trouble using MAnorm (R version) to compare sample peaks

Question

[ivoryec]

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.
StepIII: count peak read
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.

II. run command: ./MAnorm.sh sample1_peakfile[BED] sample2_peakfile[BED] sample1_readfile[BED] sample2_readfile[BED] sample1_readshift_lentgh[INT] sample2_readshift_length[INT]

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 =".

Answer 1

[Devon Ryan]

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.

[ivoryec]

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.
StepIII: count peak read
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.

[Devon Ryan]

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)

[ivoryec]

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!