Trinity tools (Part III-B): Analysis of de novo transcript/genes assembly using edgeR

Reference, Resources and Data

Nature Protocols volume 8, pages 1494-1512 (2013)

Data: http://sourceforge.net/projects/trinityrnaseq/files/misc/TrinityNatureProtocolTutorial.tgz

Machine: CentOS Linux 7 on Virtual Box

Starting directory and subdirectory contents

trinity2$ ls -l

drwxrwxr-x. 3 bdash bdash 149 Mar 24 17:58 Sp_ds

-rwxrwx—. 1 bdash bdash 175846179 Mar 24 13:01 Sp.ds.1M.left.fq

-rwxrwx—. 1 bdash bdash 175846179 Mar 24 13:01 Sp.ds.1M.right.fq

drwxrwxr-x. 3 bdash bdash 149 Mar 24 18:11 Sp_hs

-rwxrwx—. 1 bdash bdash 175736042 Mar 24 13:02 Sp.hs.1M.left.fq

-rwxrwx—. 1 bdash bdash 175736042 Mar 24 13:02 Sp.hs.1M.right.fq

drwxrwxr-x. 3 bdash bdash 149 Mar 24 18:28 Sp_log

-rwxrwx—. 1 bdash bdash 175741215 Mar 24 13:03 Sp.log.1M.left.fq

-rwxrwx—. 1 bdash bdash 175741215 Mar 24 13:03 Sp.log.1M.right.fq

drwxrwxr-x. 3 bdash bdash 149 Mar 24 18:20 Sp_plat

-rwxrwx—. 1 bdash bdash 175899533 Mar 24 13:03 Sp.plat.1M.left.fq

-rwxrwx—. 1 bdash bdash 175899533 Mar 24 13:03 Sp.plat.1M.right.fq

drwxr-xr-x. 2 bdash bdash 4096 Mar 25 11:27 trinity_out_dir

Combine RSEM-computed fragment counts into a matrix file

trinity2$ abundance_estimates_to_matrix.pl –est_method RSEM Sp_ds/RSEM.genes.results Sp_hs/RSEM.genes.results Sp_plat/RSEM.genes.results Sp_log/RSEM.ge nes.results –gene_trans_map trinity_out_dir/Trinity.fasta.gene_trans_map –name_sample_by_basedir –out_prefix genes

Some log readouts

-reading file: Sp_ds/RSEM.genes.results

-reading file: Sp_hs/RSEM.genes.results

-reading file: Sp_plat/RSEM.genes.results

-reading file: Sp_log/RSEM.genes.results

Outputting combined matrix.

/home/bdash/miniconda2/opt/trinity-2.5.1/util/support_scripts/run_TMM_scale_matrix.pl –matrix genes.isoform.TPM.not_cross_norm > genes.isoform.TMM.EXPR.matrixCMD: R –vanilla -q < __tmp_runTMM.R 1>&2

library(edgeR)

Loading required package: limma

rnaseqMatrix = read.table(“genes.isoform.TPM.not_cross_norm”, header=T, row.names=1, com=’’, check.names=F)

rnaseqMatrix = as.matrix(rnaseqMatrix)

rnaseqMatrix = round(rnaseqMatrix)

exp_study = DGEList(counts=rnaseqMatrix, group=factor(colnames(rnaseqMatrix)))

exp_study = calcNormFactors(exp_study)

exp_study$samples$eff.lib.size = exp_study$samples$lib.size * exp_study$samples$norm.factors

write.table(exp_study$samples, file=“genes.isoform.TPM.not_cross_norm.TMM_info.txt”, quote=F, sep=", row.names=F)

/home/bdash/miniconda2/opt/trinity-2.5.1/util/support_scripts/run_TMM_scale_matrix.pl –matrix genes.gene.TPM.not_cross_norm genes.gene.TMM.EXPR.matrixCMD: R –vanilla -q < __tmp_runTMM.R 1>&2

library(edgeR)

Loading required package: limma

rnaseqMatrix = read.table(“genes.gene.TPM.not_cross_norm”, header=T, row.names=1, com=’’, check.names=F)

rnaseqMatrix = as.matrix(rnaseqMatrix)

rnaseqMatrix = round(rnaseqMatrix)

exp_study = DGEList(counts=rnaseqMatrix, group=factor(colnames(rnaseqMatrix)))

exp_study = calcNormFactors(exp_study)

exp_study$samples$eff.lib.size = exp_study$samples$lib.size * exp_study$samples$norm.factors

write.table(exp_study$samples, file=“genes.gene.TPM.not_cross_norm.TMM_info.txt”, quote=F, sep=", row.names=F)

