Bedtools intersect returns 0, intersections present on IGV

Question

[kerrypop]

Hello,

I am trying to use bedtools to find intersections between H3K4me1 peaks and my regions of interest. Though I can see overlap on IGV, intersect returns 0 without any error messages.

Here is command I'm using and screenshot from IGV:

bedtools intersect -a gene.ROI -b wgEncodeBroadHistoneHuvecH3k4me1StdPk.broadPeak -u | wc -l

IGV screenshot: https://imgur.com/a/JYUzp

Is there something I'm missing?

Thank you, Kerry

[Pierre Lindenbaum]

do file use the same chromosome notation ? are the files sorted ? are both files BED files ?

[kerrypop]

Yes, same chromosome notation. Files are not sorted - I've done this analysis before and didn't sort them. Do you think sorting would overcome this? The ROI file is just a document, created using nano in terminal. The other is a broad peak file.

[dariober]

The ROI file is just a document, created using nano in terminal.

Check columns are tab separated and there are no funny, non-printable characters. If you do something like:

cat -vet gene.ROI | head

You should see columns separated by ^I, e.g.:

chr1^I67208778^I67216822^INM_032291_utr3_24_0_chr1_67208779_f^I0^I+$
chr1^I67208778^I67216822^INM_001308203_utr3_21_0_chr1_67208779_f^I0^I+$
chr1^I33585783^I33586132^INM_001301825_utr3_8_0_chr1_33585784_f^I0^I+$
chr1^I8404073^I8404227^INM_001080397_utr3_8_0_chr1_8404074_f^I0^I+$

[kerrypop]

I only used chromosome coordinates but don't see any extraneous characters

cat -vet gene.ROI | head
Chr6^I11711888^I11781280$
Chr6^I35702809^I35718690$
Chr1^I162349520^I162358608$

[Hussain Ather]

It looks like the dollar sign at the end of each line might be causing trouble. You can use this command to remove the last character from each line:

sed 's/.$//' gene.ROI > geneROI.nolast

[GenoMax]

That is because of options (-vet) used for cat command.

-e     equivalent to -vE
-E, --show-ends
              display $ at end of each line
-t     equivalent to -vT

-T, --show-tabs
          display TAB characters as ^I
-v, --show-nonprinting
              use ^ and M- notation, except for LFD and TAB

[dariober]

Your IGV screenshot seems to show the entire genome. It may be that once you zoom in into one of the ROI genes there is actually no intersection.

[kerrypop]

Here's zoomed in screenshot of the first ROI. There should be 4 intersections. https://imgur.com/a/lsiEQ

Answer 1

[harold.smith.tarheel]

It looks like your chromosome notation is NOT identical. Your IGV screenshot says 'chr6' but your gene.ROI says 'Chr6'. Linux is case-sensitive.

[kerrypop]

That did it - thank you!

Answer 2

[dariober]

I see you have Chr6, Chr1 instead of chr6, chr1 (lowercase c). That may be the problem.