Bowtie2 to align ChiPSeq Data

Question

[eilluecaf]

Hi,

I am trying to align some ChiPSeq data with bowtie2 but i am getting error. I am usin the SystemPipeR package and I am following the instruction of the tutorial. The error that I have is the following:

moduleload(modules(args)) # Skip if a module system is not used Error in system(paste0("bash -c \"module load ", module, "; export | grep '^declare -x ", : 'bash' not found

Thanks for your attention.

[Kevin Blighe]

Can you provide the link to the tutorial that you are attempting to follow?

[h.mon]

Probably this one:

NGS data analysis with R / Bioconductor: ChIP-Seq workflow

[eilluecaf]

Yes, it is the tutorial that I am following

[Kevin Blighe]

Obrigado para sua ajuda amigo - estava voltando a Europa desde Rio.

[h.mon]

Are you using Linux / Mac, or are you using Windows? Are you running on a cluster or workstation? Does your system use the Modules package to manage software availability?

[eilluecaf]

I am using Windows and I am running on workstation. My system doesn't have the Modules package, I am going to try to download this package. Sorry, I am a beginner.

[h.mon]

There is no Modules for Windows, as far as I know. And you don't necessarily need to install Modules to run SystemPipeR.

You will have some (likely a lot of) trouble running most bioinformatics software on Windows, unless you install the windows subsystem for linux.

Answer 1

[h.mon]

Windows don't have a bash shell, unless you install the windows subsystem for linux. Likewise, most bioinformatics software will not run under Windows, unless you have the windows subsystem for linux. I do not recommend cygwin, there are several quirks and very few users, you won't be able to get help when walking into problems.

[eilluecaf]

I tryed to run Bowtie 2 in Ubuntu but it is still giving me error. The error message is the following:

bash: module: no se encontró la orden

Do I need to install bash or module?

[Devon Ryan]

Ignore everything related to module in the tutorial, it's not relevant for you.

[h.mon]

Having in mind that bashis the default Ubuntu command-line shell, bash is already installed. In fact bash: error means bash is telling you about the error. What was not found was the module command, so you need to stop using the "module load" version of the commands:

moduleload(modules(args)) # Skip if a module system is not used

Modules is a software environment management, used by sysadmins of large, multi-user computers to manage installation of several, sometimes conflicting, software. You certainly don't have it, and probably don't need it.

[eilluecaf]

Okay, I skipped that step. Now I'm failure on the following line:

system("bowtie2-build ./data/tair10.fasta ./data/tair10.fasta") sh: 1: bowtie2-build: not found

I've already installed Bowtie 2 and is in the current directory. Thanks for your attention

[h.mon]

You have to set the PATH environment variable. Search for path bash. If you install bowtie2 from the Ubuntu repositories, it will already be on your PATH

[eilluecaf]

I have added the directory where is bowtie to the PATH variable. And I returns the following error:

system("./bowtie2-2.3.4.1/bowtie2-build ./data/tair10.fasta ./data/tair10.fasta")
Traceback (most recent call last):
  File "./bowtie2-2.3.4.1/bowtie2-build", line 86, in <module>
    main()
  File "./bowtie2-2.3.4.1/bowtie2-build", line 83, in main
    subprocess.call(argv)
  File "/usr/lib/python2.7/subprocess.py", line 523, in call
    return Popen(*popenargs, **kwargs).wait()
  File "/usr/lib/python2.7/subprocess.py", line 711, in __init__
    errread, errwrite)
  File "/usr/lib/python2.7/subprocess.py", line 1343, in _execute_child
    raise child_exception
OSError: [Errno 2] No such file or directory

[GenoMax]

Why did you provide the same input file name (./data/tair10.fasta) twice (or so it appears above).

[h.mon]

One is the file, the other will be the base name of the index files.

bowtie2-build [options] <reference_in> <bt2_index_base>

[GenoMax]

Ah. I generally use a new name or just the first part of fasta file name.

[h.mon]

Before starting R, did you do:

export PATH=/some/path:$PATH

My mistake, the problem now is bowtie2-build can't find the fasta file. Where is the tair10.fasta file?

[eilluecaf]

I tried this:

export PATH=$PATH:/home/usuario/chipseq/bowtie2-2.3.4.1

[eilluecaf]

The file is in /home/usuario/chipseq/data but I am using chipseq as working directory, in theory it is correct Now it returns me anothe message:

> system("./bowtie2-2.3.4.1/bowtie2-build ./data/tair10.fasta ./data/tair10.fasta") # Indexes reference genome
./bowtie2-2.3.4.1/bowtie2-build: 20: ./bowtie2-2.3.4.1/bowtie2-build: 
 Copyright 2014, Ben Langmead <[e-mail]>

 This file is part of Bowtie 2.

 Bowtie 2 is free software: you can redistribute it and/or modify
 it under the terms of the GNU General Public License as published by
 the Free Software Foundation, either version 3 of the License, or
 (at your option) any later version.

 Bowtie 2 is distributed in the hope that it will be useful,
 but WITHOUT ANY WARRANTY; without even the implied warranty of
 MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.  See the
 GNU General Public License for more details.

 You should have received a copy of the GNU General Public License
 along with Bowtie 2.  If not, see <http://www.gnu.org/licenses/>.
: not found
from: can't read /var/mail/collections
./bowtie2-2.3.4.1/bowtie2-build: 31: ./bowtie2-2.3.4.1/bowtie2-build: Syntax error: "(" unexpected
>

I think it can find bowtie2-build but there is another problem.

[h.mon]

1) if you put bowtie2 executables on the PATH, use bowtie2-build instead of ./bowtie2-2.3.4.1/bowtie2-build

2) Why don't you run the same command directly on the shell?

[eilluecaf]

I've done this and I keep getting the same error. When I apply the following lines of the tutorial I get the following

> resources <- list(walltime="1:00:00", ntasks=1, ncpus=cores(args), memory="10G")
> reg <- clusterRun(args, conffile=".BatchJobs.R", template="slurm.tmpl", Njobs=18, runid="01",
+                   resourceList=resources)
Creating dir: /home/usuario/chipseq/results/submitargs01_BJdb_7642
Saving registry: /home/usuario/chipseq/results/submitargs01_BJdb_7642/registry.RData
Adding 18 jobs to DB.
Error in system2(command = command, args = args, stdout = stdout, stderr = stderr,  : 
  error in running command
Además: There were 40 warnings (use warnings() to see them)
> waitForJobs(reg)
Error in checkRegistry(reg, writeable = FALSE) : 
  objeto 'reg' no encontrado

[GenoMax]

Can you follow @h.mon's suggestion and run the program in a terminal window directly logged into your server/cluster. Also specify a -o filt.out -e file.err so the actual SLURM error can be captured.