Additional directory/subdirectory/files created following the above run

trinity2$ ls -l

-rw-rw-r–. 1 bdash bdash 384230 Mar 25 11:31 genes.gene.counts.matrix

-rw-rw-r–. 1 bdash bdash 419995 Mar 25 11:31 genes.gene.TMM.EXPR.matrix

-rw-rw-r–. 1 bdash bdash 361306 Mar 25 11:31 genes.gene.TPM.not_cross_norm

-rw-rw-r–. 1 bdash bdash 234 Mar 25 11:31 genes.gene.TPM.not_cross_norm.TMM_info.txt

-rw-rw-r–. 1 bdash bdash 431596 Mar 25 11:31 genes.isoform.counts.matrix

-rw-rw-r–. 1 bdash bdash 475183 Mar 25 11:31 genes.isoform.TMM.EXPR.matrix

-rw-rw-r–. 1 bdash bdash 411944 Mar 25 11:31 genes.isoform.TPM.not_cross_norm

-rw-rw-r–. 1 bdash bdash 234 Mar 25 11:31 genes.isoform.TPM.not_cross_norm.TMM_info.txt

-rw-rw-r–. 1 bdash bdash 520 Mar 25 11:31 __tmp_runTMM.R

A file readout

trinity2$ head genes.isoform.counts.matrix

                Sp_ds   Sp_hs   Sp_plat Sp_log

TRINITY_DN2933_c0_g1_i1 35.00 34.00 16.00 74.00

TRINITY_DN2950_c0_g3_i1 0.00 0.00 2.00 0.00

TRINITY_DN401_c0_g1_i1 0.00 0.00 1.00 0.00

TRINITY_DN3102_c0_g1_i1 285.00 223.00 225.00 493.00

TRINITY_DN3859_c1_g2_i1 0.00 0.00 8.50 6.59

TRINITY_DN3417_c0_g1_i1 165.00 184.00 89.00 168.00

TRINITY_DN1415_c0_g1_i1 73.57 55.63 30.38 82.86

TRINITY_DN1540_c0_g1_i1 58.00 34.00 68.00 15.00

TRINITY_DN3251_c0_g4_i1 8.00 1.00 4.00 3.00

Run DE analysis for the genes: Produces volcano plots and other results

run_DE_analysis.pl –matrix isoforms.isoform.counts.matrix –output isoforms_DE –dispersion 0.1 –method edgeR

Partial readout of the log

-note, no biological replicates identified, so setting min reps = 2.

Got 4 samples, and got: 5 data fields.

Header: Sp_ds Sp_hs Sp_plat Sp_log

Next: TRINITY_DN2933_c0_g1_i1 35.00 34.00 16.00 74.00

$VAR1 = { ‘Sp_log’ => 4, ‘Sp_plat’ => 3, ‘Sp_ds’ => 1, ‘Sp_hs’ => 2 };

$VAR1 = { ‘Sp_log’ => [ ‘Sp_log’ ], ‘Sp_ds’ => [ ‘Sp_ds’ ], ‘Sp_plat’ => [ ‘Sp_plat’ ], ‘Sp_hs’ => [ ‘Sp_hs’ ] };

Contrasts to perform are: $VAR1 = [ [ ‘Sp_ds’, ‘Sp_hs’ ],

      [
        'Sp_ds',
        'Sp_log'
      ],
      
      [
        'Sp_ds',
        'Sp_plat'
      ],
      
      [
        'Sp_hs',
        'Sp_log'
      ],
      
      [
        'Sp_hs',
        'Sp_plat'
      ],
      
      [
        'Sp_log',
        'Sp_plat'
      ]
    ];

CMD: R –vanilla -q < genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.Sp_ds.vs.Sp_hs.EdgeR.Rscript

CMD: R –vanilla -q < genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.Sp_ds.vs.Sp_log.EdgeR.Rscript

CMD: R –vanilla -q < genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.Sp_ds.vs.Sp_plat.EdgeR.Rscript

CMD: R –vanilla -q < genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.Sp_hs.vs.Sp_log.EdgeR.Rscript

CMD: R –vanilla -q < genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.Sp_hs.vs.Sp_plat.EdgeR.Rscript

CMD: R –vanilla -q < genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.Sp_log.vs.Sp_plat.EdgeR.Rscript

Additional directory/subdirectory/files created following the above run

trinity2]$ ls -l

drwxrwxr-x. 2 bdash bdash 4096 Mar 25 11:38 genes_DE

trinity2$ ls -l genes_DE/

-rw-rw-r–. 1 bdash bdash 232175 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.count_matrix

-rw-rw-r–. 1 bdash bdash 744943 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results

-rw-rw-r–. 1 bdash bdash 56194 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.MA_n_Volcano.pdf

-rw-rw-r–. 1 bdash bdash 1234 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.Sp_ds.vs.Sp_hs.EdgeR.Rscript

-rw-rw-r–. 1 bdash bdash 236512 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.count_matrix

-rw-rw-r–. 1 bdash bdash 785584 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.DE_results

