trouble using MAnorm (R version) to compare sample peaks
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Entering edit mode
7.6 years ago
ivoryec ▴ 70

Hi,

I want to compare the peaks from two separate chip seq samples.

I'm trying to use MAnorm (R version) on a MacOS unix command line, but I'm having trouble.

Now the program runs through "step1" without any errors, but it doesn't seem to be making a pair of essential files: peak1.bed and peak2.bed, so all downstream steps fail.

Below, please see the head of each of my test files, and the command I ran.

If you've used this tool before, do you have any idea why I'm having problems?

Thanks very much,

$head t.* ==> t.TreatedPeaks.bed <== 1 1360 3306 1 13366 13931 1 16606 16934 1 20161 21424 1 59752 60275 1 63673 64105 1 67355 67885 1 68243 68654 1 107683 109178 1 116034 117502 ==> t.controlPeaks.bed <== 1 20618 21401 1 37881 38291 1 54003 54374 1 57362 59025 1 67346 67844 1 75598 76127 1 76181 76562 1 110638 111047 1 112394 112668 1 117123 117842 ==> t.controlReads.bed <== 1 2 52 - 1 11 61 - 1 32 82 - 1 32 82 - 1 32 82 - 1 32 82 - 1 37 87 + 1 37 87 - 1 47 97 - 1 62 112 - ==> t.treatedReads.bed <== 1 1 51 + 1 13 63 + 1 28 78 - 1 31 81 - 1 44 94 + 1 46 96 + 1 64 114 + 1 69 119 + 1 70 120 - 1 77 127 +$ MAnorm.sh t.TreatedPeaks.bed t.controlPeaks.bed t.treatedReads.bed t.controlReads.bed 266 275
StepI: clean input
StepII: classify common or unique peaks
Error: Unable to open file peak1.bed. Exiting.
Error: Unable to open file peak2.bed. Exiting.
Error: Unable to open file peak1.bed. Exiting.
Error: Unable to open file peak2.bed. Exiting.
Error: The requested file (read1.bed) could not be opened. Error message: (No such file or directory). Exiting!


Here is the usage section of the readme.txt that comes with the MAnorm:

Usage:

I. Create a folder and place in the folder MAnorm.sh, MAnorm.r, and all 4 bed files (peak file and read file for the 2 samples under comparison) to be analyzed.

ExampleCommand:

./MAnorm.sh sample1_peaks.bed sample2_peaks.bed sample1_read.bed sample2_read.bed 150 150


The first 4 parameters should be input files in bed format with no header lines
The first 2 files have ONLY 3 columns: chromosome, start, end.
The next 2 files should have 4 columns: chromosome, start, end, strand (+/-)

The last 2 parameters are the number of bp to be shifted for each read. These two parameters are found from MACS peak file *_peaks.xls after "# d =".

ChIP-Seq MAnorm • 3.7k views
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Entering edit mode
7.6 years ago

If you look at the shell script, you'll see that it's expecting UCSC chromosome names (i.e., they need to start with "chr"). So just quickly fix that and things should work.

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Entering edit mode

Thanks for reply! That is the format I had earlier, (and I changed it back to double check), and it gave this error:

StepI: clean input
awk: illegal field \$(chr1), name "1"
input record number 1, file
source line number 2

StepII: classify common or unique peaks
Error: Unable to open file peak2.bed. Exiting.
Error: Unable to open file peak2.bed. Exiting.
Error: Unable to open file peak2.bed. Exiting.
Error: Unable to open file peak2.bed. Exiting.
cat: common_peak1_count_read1: No such file or directory
...


This made me think that I was using the wrong sort of names for the chromosomes. On the upside, this way it does at least make the peak1.bed file, but still no peak2.bed, and I don't know how to remedy the error its reporting for stepI. On another post, it looked like someone was using integers for chromosome names so I thought that was the answer... apparently not.

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Entering edit mode

Ah, after trying things out a bit, the problem is an error on line 14 of MAnorm.sh. The line currently is:

{if ($$1~/chr/ && 1 !="chrM" && 1 !~/random/ && 3>2 && 2>0 && 3>0)  but should be: {if (1~/chr/ && 1 !="chrM" && 1 !~/random/ && 3>2 && 2>0 && 3>0)  ADD REPLY 0 Entering edit mode Gah! I saw at that $$ and I just thought it was some sort of notation I wasn't familiar with. Good catch! and thank you so much! I pasted that in, ran the script on my test files and now I have output files. beautiful. Thank you!