Region Fold Change Heatmap

The region_fold_change_heatmap tool generates a log2 fold change heatmap of [5’/3’/pileup] reads from a series of sequencing datasets around provided regions.

Note

This tool requires bedtools to be installed.

Usage and option summary

Usage:

PolTools region_fold_change_heatmap [-h] --numerator seq_file
                                           spike_in --denominator seq_file
                                           spike_in [-m max_log2_fc]
                                           [-r repeat_amount]
                                           [-v vertical_averaging]
                                           [-t [threads]]
                                           [--minor_ticks minor_ticks]
                                           [--major_ticks major_ticks]
                                           read type regions_file
                                           output_prefix

Required Arguments	Description
Read Type	Either five, three, or whole corresponding to 5’, 3’, or pileup reads.
Regions File	Bed formatted file containing regions of the same width.
–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 region_heatmap.tiff.

Optional Arguments	Description
-m, –max_log2_fc	Maximum value to consider as black. Default is the max value found. Decreasing this number will make the image darker.
-r, –repeat_amount	Number of pixels that should be displayed for each base pair
-v, –vertical_averaging	Number of lines to average vertically.
–minor_ticks	Distance in bp between minor tick marks. Default is no ticks.
–major_ticks	Distance in bp between major tick marks. Default is no ticks.

Behavior

region_fold_change_heatmap will generate a heatmap of the number of [5’/3’/pileup] reads within the regions from the given bed formatted regions file. When multiple numerator (or denominator) sequencing files are provided, they will be added together.

For example:

$ head regions_centered_on_max_tss.bed
chr1    959251  959261  NOC2L   46      -
chr1    960627  960637  KLHL17  27      +
chr1    966516  966526  PLEKHN1 8       +
chr1    1000092 1000102 HES4    87      -
chr1    1000290 1000300 ISG15   12      +
chr1    1020114 1020124 AGRN    35      +
chr1    1074302 1074312 RNF223  10      -
chr1    1116102 1116112 C1orf159        9       -
chr1    1231967 1231977 SDF4    321     -
chr1    1232237 1232247 B3GALT6 174     +

$ head numerator_seq_file_one.bed
chr1    11981   12023   A00876:119:HW5F5DRXX:1:2168:2248:1407   255     -
chr1    13099   13117   A00876:119:HW5F5DRXX:1:2203:31403:26757 255     -
chr1    13356   13423   A00876:119:HW5F5DRXX:1:2151:15808:7827  255     -
chr1    13435   13477   A00876:119:HW5F5DRXX:1:2273:15781:19241 255     -
chr1    13739   13772   A00876:119:HW5F5DRXX:1:2256:29966:10520 255     -
chr1    13741   13773   A00876:119:HW5F5DRXX:1:2235:4101:11882  255     -
chr1    14178   14203   A00876:119:HW5F5DRXX:1:2115:8241:31422  255     -
chr1    14734   14768   A00876:119:HW5F5DRXX:1:2165:23764:2440  255     -
chr1    14988   15012   A00876:119:HW5F5DRXX:1:2219:16134:32784 255     -
chr1    18337   18362   A00876:119:HW5F5DRXX:1:2149:32054:31328 255     -

$ PolTools region_heatmap five regions_centered_on_max_tss.bed --numerator numerator_seq_file_one.bed 1 \
  --denominator denominator_seq_file_one.bed 0.95 five_heatmap -r 20 -m 10 --minor_ticks 10 --major_ticks 50