-rw-rw-r–. 1 bdash bdash 58462 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.DE_results.MA_n_Volcano.pdf

-rw-rw-r–. 1 bdash bdash 1240 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.Sp_ds.vs.Sp_log.EdgeR.Rscript

-rw-rw-r–. 1 bdash bdash 238730 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.count_matrix

-rw-rw-r–. 1 bdash bdash 750585 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.DE_results

-rw-rw-r–. 1 bdash bdash 54153 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.DE_results.MA_n_Volcano.pdf

-rw-rw-r–. 1 bdash bdash 1246 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.Sp_ds.vs.Sp_plat.EdgeR.Rscript

-rw-rw-r–. 1 bdash bdash 231199 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.count_matrix

-rw-rw-r–. 1 bdash bdash 743784 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.DE_results

-rw-rw-r–. 1 bdash bdash 55135 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.DE_results.MA_n_Volcano.pdf

-rw-rw-r–. 1 bdash bdash 1240 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.Sp_hs.vs.Sp_log.EdgeR.Rscript

-rw-rw-r–. 1 bdash bdash 235014 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.count_matrix

-rw-rw-r–. 1 bdash bdash 795142 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.DE_results

-rw-rw-r–. 1 bdash bdash 58927 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.DE_results.MA_n_Volcano.pdf

-rw-rw-r–. 1 bdash bdash 1246 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.Sp_hs.vs.Sp_plat.EdgeR.Rscript

-rw-rw-r–. 1 bdash bdash 240471 Mar 25 11:38 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.count_matrix

-rw-rw-r–. 1 bdash bdash 833522 Mar 25 11:38 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results

-rw-rw-r–. 1 bdash bdash 62074 Mar 25 11:38 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results.MA_n_Volcano.pdf

-rw-rw-r–. 1 bdash bdash 1252 Mar 25 11:38 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.Sp_log.vs.Sp_plat.EdgeR.Rscript

“png”, “jpeg”, “jpg”, “tiff”, “pnm”

pdf_convert('genes_DE/genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.MA_n_Volcano.pdf', format = "tiff", pages = NULL, filenames = NULL, dpi = 300, antialias = TRUE, opw = "", upw = "", verbose = TRUE)

Converting page 1 to genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.MA_n_Volcano_1.tiff... done!
Converting page 2 to genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.MA_n_Volcano_2.tiff... done!
[1] "genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.MA_n_Volcano_1.tiff"
[2] "genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.MA_n_Volcano_2.tiff"

Analyze differentail expression of genes: Produces heatmap and other results

genes_DE$analyze_diff_expr.pl –matrix ../genes.isoform.TMM.EXPR.matrix –output DE_genes

** Found 568 features as differentially expressed.

CMD: /home/bdash/miniconda2/opt/trinity-2.5.1/Analysis/DifferentialExpression/PtR -m DE_genes.matrix –log2 –heatmap –min_colSums 0 –min_rowSums 0 –gene_dist euclidean –sample_dist euclidean –sample_cor_matrix –center_rows –save CMD: R –vanilla -q < DE_genes.matrix.R

library(cluster)

library(Biobase)

……………………

library(qvalue)

……………………

source(“/home/bdash/miniconda2/opt/trinity-2.5.1/Analysis/DifferentialExpression/R/heatmap.3.R”)

source(“/home/bdash/miniconda2/opt/trinity-2.5.1/Analysis/DifferentialExpression/R/misc_rnaseq_funcs.R”)

source(“/home/bdash/miniconda2/opt/trinity-2.5.1/Analysis/DifferentialExpression/R/pairs3.R”)

source(“/home/bdash/miniconda2/opt/trinity-2.5.1/Analysis/DifferentialExpression/R/vioplot2.R”)

Additional directrories/subdirectories/files crated following aobe run

genes_DE$ ls -l

-rw-rw-r–. 1 bdash bdash 107 Mar 25 11:44 DE_feature_counts.P0.001_C2.matrix

-rw-rw-r–. 1 bdash bdash 30267 Mar 25 11:44 DE_genes.matrix

-rw-rw-r–. 1 bdash bdash 53939 Mar 25 11:44 DE_genes.matrix.log2.centered.dat

-rw-rw-r–. 1 bdash bdash 36613 Mar 25 11:44 DE_genes.matrix.log2.centered.genes_vs_samples_heatmap.pdf

-rw-rw-r–. 1 bdash bdash 290 Mar 25 11:44 DE_genes.matrix.log2.centered.sample_cor.dat

-rw-rw-r–. 1 bdash bdash 5864 Mar 25 11:44 DE_genes.matrix.log2.centered.sample_cor_matrix.pdf

