Gene-level RNA-Seq Data Analysis (lagacy)
  • Introduction
  • RNA-Seq Analysis Workflow
    • Login to server
    • Obtain data and software
    • Create mapping indices
    • Mapping with STAR
    • Quantification using RSEM
  • De novo assembly using Trinity
    • De novo assembly of RNA-Seq reads
    • Compare de novo reconstructed transcripts to reference annotations
    • Quantification using RSEM
  • Differential expression analysis using R
    • Install R libraries
    • Perform DE analysis
    • Perform DE analysis (Trinity)
    • Extracting DE transcripts and generating heatmaps (Trinity)
  • Visualization
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  • Option
  • Output

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  1. Differential expression analysis using R

Extracting DE transcripts and generating heatmaps (Trinity)

The Trinity package includes scripts that facilitate the extracting of differentially expressed (DE) transcripts (by FDR and/or log2 fold change) and generating heatmaps and plots for visualization.

Execution

Below command will extract DE transcripts that are at least 4-fold DE at a significance of <= 0.001 in any of the pairwise sample comparisons.

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

cd ~/LSLNGS2015/Trinity/edgeR_trans

PATH_TO_TRINITY/Analysis/DifferentialExpression/analyze_diff_expr.pl \
--matrix ../Trinity_trans.TMM.EXPR.matrix -P 1e-3 -C 2

Option

--matrix location of the matrix.normalized.FPKM file

-P p-value cutoff for FDR (default: 0.001)

-C min abs(log2 fold change) (default: 2, i.e. meaning 2^(2) or 4-fold).

--output prefix for output file prefix for output file (default: diffExpr.P${Pvalue}_C${C})

Output

ls -la diffExpr.*

-rw-rw-r-- 1 ycl6 ycl6  3141 Nov  1 16:02 diffExpr.P1e-3_C2.matrix
-rw-rw-r-- 1 ycl6 ycl6  5119 Nov  1 16:02 diffExpr.P1e-3_C2.matrix.log2.centered.dat
-rw-rw-r-- 1 ycl6 ycl6  9026 Nov  1 16:02 diffExpr.P1e-3_C2.matrix.log2.centered.genes_vs_samples_heatmap.pdf
-rw-rw-r-- 1 ycl6 ycl6  4734 Nov  1 16:02 diffExpr.P1e-3_C2.matrix.log2.dat
-rw-rw-r-- 1 ycl6 ycl6   278 Nov  1 16:02 diffExpr.P1e-3_C2.matrix.log2.sample_cor.dat
-rw-rw-r-- 1 ycl6 ycl6  5620 Nov  1 16:02 diffExpr.P1e-3_C2.matrix.log2.sample_cor_matrix.pdf
-rw-rw-r-- 1 ycl6 ycl6  4073 Nov  1 16:02 diffExpr.P1e-3_C2.matrix.R
-rw-rw-r-- 1 ycl6 ycl6 31811 Nov  1 16:02 diffExpr.P1e-3_C2.matrix.RData

The R script diffExpr.P1e-3_C2.matrix.R is generated to include all the R commands used.

We use head to examine the first 10 lines of the file containing the DE transcripts at the specified thresholds

head diffExpr.P1e-3_C2.matrix

        Sp_ds   Sp_hs   Sp_log  Sp_plat
TRINITY_DN12_c0_g2_i1   709.850 85.668  115.982 1045.466
TRINITY_DN210_c0_g1_i1  141.757 461.640 1326.107        247.219
TRINITY_DN28_c0_g1_i1   7488.551        55125.342       918.115 3457.890
TRINITY_DN45_c0_g1_i1   53513.610       23278.810       804.181 31629.110
TRINITY_DN62_c0_g1_i1   369.557 17.474  160.725 4146.868
TRINITY_DN50_c0_g1_i1   46416.667       21292.999       146.745 9106.154
TRINITY_DN261_c0_g1_i1  19545.983       347.357 105.047 25441.557
TRINITY_DN16_c0_g2_i1   1615.428        732.431 77.323  1950.319
TRINITY_DN39_c0_g1_i1   241.880 291.168 0.000   0.000

Use WinSCP to download the PDF file and view it on your PC or laptop.

The diffExpr.P1e-3_C2.matrix.log2.centered.genes_vs_samples_heatmap.pdf shows the clustering result of transcripts along the vertical axis and samples along the horizontal axis.

The diffExpr.P1e-3_C2.matrix.log2.sample_cor_matrix.pdf shows the pairwise sample correlation matrix.

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Last updated 5 years ago

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genes vs. samples heatmap
sample correlation matrix