no out put in output.clipping_profile.r

[ASHWINI YADAV]

Hi ,

I am running clipping_profile.py but I am not getting any out in output.clipping_profile.xls and output.clipping_profile.r

Can you please help me with that

Thanks,
Ashwini

[Liguo Wang]

This is probably because your aligner does not support clipped mapping. 

see our manual: http://rseqc.sourceforge.net/

"Note that to use this funciton, CIGAR strings within SAM/BAM file should have ‘S’ operation (This means your reads aligner should support clipped mapping)."

Liguo

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[ASHWINI KUMAR]

Hi Liguo,

Thanks for the explanation. I want to know CIGAR string is only useful for clipping_profile.py or other RSeqc functions also need CIGAR string.

Which aligner would you recommend that supports clipped mapping.

Best regards,
Ashwini

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Ashwini Kumar

PhD Student

Institute for Molecular Medicine Finland (FIMM)

Nordic EMBL Partnership for Molecular Medicine

Biomedicum Helsinki 2U (D304b)

P.O. Box 20 (Tukholmankatu 8)

FI-00014 University of Helsinki, Finland

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[Liguo Wang]

Other functions in RSeQC do not require clipped mapping. MapSplice supports clipped mapping, but of course there are many others, please do you own research.

Liguo

[ASHWINI KUMAR]

Hi Liguo,

Thanks for the info. I tried with geneBody_coverage.py and I got this error and both output file are empty - 1. output.geneBodyCoverage_plot.r 2. output.geneBodyCoverage.txt

ERROR-
Load BAM file ... /opt/gridengine/default/spool/c19/job_scripts/9354807: line 30: 28529 Segmentation fault      (core dumped) geneBody_coverage.py -r /homes/akumar/RseQc_Results/Ensemble/hg19_Ensembl.bed -i /vault/rnaseq/fhrb_pm/FHRB.542/542_16052012_1200_BM/rna_seq/rnaseq_production_run_2.3_v67/geneExpression/alignment/tophat_paired.bam

Can you please have a look on the error.

Best regards,
Ashwini

[Liguo Wang]

Well, I guess you do not have enough memory. geneBody_coverage.py requires huge amount of RAM (up to 20Gb). If this exceed the  available RAM,  Convert BAM into bigwig then run geneBody_coverage2.py.

Thanks

Liguo

[ASHWINI KUMAR]

Hi Liguo,

I am using RSeQc and it's working well expect these two- 1 Splice Events, 2 gene body coverage are not working well, can you please have a look on the attached files. These are  same sample but their library was prepared using two different methods first is ribodeplition and second is polyA enrichment.

I am comparing these two libraries preparation methods

Thank you

Best regards,

Ashwini

On 19 February 2014 21:52, Liguo Wang <wangl...@gmail.com> wrote:

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[Liguo Wang]

I couldn't see any issues from these plots. For the junction annotation, you have very few novel junctions (complete novel + partial novel), therefore these two pieces are squeezed together in piechart.

the coverage plot also seems OK. Keep in mind, the coverage profile is depending on the gene models provided (i.e. a few extremely high expression genes such as microRNA might dominate your results). You might want to use hundreds of house keeping genes to get more reliable results.

Liguo

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[ASHWINI KUMAR]

Thank you Liguo,

Actually I am comparing two different library preparation protocols those are used for RNA-Seq library preparation. First is rRNA depletion and second is Poly A enrichment method. I was expecting 5' bias in rRNA depletion method and 3' bias in Poly A enrichment method but they both are looking same. I have attached gene body coverage output from another sample as well. Please have a look

I have used Ensemble gene model - should I try with different gene model?

wget http://dldcc-web.brc.bcm.edu/lilab/liguow/RSeQC/dat/hg19_Ensembl.bed.gz

Thank You

Ashwini

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[Liguo Wang]

The polyA selection protocol has a little bit 3' bias. The coverage was also affected by your RNA quality (measured by RIN etc).  Using Refseq or house keeping gene list might give you more reliable results.

Liguo