-rw-rw-r–. 1 bdash bdash 3857 Mar 25 11:44 DE_genes.matrix.R

-rw-rw-r–. 1 bdash bdash 1342218 Mar 25 11:44 DE_genes.matrix.RData

-rw-rw-r–. 1 bdash bdash 10031 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.P0.001_C2.DE.subset

-rw-rw-r–. 1 bdash bdash 5830 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.P0.001_C2.Sp_ds-UP.subset

-rw-rw-r–. 1 bdash bdash 4268 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.P0.001_C2.Sp_hs-UP.subset

-rw-rw-r–. 1 bdash bdash 24 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.samples

-rw-rw-r–. 1 bdash bdash 28526 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.DE_results.P0.001_C2.DE.subset

-rw-rw-r–. 1 bdash bdash 19319 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.DE_results.P0.001_C2.Sp_ds-UP.subset

-rw-rw-r–. 1 bdash bdash 9274 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.DE_results.P0.001_C2.Sp_log-UP.subset

-rw-rw-r–. 1 bdash bdash 26 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.DE_results.samples

-rw-rw-r–. 1 bdash bdash 6892 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.DE_results.P0.001_C2.DE.subset

-rw-rw-r–. 1 bdash bdash 2225 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.DE_results.P0.001_C2.Sp_ds-UP.subset

-rw-rw-r–. 1 bdash bdash 4734 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.DE_results.P0.001_C2.Sp_plat-UP.subset

-rw-rw-r–. 1 bdash bdash 28 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.DE_results.samples

-rw-rw-r–. 1 bdash bdash 21828 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.DE_results.P0.001_C2.DE.subset

-rw-rw-r–. 1 bdash bdash 17739 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.DE_results.P0.001_C2.Sp_hs-UP.subset

-rw-rw-r–. 1 bdash bdash 4156 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.DE_results.P0.001_C2.Sp_log-UP.subset

-rw-rw-r–. 1 bdash bdash 26 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.DE_results.samples

-rw-rw-r–. 1 bdash bdash 35385 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.DE_results.P0.001_C2.DE.subset

-rw-rw-r–. 1 bdash bdash 17568 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.DE_results.P0.001_C2.Sp_hs-UP.subset

-rw-rw-r–. 1 bdash bdash 17884 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.DE_results.P0.001_C2.Sp_plat-UP.subset

-rw-rw-r–. 1 bdash bdash 28 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.DE_results.samples

-rw-rw-r–. 1 bdash bdash 52861 Mar 25 11:44 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results.P0.001_C2.DE.subset

-rw-rw-r–. 1 bdash bdash 19495 Mar 25 11:44 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results.P0.001_C2.Sp_log-UP.subset

-rw-rw-r–. 1 bdash bdash 33433 Mar 25 11:44 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results.P0.001_C2.Sp_plat-UP.subset

-rw-rw-r–. 1 bdash bdash 30 Mar 25 11:44 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results.samples

library(pdftools)

pdf_convert('genes_DE/DE_genes.matrix.log2.centered.genes_vs_samples_heatmap.pdf', format = "jpeg", pages = NULL, filenames = NULL, dpi = 300, antialias = TRUE, opw = "", upw = "", verbose = TRUE)

Converting page 1 to DE_genes.matrix.log2.centered.genes_vs_samples_heatmap_1.jpeg... done!
[1] "DE_genes.matrix.log2.centered.genes_vs_samples_heatmap_1.jpeg"

DE_genes.matrix.heatmap

pdf_convert('genes_DE/DE_genes.matrix.log2.centered.sample_cor_matrix.pdf', format = "jpeg", pages = NULL, filenames = NULL, dpi = 300, antialias = TRUE, opw = "", upw = "", verbose = TRUE)

Converting page 1 to DE_genes.matrix.log2.centered.sample_cor_matrix_1.jpeg... done!
[1] "DE_genes.matrix.log2.centered.sample_cor_matrix_1.jpeg"

DE_genes.cor_matrix

---
title: "Trinity tools (Part III-B): Analysis of de novo transcript/genes assembly using edgeR"
author: "Bhagirathi Dash"
date: "March 19, 2019"
output: html_notebook
---

