TES Fold Change Heatmap

The TES_heatmap tool generates a heatmap plotting the position and quantity of 3’ end reads sorted by gene length centered on the TES.

Note

This tool requires bedtools to be installed.

Usage

Usage:

PolTools TES_fold_change_heatmap [-h] --numerator seq_file spike_in
                                        --denominator seq_file spike_in
                                        [-w width] [-e height]
                                        [-d downstream_distance]
                                        [-u upstream_distance] [-b bp_width]
                                        [-m max_log2_fc]
                                        [--minor_ticks minor_ticks]
                                        [--major_ticks major_ticks]
                                        [-t [threads]]
                                        truQuant_output_file output_prefix

Required Arguments	Description
truQuant Output File	File ending in -truQuant_output.txt generated from truQuant
–numerator seq_file spike_in	Sequencing file and its accompanying normalization factor to be used as the numerator of the heatmap. Additional files can be provided with multiple –numerator arguments
–denominator seq_file spike_in	Sequencing file and its accompanying normalization factor to be used as the denominator of the heatmap. Additional files can be provided with multiple –denominator arguments
Output Prefix	Output filename will begin with the output prefix and also contain the run parameters and ends in gene_body_heatmap.tiff.

Optional Arguments	Description
-u, –upstream_distance	Distance upstream of the max TSS from truQuant to show on the heatmap. Default is 50,000 bp.
-d, –distance_past_tes	Distance past the TES to show on the heatmap. Default is 50,000 bp.
-b, –bp_width	Total distance shown on the heatmap. Default is 400,000 bp.
-w, –width	Width of the heatmap in pixels. Default is 2,000 px.
-e, –height	Height of the heatmap in pixels. Default is 2,000 px.
-m, –max_black	Maximum value to consider as black. Default is the max value found. Decreasing this number will make the image darker.
–minor_ticks	Distance in bp between minor tick marks. Default is 10,000 bp.
–major_ticks	Distance in bp between major tick marks. Default is 50,000 bp.

Behavior

TES_heatmap will create a file starting with the output prefix which contains the run parameters and ends in TES_heatmap.tiff. First, regions to be quantified are designed from a distance upstream of the max TSS to a distance downstream of the TES (default is 50 kb away from each position) for each gene in the truQuant file. Then, the tsrFinder run associated with truQuant is used to find regions to blacklist (TSRs with at least 30% of the number of 5’ ends found in the pause region of the gene). These regions are blacklisted in the sequencing files and the 3’ ends are quantified in each location of the previously defined regions. The 3’ end reads are normalized according to the correction factors. The two numerator datasets are added together and divided by the sum of the denominator datasets. The resulting division matrix is subjected to a log2 fold change transformation, to generate the heatmap.

For example:

$ head seq_file-truQuant_output.txt
Gene    Chromosome      Pause Region Left       Pause Region Right      Strand  Total 5' Reads  MaxTSS  MaxTSS 5' Reads Weighted Pause Region Center    STDEV of TSSs   Gene Body Left  Gene Body Right Gene Body Distance      seq_file.bed Pause Region   seq_file.bed Gene Body
NOC2L   chr1    959177  959327  -       194     959255  46      959250  13.306459171023036      944203  959177  14974   194     18
KLHL17  chr1    960552  960702  +       234     960632  27      960626  25.417791063821863      960702  965719  5017    234     17
PLEKHN1 chr1    966439  966589  +       25      966521  8       966513  19.47408534437497       966589  975865  9276    25      11
HES4    chr1    1000013 1000163 -       239     1000096 87      1000086 27.14758979723915       998962  1000013 1051    239     68
ISG15   chr1    1000204 1000354 +       160     1000295 12      1000278 36.24344768368484       1000354 1014540 14186   160     111
AGRN    chr1    1020042 1020192 +       112     1020119 35      1020116 25.189637892253575      1020192 1056118 35926   112     76
RNF223  chr1    1074208 1074358 -       32      1074306 10      1074284 32.567238138964136      1070967 1074208 3241    32      8
C1orf159        chr1    1116028 1116178 -       51      1116106 9       1116103 19.81136532595448       1081818 1116028 34210   51      11
SDF4    chr1    1231907 1232057 -       1105    1231971 321     1231978 23.701136922154493      1216908 1231907 14999   1097    177

$ PolTools TES_fold_change_heatmap seq_file-truQuant_output.txt --numerator flavo.bed 1 --denominator dmso.bed 1 treatment -m 2