error with htseq count and bed tool

[meghavarshney]

hello every one,

as i am working on count based differential analysis and for that i am trying to use HTSEQ and bedtools
1. I am using Bedtool to count read in as

bedtools multicov -bams sample1.bam sample2.bam sample3.bam -bed all.gff3

here in spite of bed file i am using reference genome file all.gff3. is it correct to use it ?
Because I am not sure that I will get proper result with these gff3 file or not
2. I am getting error with ht-seq as it is already installed in my machine but still it is showing that

[root@localhost ~]# htseq-count
bash: htseq-count: command not found...

Kindly reply me

Thanks

[dpryan]

1) It may be installed, but it's not in your path. Look at where it was installed and either change your $PATH variable or use the full path to specify the executable.
2) You shouldn't be running things like this as root.

[meghavarshney]

I had installed it in this location and still m not able to access it

[root@localhost HTSeq-0.6.1]# python setup.py install --user
running install
running bdist_egg
running egg_info
writing HTSeq.egg-info/PKG-INFO
writing top-level names to HTSeq.egg-info/top_level.txt
writing dependency_links to HTSeq.egg-info/dependency_links.txt
reading manifest file 'HTSeq.egg-info/SOURCES.txt'
reading manifest template 'MANIFEST.in'
no previously-included directories found matching 'example_data'
no previously-included directories found matching 'test'
warning: no previously-included files found matching 'todo.txt'
writing manifest file 'HTSeq.egg-info/SOURCES.txt'
installing library code to build/bdist.linux-x86_64/egg
running install_lib
running build_py
running build_ext
creating build/bdist.linux-x86_64/egg
creating build/bdist.linux-x86_64/egg/HTSeq
copying build/lib.linux-x86_64-2.7/HTSeq/__init__.py -> build/bdist.linux-x86_64/egg/HTSeq
copying build/lib.linux-x86_64-2.7/HTSeq/_HTSeq_internal.py -> build/bdist.linux-x86_64/egg/HTSeq
copying build/lib.linux-x86_64-2.7/HTSeq/StepVector.py -> build/bdist.linux-x86_64/egg/HTSeq
copying build/lib.linux-x86_64-2.7/HTSeq/_version.py -> build/bdist.linux-x86_64/egg/HTSeq
creating build/bdist.linux-x86_64/egg/HTSeq/scripts
copying build/lib.linux-x86_64-2.7/HTSeq/scripts/__init__.py -> build/bdist.linux-x86_64/egg/HTSeq/scripts
copying build/lib.linux-x86_64-2.7/HTSeq/scripts/qa.py -> build/bdist.linux-x86_64/egg/HTSeq/scripts
copying build/lib.linux-x86_64-2.7/HTSeq/scripts/count.py -> build/bdist.linux-x86_64/egg/HTSeq/scripts
copying build/lib.linux-x86_64-2.7/HTSeq/_HTSeq.so -> build/bdist.linux-x86_64/egg/HTSeq
copying build/lib.linux-x86_64-2.7/HTSeq/_StepVector.so -> build/bdist.linux-x86_64/egg/HTSeq
byte-compiling build/bdist.linux-x86_64/egg/HTSeq/__init__.py to __init__.pyc
byte-compiling build/bdist.linux-x86_64/egg/HTSeq/_HTSeq_internal.py to _HTSeq_internal.pyc
byte-compiling build/bdist.linux-x86_64/egg/HTSeq/StepVector.py to StepVector.pyc
byte-compiling build/bdist.linux-x86_64/egg/HTSeq/_version.py to _version.pyc
byte-compiling build/bdist.linux-x86_64/egg/HTSeq/scripts/__init__.py to __init__.pyc
byte-compiling build/bdist.linux-x86_64/egg/HTSeq/scripts/qa.py to qa.pyc
byte-compiling build/bdist.linux-x86_64/egg/HTSeq/scripts/count.py to count.pyc
creating stub loader for HTSeq/_HTSeq.so
creating stub loader for HTSeq/_StepVector.so
byte-compiling build/bdist.linux-x86_64/egg/HTSeq/_HTSeq.py to _HTSeq.pyc
byte-compiling build/bdist.linux-x86_64/egg/HTSeq/_StepVector.py to _StepVector.pyc
creating build/bdist.linux-x86_64/egg/EGG-INFO
installing scripts to build/bdist.linux-x86_64/egg/EGG-INFO/scripts
running install_scripts
running build_scripts
creating build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/htseq-qa -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
copying build/scripts-2.7/htseq-count -> build/bdist.linux-x86_64/egg/EGG-INFO/scripts
changing mode of build/bdist.linux-x86_64/egg/EGG-INFO/scripts/htseq-qa to 755
changing mode of build/bdist.linux-x86_64/egg/EGG-INFO/scripts/htseq-count to 755
copying HTSeq.egg-info/PKG-INFO -> build/bdist.linux-x86_64/egg/EGG-INFO
copying HTSeq.egg-info/SOURCES.txt -> build/bdist.linux-x86_64/egg/EGG-INFO
copying HTSeq.egg-info/dependency_links.txt -> build/bdist.linux-x86_64/egg/EGG-INFO
copying HTSeq.egg-info/top_level.txt -> build/bdist.linux-x86_64/egg/EGG-INFO
writing build/bdist.linux-x86_64/egg/EGG-INFO/native_libs.txt
zip_safe flag not set; analyzing archive contents...
HTSeq.StepVector: module references __file__
creating 'dist/HTSeq-0.6.1-py2.7-linux-x86_64.egg' and adding 'build/bdist.linux-x86_64/egg' to it
removing 'build/bdist.linux-x86_64/egg' (and everything under it)
Processing HTSeq-0.6.1-py2.7-linux-x86_64.egg
removing '/root/.local/lib/python2.7/site-packages/HTSeq-0.6.1-py2.7-linux-x86_64.egg' (and everything under it)
creating /root/.local/lib/python2.7/site-packages/HTSeq-0.6.1-py2.7-linux-x86_64.egg
Extracting HTSeq-0.6.1-py2.7-linux-x86_64.egg to /root/.local/lib/python2.7/site-packages
Removing HTSeq 0.6.1p1 from easy-install.pth file
Adding HTSeq 0.6.1 to easy-install.pth file
Installing htseq-qa script to /root/.local/bin
Installing htseq-count script to /root/.local/bin