## Reference, Resources and Data

### Nature Protocols volume 8, pages 1494-1512 (2013)

### Data: http://sourceforge.net/projects/trinityrnaseq/files/misc/TrinityNatureProtocolTutorial.tgz

### Machine: CentOS Linux 7 on Virtual Box 


## Starting directory and subdirectory contents

### trinity2$ ls -l


drwxrwxr-x. 3 bdash bdash       149 Mar 24 17:58 Sp_ds

-rwxrwx---. 1 bdash bdash 175846179 Mar 24 13:01 Sp.ds.1M.left.fq

-rwxrwx---. 1 bdash bdash 175846179 Mar 24 13:01 Sp.ds.1M.right.fq

drwxrwxr-x. 3 bdash bdash       149 Mar 24 18:11 Sp_hs

-rwxrwx---. 1 bdash bdash 175736042 Mar 24 13:02 Sp.hs.1M.left.fq

-rwxrwx---. 1 bdash bdash 175736042 Mar 24 13:02 Sp.hs.1M.right.fq

drwxrwxr-x. 3 bdash bdash       149 Mar 24 18:28 Sp_log

-rwxrwx---. 1 bdash bdash 175741215 Mar 24 13:03 Sp.log.1M.left.fq

-rwxrwx---. 1 bdash bdash 175741215 Mar 24 13:03 Sp.log.1M.right.fq

drwxrwxr-x. 3 bdash bdash       149 Mar 24 18:20 Sp_plat

-rwxrwx---. 1 bdash bdash 175899533 Mar 24 13:03 Sp.plat.1M.left.fq

-rwxrwx---. 1 bdash bdash 175899533 Mar 24 13:03 Sp.plat.1M.right.fq

drwxr-xr-x. 2 bdash bdash      4096 Mar 25 11:27 trinity_out_dir




## Combine RSEM-computed fragment counts into a matrix file


### trinity2$ abundance_estimates_to_matrix.pl --est_method RSEM Sp_ds/RSEM.genes.results Sp_hs/RSEM.genes.results Sp_plat/RSEM.genes.results Sp_log/RSEM.ge  nes.results --gene_trans_map trinity_out_dir/Trinity.fasta.gene_trans_map --name_sample_by_basedir --out_prefix genes

#### Some log readouts

-reading file: Sp_ds/RSEM.genes.results

-reading file: Sp_hs/RSEM.genes.results

-reading file: Sp_plat/RSEM.genes.results

-reading file: Sp_log/RSEM.genes.results


* Outputting combined matrix.

/home/bdash/miniconda2/opt/trinity-2.5.1/util/support_scripts/run_TMM_scale_matrix.pl --matrix genes.isoform.TPM.not_cross_norm > genes.isoform.TMM.EXPR.matrixCMD: R --vanilla -q < __tmp_runTMM.R 1>&2 

> library(edgeR)

Loading required package: limma

> rnaseqMatrix = read.table("genes.isoform.TPM.not_cross_norm", header=T, row.names=1, com='', check.names=F)

> rnaseqMatrix = as.matrix(rnaseqMatrix)

> rnaseqMatrix = round(rnaseqMatrix)

> exp_study = DGEList(counts=rnaseqMatrix, group=factor(colnames(rnaseqMatrix)))

> exp_study = calcNormFactors(exp_study)

> exp_study$samples$eff.lib.size = exp_study$samples$lib.size * exp_study$samples$norm.factors

> write.table(exp_study$samples, file="genes.isoform.TPM.not_cross_norm.TMM_info.txt", quote=F, sep="\t", row.names=F)

> 
/home/bdash/miniconda2/opt/trinity-2.5.1/util/support_scripts/run_TMM_scale_matrix.pl --matrix genes.gene.TPM.not_cross_norm 
> genes.gene.TMM.EXPR.matrixCMD: R --vanilla -q < __tmp_runTMM.R 1>&2 

> library(edgeR)

Loading required package: limma

> rnaseqMatrix = read.table("genes.gene.TPM.not_cross_norm", header=T, row.names=1, com='', check.names=F)

> rnaseqMatrix = as.matrix(rnaseqMatrix)

> rnaseqMatrix = round(rnaseqMatrix)

> exp_study = DGEList(counts=rnaseqMatrix, group=factor(colnames(rnaseqMatrix)))

> exp_study = calcNormFactors(exp_study)

> exp_study$samples$eff.lib.size = exp_study$samples$lib.size * exp_study$samples$norm.factors

> write.table(exp_study$samples, file="genes.gene.TPM.not_cross_norm.TMM_info.txt", quote=F, sep="\t", row.names=F)


### Additional directory/subdirectory/files created following the above run

#### trinity2$ ls -l

-rw-rw-r--. 1 bdash bdash    384230 Mar 25 11:31 genes.gene.counts.matrix

-rw-rw-r--. 1 bdash bdash    419995 Mar 25 11:31 genes.gene.TMM.EXPR.matrix

-rw-rw-r--. 1 bdash bdash    361306 Mar 25 11:31 genes.gene.TPM.not_cross_norm

-rw-rw-r--. 1 bdash bdash       234 Mar 25 11:31 genes.gene.TPM.not_cross_norm.TMM_info.txt

**-rw-rw-r--. 1 bdash bdash    431596 Mar 25 11:31 genes.isoform.counts.matrix**

**-rw-rw-r--. 1 bdash bdash    475183 Mar 25 11:31 genes.isoform.TMM.EXPR.matrix**

-rw-rw-r--. 1 bdash bdash    411944 Mar 25 11:31 genes.isoform.TPM.not_cross_norm

