Question: if my data sets are strand-spesific RNA-seq
1
gravatar for F
3.0 years ago by
F3.2k
Iran
F3.2k wrote:

hi,

how I could know whether my data sets coming from RNA-seq are strand-specific or not????? I emailed the company but they are in Chinese new year and could not help me yet but I should start the analysis.

thank you 

ADD COMMENTlink modified 3.0 years ago by Devon Ryan88k • written 3.0 years ago by F3.2k
1
gravatar for Devon Ryan
3.0 years ago by
Devon Ryan88k
Freiburg, Germany
Devon Ryan88k wrote:

Align a million or so reads (using the unstranded mode, if your aligner has one) and then use RSeQC to infer the experiment type.

ADD COMMENTlink modified 3.0 years ago • written 3.0 years ago by Devon Ryan88k
2

Just to complete your suggestion with a link =) 

http://rseqc.sourceforge.net/#infer-experiment-py

ADD REPLYlink written 3.0 years ago by Parham1.4k
1

thank you, then I need both bam and bed files? but in UCSC Table Browser there is no such a bed for Arabidopsis, then what to do?

ADD REPLYlink modified 3.0 years ago • written 3.0 years ago by F3.2k
2

Get the bed from BioMart (Ensembl plant): http://plants.ensembl.org/Arabidopsis_thaliana/Info/Index

Make sure the genome build matches.

ADD REPLYlink written 3.0 years ago by genomax62k
1

thank you, for aligning I used gtf coming from ensembl but there is gff3. no problem???

the main problem is this, I tried to convert my gtf to bed by bedops but some bug that I could not solve then I tried galaxy but in galaxy there is only gff to bed then I used my gtf instead of gff but

[izadi@lbox161 bin]$ infer_experiment.py -r Galaxy142-[GFF-to-BED_on_data_141].bed -i 204.bam
infer_experiment.py: No match.
[izadi@lbox161 bin]$ 

then do you know another solution please?

 

ADD REPLYlink modified 3.0 years ago • written 3.0 years ago by F3.2k
1
./infer_experiment.py -r Galaxy142-[GFF-to-BED_on_data_141].bed -i 204.bam
ADD REPLYlink written 3.0 years ago by Devon Ryan88k
1

Looks like you need to provide the path for infer_experiment.py since that does not seem to be in that bin directory.

ADD REPLYlink written 3.0 years ago by genomax62k

thank you but please consider

[izadi@lbox161 bin]$ pwd
/usr/data/nfs6/izadi/RSeQC-2.6.3/usr/bin

[izadi@lbox161 bin]$ ls
204.bam                                 junction_saturation.py
bam2fq.py                               mismatch_profile.py
bam2wig.py                              normalize_bigwig.py
bam_stat.py                             overlay_bigwig.py
clipping_profile.py                     read_distribution.py
deletion_profile.py                     read_duplication.py
divide_bam.py                           read_GC.py
FPKM_count.py                           read_hexamer.py
Galaxy142-[GFF-to-BED_on_data_141].bed  read_NVC.py
geneBody_coverage2.py                   read_quality.py
geneBody_coverage.py                    RNA_fragment_size.py
infer_experiment.py                     RPKM_saturation.py
inner_distance.py                       split_bam.py
insertion_profile.py                    split_paired_bam.py
junction_annotation.py                  tin.py
[izadi@lbox161 bin]$ 

 

ADD REPLYlink written 3.0 years ago by F3.2k
1

Try this in case Devon's suggestion did not work

$ python ./infer_experiment.py -r Galaxy142-[GFF-to-BED_on_data_141].bed -i 204.bam
ADD REPLYlink modified 3.0 years ago • written 3.0 years ago by genomax62k
1

thank you,

[izadi@lbox161 bin]$ python infer_experiment.py -r Galaxy142-[GFF-to-BED_on_data_141].bed -i 204.bam
python: No match.
[izadi@lbox161 bin]$ 

:(

 

ADD REPLYlink written 3.0 years ago by F3.2k
1

You don't have python installed on this machine? What OS are you using?

