created for gene-level count matrix:
ls -la Trinity_genes*
Files created for transcript-level count matrix:
ls -la Trinity_trans*
We use head to examine the first 10 lines of the counts matrix
head Trinity_genes.counts.matrix
head Trinity_trans.counts.matrix
Perform pairwise sample comparisons using edgeR
Locate Analysis/DifferentialExpression/run_DE_analysis.pl in the trinityrnaseq-2.2.0 distribution, and run
Options
--matrix matrix of raw read counts, not the normalized values
--method choose one of the three methods: edgeR, DESeq, DESeq2
--dispersion edgeR dispersion value (default: 0.1)
--output name of directory to place outputs
Output
The edgeR_genes and edgeR_trans folders contain the pairwise sample comparisons results (*.edgeR.DE_results) and M-A and Volcano plots (*.MA_n_Volcano.pdf) for the gene-level and transcript-level analysis respectively.
For example, ls -la edgeR_genes
Use head to examine one of the *.DE_results file.
head edgeR_genes/Trinity_genes.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results
Use WinSCP to download the PDF file and view it on your PC or laptop.
Below shows the M-A and Volcano plots in the PDF file named Trinity_genes.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results.MA_n_Volcano.pdf
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