-rw-rw-r--. 1 bdash bdash       234 Mar 25 11:31 genes.isoform.TPM.not_cross_norm.TMM_info.txt

-rw-rw-r--. 1 bdash bdash       520 Mar 25 11:31 __tmp_runTMM.R



### A file readout

#### trinity2$ head genes.isoform.counts.matrix 

	                Sp_ds	Sp_hs	Sp_plat	Sp_log
	
TRINITY_DN2933_c0_g1_i1	35.00	34.00	16.00	74.00

TRINITY_DN2950_c0_g3_i1	0.00	0.00	2.00	0.00

TRINITY_DN401_c0_g1_i1	0.00	0.00	1.00	0.00

TRINITY_DN3102_c0_g1_i1	285.00	223.00	225.00	493.00

TRINITY_DN3859_c1_g2_i1	0.00	0.00	8.50	6.59

TRINITY_DN3417_c0_g1_i1	165.00	184.00	89.00	168.00

TRINITY_DN1415_c0_g1_i1	73.57	55.63	30.38	82.86

TRINITY_DN1540_c0_g1_i1	58.00	34.00	68.00	15.00

TRINITY_DN3251_c0_g4_i1	8.00	1.00	4.00	3.00



## Run DE analysis for the genes: Produces volcano plots and other results

### run_DE_analysis.pl --matrix isoforms.isoform.counts.matrix --output isoforms_DE --dispersion 0.1 --method edgeR


### Partial readout of the log

-note, no biological replicates identified, so setting min reps = 2.

Got 4 samples, and got: 5 data fields.

Header: Sp_ds	Sp_hs	Sp_plat	Sp_log

Next: TRINITY_DN2933_c0_g1_i1	35.00	34.00	16.00	74.00

$VAR1 = {
          'Sp_log' => 4,
          'Sp_plat' => 3,
          'Sp_ds' => 1,
          'Sp_hs' => 2
        };
        
$VAR1 = {
          'Sp_log' => [
                        'Sp_log'
                      ],
          'Sp_ds' => [
                       'Sp_ds'
                     ],
          'Sp_plat' => [
                         'Sp_plat'
                       ],
          'Sp_hs' => [
                       'Sp_hs'
                     ]
        };
        
Contrasts to perform are: $VAR1 = [
          [
            'Sp_ds',
            'Sp_hs'
          ],
          
          [
            'Sp_ds',
            'Sp_log'
          ],
          
          [
            'Sp_ds',
            'Sp_plat'
          ],
          
          [
            'Sp_hs',
            'Sp_log'
          ],
          
          [
            'Sp_hs',
            'Sp_plat'
          ],
          
          [
            'Sp_log',
            'Sp_plat'
          ]
        ];

CMD: R --vanilla -q < genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.Sp_ds.vs.Sp_hs.EdgeR.Rscript

CMD: R --vanilla -q < genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.Sp_ds.vs.Sp_log.EdgeR.Rscript

CMD: R --vanilla -q < genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.Sp_ds.vs.Sp_plat.EdgeR.Rscript

CMD: R --vanilla -q < genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.Sp_hs.vs.Sp_log.EdgeR.Rscript

CMD: R --vanilla -q < genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.Sp_hs.vs.Sp_plat.EdgeR.Rscript

CMD: R --vanilla -q < genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.Sp_log.vs.Sp_plat.EdgeR.Rscript


### Additional directory/subdirectory/files created following the above run

#### trinity2]$ ls -l

**drwxrwxr-x. 2 bdash bdash      4096 Mar 25 11:38 genes_DE**


#### trinity2$ ls -l genes_DE/

-rw-rw-r--. 1 bdash bdash 232175 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.count_matrix

-rw-rw-r--. 1 bdash bdash 744943 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results

-rw-rw-r--. 1 bdash bdash  56194 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.MA_n_Volcano.pdf

-rw-rw-r--. 1 bdash bdash   1234 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.Sp_ds.vs.Sp_hs.EdgeR.Rscript

-rw-rw-r--. 1 bdash bdash 236512 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.count_matrix

-rw-rw-r--. 1 bdash bdash 785584 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.DE_results

-rw-rw-r--. 1 bdash bdash  58462 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.DE_results.MA_n_Volcano.pdf

-rw-rw-r--. 1 bdash bdash   1240 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.Sp_ds.vs.Sp_log.EdgeR.Rscript

-rw-rw-r--. 1 bdash bdash 238730 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.count_matrix

-rw-rw-r--. 1 bdash bdash 750585 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.DE_results

-rw-rw-r--. 1 bdash bdash  54153 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.DE_results.MA_n_Volcano.pdf

-rw-rw-r--. 1 bdash bdash   1246 Mar 25 11:38 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.Sp_ds.vs.Sp_plat.EdgeR.Rscript