$ which python
ADD REPLYlink modified 3.0 years ago • written 3.0 years ago by genomax62k
1

fedora22

[izadi@lbox161 bin]$ which python
/bin/python
[izadi@lbox161 bin]$ 

 

ADD REPLYlink written 3.0 years ago by F3.2k
1

Looks like python/infer_experiment.py does not like that atrocious BED file name. Can you move/copy it to something else and try.

$ cp Galaxy142-[GFF-to-BED_on_data_141].bed simple.bed

$ ./infer_experiment.py -r simple.bed -i 204.bam
ADD REPLYlink written 3.0 years ago by genomax62k
1

thank you, 

[izadi@lbox161 bin]$ infer_experiment.py -r simple.bed -i 204.bam
Traceback (most recent call last):
  File "infer_experiment.py", line 49, in <module>
    from bx.bitset import *
ImportError: No module named bx.bitset
[izadi@lbox161 bin]$ 

ADD REPLYlink written 3.0 years ago by F3.2k
1

http://askubuntu.com/questions/631250/python-related-errors

ADD REPLYlink written 3.0 years ago by genomax62k
1

thank you

[izadi@lbox161 RSeQC-2.6.3]$ python setup.py build
running build
running build_py

running build_ext
running build_scripts
[izadi@lbox161 RSeQC-2.6.3]$ 
[izadi@lbox161 RSeQC-2.6.3]$ sudo python setup.py install
[sudo] password for izadi: 
Sorry, user izadi is not allowed to execute '/bin/python setup.py install' as root on lbox161.mpikg.mpg.de.
[izadi@lbox161 RSeQC-2.6.3]$ 

ADD REPLYlink written 3.0 years ago by F3.2k
1

python setup.py install --root=/home/user/XXX/

ADD REPLYlink written 2.4 years ago by F3.2k

You could ask your sys admins to install the modules or try to install them in your own directory: https://www.seas.upenn.edu/cets/answers/install-python-module.html

It is a bit late in this thread but can't you ask whoever produced this data if they used a "strand-specific" protocol?

ADD REPLYlink written 3.0 years ago by genomax62k
1

thank you

I emailed the company but they are in holliday

ADD REPLYlink modified 3.0 years ago • written 3.0 years ago by F3.2k

Not ideal but you could look at the alignments in IGV/Tablet to see if you can see an identifiable pattern in the alignment of reads.

ADD REPLYlink written 3.0 years ago by genomax62k
1

thank you so much

in a biostars post I read something about sam flags

then i saw 

abundances of 83, 99, 147 and 163 flags of my sam file is the same=1 , then might library IS sequenced strand-specific

anyway I think they will back from holiday Feb 14 and may they reply my email

ADD REPLYlink modified 3.0 years ago • written 3.0 years ago by F3.2k
1

sorry, I installed Python then this is my result

Reading reference gene model simple.bed ... Done
Loading SAM/BAM file ...  Total 200000 usable reads were sampled


This is PairEnd Data
Fraction of reads failed to determine: 0.0068
Fraction of reads explained by "1++,1--,2+-,2-+": 0.4992
Fraction of reads explained by "1+-,1-+,2++,2--": 0.4940

my library was sequenced in strand-specific mode? sorry if so first or second strand?

thank you

ADD REPLYlink written 3.0 years ago by F3.2k
1

No it was not strand-specific. See the explanation here: http://rseqc.sourceforge.net/#infer-experiment-py

ADD REPLYlink written 3.0 years ago by genomax62k
1

thank youuu

ADD REPLYlink written 3.0 years ago by F3.2k

Eeek, I had a few missing words in there too! Thanks for adding the link!

ADD REPLYlink written 3.0 years ago by Devon Ryan88k
1

thank you, 

I aligned but totally on transcriptom and  I have a accepted_hits.bam now. 

now I am in this path

/usr/data/nfs6/izadi/RSeQC-2.6.3/usr/bin

then how I can detect if is strand-specific?

 

 

ADD REPLYlink written 3.0 years ago by F3.2k
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