search

Perform DE analysis (Trinity)

hashtag
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.results

hashtag
Options

--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)

hashtag
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

hashtag
Perform pairwise sample comparisons using edgeR

Locate Analysis/DifferentialExpression/run_DE_analysis.pl in the trinityrnaseq-2.2.0 distribution, and run

hashtag
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

hashtag
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

MA_n_Volcano
PreviousPerform DE analysisNextExtracting DE transcripts and generating heatmaps (Trinity)

Last updated