-rw-rw-r--. 1 bdash bdash 231199 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.count_matrix

-rw-rw-r--. 1 bdash bdash 743784 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.DE_results

-rw-rw-r--. 1 bdash bdash  55135 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.DE_results.MA_n_Volcano.pdf

-rw-rw-r--. 1 bdash bdash   1240 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.Sp_hs.vs.Sp_log.EdgeR.Rscript

-rw-rw-r--. 1 bdash bdash 235014 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.count_matrix

-rw-rw-r--. 1 bdash bdash 795142 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.DE_results

-rw-rw-r--. 1 bdash bdash  58927 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.DE_results.MA_n_Volcano.pdf

-rw-rw-r--. 1 bdash bdash   1246 Mar 25 11:38 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.Sp_hs.vs.Sp_plat.EdgeR.Rscript

-rw-rw-r--. 1 bdash bdash 240471 Mar 25 11:38 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.count_matrix

-rw-rw-r--. 1 bdash bdash 833522 Mar 25 11:38 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results

-rw-rw-r--. 1 bdash bdash  62074 Mar 25 11:38 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results.MA_n_Volcano.pdf

-rw-rw-r--. 1 bdash bdash   1252 Mar 25 11:38 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.Sp_log.vs.Sp_plat.EdgeR.Rscript


##  “png”, “jpeg”, “jpg”, “tiff”, “pnm”

```{r}
pdf_convert('genes_DE/genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.MA_n_Volcano.pdf', format = "tiff", pages = NULL, filenames = NULL, dpi = 300, antialias = TRUE, opw = "", upw = "", verbose = TRUE)
```




















## Analyze differentail expression of genes: Produces heatmap and other results



### genes_DE$analyze_diff_expr.pl --matrix ../genes.isoform.TMM.EXPR.matrix --output DE_genes


** Found 568 features as differentially expressed.

CMD: /home/bdash/miniconda2/opt/trinity-2.5.1/Analysis/DifferentialExpression/PtR -m DE_genes.matrix --log2 --heatmap --min_colSums 0 --min_rowSums 0 --gene_dist euclidean --sample_dist euclidean --sample_cor_matrix --center_rows --save 
CMD: R --vanilla -q < DE_genes.matrix.R

> library(cluster)

> library(Biobase)


........................

> library(qvalue)

........................

> source("/home/bdash/miniconda2/opt/trinity-2.5.1/Analysis/DifferentialExpression/R/heatmap.3.R")

> source("/home/bdash/miniconda2/opt/trinity-2.5.1/Analysis/DifferentialExpression/R/misc_rnaseq_funcs.R")

> source("/home/bdash/miniconda2/opt/trinity-2.5.1/Analysis/DifferentialExpression/R/pairs3.R")

> source("/home/bdash/miniconda2/opt/trinity-2.5.1/Analysis/DifferentialExpression/R/vioplot2.R")


### Additional directrories/subdirectories/files crated following aobe run

genes_DE$ ls -l

-rw-rw-r--. 1 bdash bdash     107 Mar 25 11:44 DE_feature_counts.P0.001_C2.matrix

-rw-rw-r--. 1 bdash bdash   30267 Mar 25 11:44 DE_genes.matrix

-rw-rw-r--. 1 bdash bdash   53939 Mar 25 11:44 DE_genes.matrix.log2.centered.dat

-rw-rw-r--. 1 bdash bdash   36613 Mar 25 11:44 DE_genes.matrix.log2.centered.genes_vs_samples_heatmap.pdf

-rw-rw-r--. 1 bdash bdash     290 Mar 25 11:44 DE_genes.matrix.log2.centered.sample_cor.dat

-rw-rw-r--. 1 bdash bdash    5864 Mar 25 11:44 DE_genes.matrix.log2.centered.sample_cor_matrix.pdf

-rw-rw-r--. 1 bdash bdash    3857 Mar 25 11:44 DE_genes.matrix.R

-rw-rw-r--. 1 bdash bdash 1342218 Mar 25 11:44 DE_genes.matrix.RData

-rw-rw-r--. 1 bdash bdash   10031 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.P0.001_C2.DE.subset

-rw-rw-r--. 1 bdash bdash    5830 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.P0.001_C2.Sp_ds-UP.subset

-rw-rw-r--. 1 bdash bdash    4268 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.P0.001_C2.Sp_hs-UP.subset

-rw-rw-r--. 1 bdash bdash      24 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_hs.edgeR.DE_results.samples

-rw-rw-r--. 1 bdash bdash   28526 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.DE_results.P0.001_C2.DE.subset

-rw-rw-r--. 1 bdash bdash   19319 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.DE_results.P0.001_C2.Sp_ds-UP.subset

-rw-rw-r--. 1 bdash bdash    9274 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.DE_results.P0.001_C2.Sp_log-UP.subset

