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heatmap for HTSeq data in R

Hi friends

I try to plot heatmap for significant genes with cut off FDR < 0.05 and Fold change abs(2).

I have two question:

  1. I want to have range of my colors -3 to 3 as my minimum and maximum values are in the file. I dont know why are change it to -4 to 4. How can I set this?
  2. How can I cluster columns inside each group (not all together). I mean blue groups together and yellow groups together.

Please help me with this.

my data file looks like this:

TCGA-DV-A4W0    TCGA-B0-5812    TCGA-CZ-4858    TCGA-CZ-5985    TCGA-B0-5706
ENSG00000136546 0.181830391 0.694867529 0.215741062 -1.185141681    -1.237408974
ENSG00000115507 -0.361309104    1.684467761 1.044270623 0.157551622 0.004584822
ENSG00000163207 -0.147714241    -0.147823434    -0.149200113    -0.150367088    -0.14789331
ENSG00000123560 -0.34272044 -0.687421791    0.05798495  -0.876653053    -1.48726563
ENSG00000178462 -1.063056021    0.040025267 -1.35758621 -1.075000431    -1.090410434
ENSG00000071991 -0.174786987    -0.174956939    -0.169668498    -0.179650013    -0.179436852
ENSG00000177354 -0.33433836 -0.346731513    -0.555600129    -0.928440339    0.259568992
ENSG00000081051 1.218425978 -0.058534672    -1.104272404    -0.046479571    -0.81553282
ENSG00000178919 -0.170636704    -0.184535399    -0.147903288    -0.185249758    -0.184555276
ENSG00000144227 -0.339595559    0.721073074 -0.557406478    -0.937305769    -0.35982469
ENSG00000186910 -0.405019206    -0.423136465    0.976417264 -1.308551699    -0.435060224
ENSG00000043355 1.234927266 0.663318914 2.980944167 -0.092653328    -1.004937028
ENSG00000164756 -0.131731478    -0.13173574 -0.131788961    -0.13147134 -0.131738464
ENSG00000140557 1.608839856 -0.206466577    -0.520509236    -1.871781298    -1.06273877
ENSG00000163825 -0.759650728    0.149011499 -0.194730763    0.087241581 -0.791364476
ENSG00000131771 -0.550962856    0.299444596 -0.816868438    -1.224446647    -1.311569538
ENSG00000148965 -1.029686968    -1.050990295    -1.410026967    2.414698793 -1.065010944
ENSG00000138675 0.564811739 -1.33506518 -0.834581288    -1.630498742    -1.340582556
ENSG00000129654 -0.423541548    0.663408733 -1.563152043    -1.719406198    -1.444017051

This is my heatmap

https://ibb.co/0FzL8DZ

This is the code I used:

data <- read.table("mydata.txt", sep = '\t',
                  header = TRUE, row.names = 1, stringsAsFactors = FALSE, check.names=FALSE)
mat <- as.matrix (data, stringsAsFactors = FALSE)

metadata <- read.table("metadata.txt", sep = '\t',
                       header = TRUE, row.names = 1, stringsAsFactors = FALSE)


sig_genes <- read.table("siggenes.txt", sep = '\t',
                        header = FALSE, stringsAsFactors = FALSE)[,1]


myCol <- colorRampPalette(c('dodgerblue', 'black', 'yellow'))(100)
myBreaks <- seq(-3, 3, length.out = 100) ## this line I set to -3 to 3 but it didnot help and the color range is -4 and 4 in resulted heatmap


pamClusters <- cluster::pam(mat, k = 2)
pamClusters$clustering <- paste0('Cluster ', pamClusters$clustering)

# order of the clusters top to bottom
pamClusters$clustering <- factor(pamClusters$clustering,
                                 levels = c('Cluster 1', 'Cluster 2'))



##create the actual heatmap object
hmap <- Heatmap(mat,


                cluster_row_slices = FALSE,
                name = 'Gene\nZ-\nscore',
                col = colorRamp2(myBreaks, myCol),

