Heat map construction from TMM normalized FPKM value

[Yogesh Gupta]

Dear All,

Is it possible to construct heat map from excel sheet having FPKM value at different development stages for analysis of differential gene expression.

Thanks

Yogesh

--

Yogesh Gupta

Senior Research Fellow

National Agri-Food Biotechnology Institute
(Departement of Biotechnology, Government of India)

C-127, Industrial Area, Phase 8, SAS Nagar

Mohali-160071 Punjab, India

Cell No. +91-9041074172

[Ken Field]

Yogesh-

You really should do that within R, after importing the data from a CSV file that you saved in excel. I like to use the Trinity code that gets generated for you when you run:

$TRINITY_HOME/Analysis/DifferentialExpression/run_DE_analysis.pl

If you would like to do it in excel, you can use Conditional Formating to color code cells based on their values. For example, the following row of TMM-normalized fpkm values have been highlighted with a color scale to show their relative values:

0.049

0.032

0.202

0.063

0.026

149.837

80.964

56.518

58.63

152.937

26.019

Note that if you want to highlight each row with its own scale, you have to format each row separately. If you format the whole table at once, it takes the maximum and minimum values for the entire table to set the color scale.

Ken

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Ken Field, Ph.D.

Associate Professor of Biology

Program in Cell Biology/Biochemistry

Bucknell University

Room 203A Biology Building

[Brian Haas]

If you have a matrix file containing expression values, you can use the Trinity-included PtR script for generating a heatmap:

  trinityrnaseq/Analysis/DifferentialExpression/PtR   --matrix  my_matrix.fpkm  --log2  --heatmap

[Brian Haas]

Hi Yogesh,

Try saving the xlsx file as tab-delimited.  Then, do this to get rid of the cntrl-M characters that excel puts in there:

    cat my_matrix.fpkm.txt | perl -lane 's/\cM/\n/g; print;' > file.tab.txt

and then make your heatmap:

   trinityrnaseq/Analysis/DifferentialExpression/PtR -m  file.tab.txt --log2 --heatmap --center_rows

I'll send you the row-centered as well as the one w/o the row-centering separately.  Row-centering is more useful when looking for overall patterns.

best,

~b

On Mon, Jul 20, 2015 at 4:52 AM, Yogesh Gupta <yoges...@gmail.com> wrote:

Dear All ,

I am getting error in heat map contruction using trinityrnaseq/Analysis/DifferentialExpression/PtR   --matrix  my_matrix.fpkm  --log2  --heatmap

EXCEL SHEET WHICH I AM USING IS ATTACHED.

CAMMAND LINE IS :

 /home/yogesh/softwares/TRINITY_HOME/Analysis/DifferentialExpression/PtR --matrix /sataslave/annona_illumina/hybrid_data/blast_artf_hormone_seedgene/my_matrix.fpkm --log2 --heatmap

Thanks in Advance

Yogesh

Error is :

CMD: R --vanilla -q < my_matrix.fpkm.R

> library(cluster)

> library(Biobase)

Loading required package: BiocGenerics

Loading required package: parallel

Attaching package: ‘BiocGenerics’

The following objects are masked from ‘package:parallel’:

    clusterApply, clusterApplyLB, clusterCall, clusterEvalQ,

    clusterExport, clusterMap, parApply, parCapply, parLapply,

    parLapplyLB, parRapply, parSapply, parSapplyLB

The following object is masked from ‘package:stats’:

    xtabs

The following objects are masked from ‘package:base’:

    anyDuplicated, append, as.data.frame, as.vector, cbind, colnames,

    do.call, duplicated, eval, evalq, Filter, Find, get, intersect,

    is.unsorted, lapply, Map, mapply, match, mget, order, paste, pmax,

    pmax.int, pmin, pmin.int, Position, rank, rbind, Reduce, rep.int,

    rownames, sapply, setdiff, sort, table, tapply, union, unique,

    unlist, unsplit

Welcome to Bioconductor

    Vignettes contain introductory material; view with

    'browseVignettes()'. To cite Bioconductor, see

    'citation("Biobase")', and for packages 'citation("pkgname")'.

> library(qvalue)

> NO_REUSE = F

>

> # try to reuse earlier-loaded data if possible

> if (file.exists("my_matrix.fpkm.RData") && ! NO_REUSE) {

+     print('RESTORING DATA FROM EARLIER ANALYSIS')

+     load("my_matrix.fpkm.RData")

+ } else {

+     print('Reading matrix file.')

+     primary_data = read.table("/sataslave/annona_illumina/hybrid_data/blast_artf_hormone_seedgene/my_matrix.fpkm", header=T, com='', sep="\t", row.names=1, check.names=F)

+     primary_data = as.matrix(primary_data)

+ }

[1] "Reading matrix file."

Error in scan(file, what, nmax, sep, dec, quote, skip, nlines, na.strings,  :

  line 2 did not have 2 elements

Calls: read.table -> scan

In addition: Warning messages:

1: In read.table("/sataslave/annona_illumina/hybrid_data/blast_artf_hormone_seedgene/my_matrix.fpkm",  :

  line 1 appears to contain embedded nulls

2: In read.table("/sataslave/annona_illumina/hybrid_data/blast_artf_hormone_seedgene/my_matrix.fpkm",  :

  line 2 appears to contain embedded nulls

3: In read.table("/sataslave/annona_illumina/hybrid_data/blast_artf_hormone_seedgene/my_matrix.fpkm",  :

  line 4 appears to contain embedded nulls

Execution halted

Error, cmd: R --vanilla -q < my_matrix.fpkm.R died with ret 256 at /home/yogesh/softwares/TRINITY_HOME/Analysis/DifferentialExpression/PtR line 1568.

On Mon, Jul 20, 2015 at 6:25 AM, Brian Haas <bh...@broadinstitute.org> wrote:

If you have a matrix file containing expression values, you can use the Trinity-included PtR script for generating a heatmap:

  trinityrnaseq/Analysis/DifferentialExpression/PtR   --matrix  my_matrix.fpkm  --log2  --heatmap

--

Yogesh Gupta

Senior Research Fellow

National Agri-Food Biotechnology Institute
(Departement of Biotechnology, Government of India)

C-127, Industrial Area, Phase 8, SAS Nagar

Mohali-160071 Punjab, India

Cell No. +91-9041074172

--

--
Brian J. Haas
The Broad Institute
http://broadinstitute.org/~bhaas

 

[Yogesh Gupta]

Dear Brian,

I am making heat map as suggested, but in some cases no of genes in matrix is more and it is making heat map for less genes.

Please give suggestions.

Thanks in Advance

Yogesh

On Mon, Jul 20, 2015 at 9:14 PM, Yogesh Gupta <yoges...@gmail.com> wrote:

 --heatmap_colorscheme "green,black,red"

On Mon, Jul 20, 2015 at 8:29 PM, Brian Haas <bh...@broadinstitute.org> wrote:

pdfs attached

--

[Yogesh Gupta]

Dear Brian,

I have some queries regarding heat map. 

Can we make heat map without clustering?

In some cases no. of genes is more in input file and heat map shows less gene. I am attaching one for which I constructed heat map, there is only 32 gene in heat map whereas input file has 39 genes.

Thanks in advance.

Yogesh

[Brian Haas]

Hi Yogesh,

If you're running PtR to generate the heatmap, then include

 --min_rowSums 0

and it should include all the data points.  By default, it filters out those rows that have less than a sum of 10 in the row.

best,

~b