-rw-rw-r--. 1 bdash bdash      26 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_log.edgeR.DE_results.samples

-rw-rw-r--. 1 bdash bdash    6892 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.DE_results.P0.001_C2.DE.subset

-rw-rw-r--. 1 bdash bdash    2225 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.DE_results.P0.001_C2.Sp_ds-UP.subset

-rw-rw-r--. 1 bdash bdash    4734 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.DE_results.P0.001_C2.Sp_plat-UP.subset

-rw-rw-r--. 1 bdash bdash      28 Mar 25 11:44 genes.isoform.counts.matrix.Sp_ds_vs_Sp_plat.edgeR.DE_results.samples

-rw-rw-r--. 1 bdash bdash   21828 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.DE_results.P0.001_C2.DE.subset

-rw-rw-r--. 1 bdash bdash   17739 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.DE_results.P0.001_C2.Sp_hs-UP.subset

-rw-rw-r--. 1 bdash bdash    4156 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.DE_results.P0.001_C2.Sp_log-UP.subset

-rw-rw-r--. 1 bdash bdash      26 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_log.edgeR.DE_results.samples

-rw-rw-r--. 1 bdash bdash   35385 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.DE_results.P0.001_C2.DE.subset

-rw-rw-r--. 1 bdash bdash   17568 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.DE_results.P0.001_C2.Sp_hs-UP.subset

-rw-rw-r--. 1 bdash bdash   17884 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.DE_results.P0.001_C2.Sp_plat-UP.subset

-rw-rw-r--. 1 bdash bdash      28 Mar 25 11:44 genes.isoform.counts.matrix.Sp_hs_vs_Sp_plat.edgeR.DE_results.samples

-rw-rw-r--. 1 bdash bdash   52861 Mar 25 11:44 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results.P0.001_C2.DE.subset

-rw-rw-r--. 1 bdash bdash   19495 Mar 25 11:44 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results.P0.001_C2.Sp_log-UP.subset

-rw-rw-r--. 1 bdash bdash   33433 Mar 25 11:44 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results.P0.001_C2.Sp_plat-UP.subset

-rw-rw-r--. 1 bdash bdash      30 Mar 25 11:44 genes.isoform.counts.matrix.Sp_log_vs_Sp_plat.edgeR.DE_results.samples



```{r}
library(pdftools)
```


```{r}
pdf_convert('genes_DE/DE_genes.matrix.log2.centered.genes_vs_samples_heatmap.pdf', format = "jpeg", pages = NULL, filenames = NULL, dpi = 300, antialias = TRUE, opw = "", upw = "", verbose = TRUE)
```


![DE_genes.matrix.heatmap](DE_genes.matrix.log2.centered.genes_vs_samples_heatmap_1.jpeg)


```{r}
pdf_convert('genes_DE/DE_genes.matrix.log2.centered.sample_cor_matrix.pdf', format = "jpeg", pages = NULL, filenames = NULL, dpi = 300, antialias = TRUE, opw = "", upw = "", verbose = TRUE)
```

![DE_genes.cor_matrix](DE_genes.matrix.log2.centered.sample_cor_matrix_1.jpeg)









Trinity tools (Part III-B): Analysis of de novo transcript/genes assembly using edgeR

Bhagirathi Dash

March 19, 2019

Reference, Resources and Data

Nature Protocols volume 8, pages 1494-1512 (2013)

Data: http://sourceforge.net/projects/trinityrnaseq/files/misc/TrinityNatureProtocolTutorial.tgz

Machine: CentOS Linux 7 on Virtual Box

Starting directory and subdirectory contents

trinity2$ ls -l

Combine RSEM-computed fragment counts into a matrix file

trinity2$ abundance_estimates_to_matrix.pl –est_method RSEM Sp_ds/RSEM.genes.results Sp_hs/RSEM.genes.results Sp_plat/RSEM.genes.results Sp_log/RSEM.ge nes.results –gene_trans_map trinity_out_dir/Trinity.fasta.gene_trans_map –name_sample_by_basedir –out_prefix genes

Some log readouts

Additional directory/subdirectory/files created following the above run

trinity2$ ls -l

A file readout

trinity2$ head genes.isoform.counts.matrix

Run DE analysis for the genes: Produces volcano plots and other results

run_DE_analysis.pl –matrix isoforms.isoform.counts.matrix –output isoforms_DE –dispersion 0.1 –method edgeR

Partial readout of the log

Additional directory/subdirectory/files created following the above run

trinity2]$ ls -l

trinity2$ ls -l genes_DE/

“png”, “jpeg”, “jpg”, “tiff”, “pnm”

Analyze differentail expression of genes: Produces heatmap and other results

genes_DE$analyze_diff_expr.pl –matrix ../genes.isoform.TMM.EXPR.matrix –output DE_genes

Additional directrories/subdirectories/files crated following aobe run