Installed /root/.local/lib/python2.7/site-packages/HTSeq-0.6.1-py2.7-linux-x86_64.egg
Processing dependencies for HTSeq==0.6.1
Finished processing dependencies for HTSeq==0.6.1

as i had tried it in these ways

[pinky@localhost HTSeq-0.6.1]$ htseq-count
bash: htseq-count: command not found...
[pinky@localhost HTSeq-0.6.1]$ python htseq-count
python: can't open file 'htseq-count': [Errno 2] No such file or directory
[pinky@localhost HTSeq-0.6.1]$ python /home/pinky/Downloads/HTSeq-0.6.1/htseq-count
python: can't open file '/home/pinky/Downloads/HTSeq-0.6.1/htseq-count': [Errno 2] No such file or directory

but still the problem remains same.

[dpryan]

Normally python programs end up in ~/.local/bin when you install them like this. Have a look there and add that to your path.

[meghavarshney]

i had checked but still i didn't find

[pinky@localhost bin]$ htseq-count
bash: htseq-count: command not found...

as from root this folder contain

[root@localhost bin]# ls
annotateBed                                  getOverlap
bam2bed                                      gff2bed
bam2bed_gnuParallel                          gff2starch
bam2bed_sge                                  groupBy
bam2starch                                   gtf2bed
bam2starch_gnuParallel                       gtf2starch
bam2starch_sge                               gvf2bed
bamToBed                                     gvf2starch
bamToFastq                                   intersectBed
bed12ToBed6                                  linksBed
bedextract                                   mapBed
bedmap                                       maskFastaFromBed
bedops                                       mergeBed
bedpeToBam                                   multiBamCov
bedToBam                                     multiIntersectBed
bedToIgv                                     nucBed
bedtools                                     pairToBed
closestBed                                   pairToPair
closest-features                             psl2bed
clusterBed                                   psl2starch
complementBed                                R
convert2bed                                  randomBed
coverageBed                                  rmsk2bed
expandCols                                   rmsk2starch
fasta_clipping_histogram.pl                  Rscript
fasta_formatter                              sam2bed
fastaFromBed                                 sam2starch
fasta_nucleotide_changer                     shuffleBed
fastq_masker                                 slopBed
fastq_quality_boxplot_graph.sh               sort-bed
fastq_quality_converter                      sortBed
fastq_quality_filter                         starch
fastq_quality_trimmer                        starchcat
fastq_to_fasta                               starchcluster_gnuParallel
fastx_artifacts_filter                       starchcluster_sge
fastx_barcode_splitter.pl                    subtractBed
fastx_clipper                                tagBam
fastx_collapser                              trimmomatic-0.30.jar
fastx_nucleotide_distribution_graph.sh       unionBedGraphs
fastx_nucleotide_distribution_line_graph.sh  unstarch
fastx_quality_stats                          vcf2bed
fastx_renamer                                vcf2starch
fastx_reverse_complement                     wig2bed
fastx_trimmer                                wig2starch
fastx_uncollapser                            windowBed
flankBed                                     windowMaker
genomeCoverageBed
[root@localhost bin]#

so is their any other way to tackle this problem.

[dpryan]

That looks like ~/bin, not ~/.local/bin. The installation log you posted even showed that that's where it put the executables.

[dariober]

I had installed it in this location and still m not able to access it

[root@localhost HTSeq-0.6.1]# python setup.py install --user
...
Installing htseq-qa script to /root/.local/bin
Installing htseq-count script to /root/.local/bin

I think you have done something weird here: You have installed the packages as root using the option --user. This installed htseq-count in the root home directory,/root/.local/bin. As regular user you can't access /root/.local/bin as it's not on path.

If you execute python setup.py install --user as regular user you should be fine and see htseq-count in ~/.local/bin/.

[meghavarshney]

thnkxx

thnkx to both of you . i got solution as for this i reinstalled HTSeq again as a regular user and now its properly working for me .


thankx again