                # colour-bar that represents gradient of expression
                heatmap_legend_param = list(
                  color_bar = 'continuous',
                  legend_direction = 'vertical',
                  legend_width = unit(8, 'cm'),
                  legend_height = unit(5.0, 'cm'),
                  title_position = 'topcenter'),
                  #title_gp=gpar(fontsize = 12, fontface = 'bold'),
                  #labels_gp=gpar(fontsize = 12, fontface = 'bold')),
                cluster_columns = FALSE,
                # row (gene) parameters
                cluster_rows = TRUE,
                show_row_dend = TRUE,
                #row_title = 'Statistically significant genes',
                row_title_side = 'left',
                row_title_gp = gpar(fontsize = 12,  fontface = 'bold'),
                row_title_rot = 90,
                show_row_names = FALSE,
                row_names_gp = gpar(fontsize = 10, fontface = 'bold'),
                row_names_side = 'left',
                row_dend_width = unit(25,'mm'),

                # cluster methods for rows 
                clustering_distance_rows = function(x) as.dist(1 - cor(t(x))),
                clustering_method_rows = 'ward.D2',

                top_annotation = colAnn)


draw(hmap + genelabels,
     heatmap_legend_side = 'left',
     annotation_legend_side = "right", row_sub_title_side = 'left')
rna-seq
• 2,033 views • link
updated by Ram Ram • written by Rob • Rob

Hello rhasanvandj!

Questions similar to yours can already be found at:

We have closed your question to allow us to keep similar content in the same thread.

If you disagree with this please tell us why in a reply below. We'll be happy to talk about it.

Cheers!

• 0 views • link
written by ATpoint ATpoint

Hello ATpoint I believe that my questions cannot be found in previous questions. these questions may look similar to previous one but actually they are different. I could not find this in the website. Can you please not close my post as it is very important for me and I still did not find answer to my questions.

Thanks

• 0 views • link
written by Rob • Rob

Fair enough, I think it is 99.9% identical to the previous one but be it. Just as advise, if you keep posting identical questions you rather repel users as they might get tired to basically read the same question from the same user twice. You might also want to consider removing that wall of code by trimming it to the absolute relevant part that is necessary to understand the problem. All these md5 and normalization things are obsolete if you simply provide an example of the data that are actually being passed to the heatmap with dput().

• 0 views • link
written by ATpoint ATpoint

Thank you for your advice. I edited my post.

• 0 views • link
written by Rob • Rob

And messed it up. I've fixed the formatting. Your data is not the output of dput by the way. Try running dput(data) in your R session to see what that looks like.

• 0 views • link
written by Ram Ram

You've messed it up yet again. Please stop editing the post.

• 0 views • link
written by Ram Ram

Oh sorry sure I did not change it again

• 0 views • link
written by Rob • Rob

The legend breaks won't restrict min/max values in the legend because those are based on the data being plotted.

What is the output of max(data) and min(data)? You may have to manually set all values <-3 to -3 and all values >3 to 3.

• 0 views • link
written by Ram Ram

in the data file min value is -3 and max is 3. I want my range to be the same. but here R changes it to -4 to 4. how can I set the range manually?

• 0 views • link
written by Rob • Rob

If the minimum is -3 and the maximum is +3, there is no reason the legend should show -4 -> +4

Can you try running mat2<-mat; mat2[mat2 < -3] <- -3; mat2[mat2 > 3] <- 3 and then use mat2 to plot the heatmap?

• 0 views • link
written by Ram Ram

Thanks. It did not help. the range is still -4 to 4

• 0 views • link
written by Rob • Rob

I just noticed that you're creating myBreaks but not using it in the code. Try adding at = myBreaks to the heatmap_legend_param list. Change the length.out in myBreaks to something sensible like 5 or 7 instead of 100.

• 0 views • link
written by Ram Ram

Thanks I am trying to fix this. Also I am trying to cluster column inside each group separately. How can I do this? I don't want to mix clustering two groups.

• 0 views • link
written by Rob • Rob

You can't do that, or the heatmap won't make sense. If you need both rows and columns to be clustered independently on the split, use 2 heatmaps.

• 0 views • link
written by Ram Ram

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