Question

Cufflinks: Why in the skipped gtf file showed a portion of genome masked

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9.9 years ago

mjoyraj ▴ 80

I mapped my RNA-se reads with Tophat with the commands

Tophat -p 8 -o output -I 20000 -g 5 -G gtf_file Genome Fastq1 fastq2

Then I assembled the aligned tophat bam file using cufflinks with the commands

Cufflinks -p 20 -o output accepted.bam file

I found some of the genes are not assembled and they are masked as can be seen in skipped.grf.

I wonder why this has happened. Can anybody suggest something

Cufflinks Assembly RNA-Seq • 3.4k views

ADD COMMENT • link updated 2.7 years ago by Ram 44k • written 9.9 years ago by mjoyraj ▴ 80

Ram · Accepted Answer · 2015-01-14

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9.9 years ago

Devon Ryan 104k

That tophat command is appropriate for PE mapping. You need to separate the fastq files with commas (no spaces!).
If coverage is too high then cufflinks will skip a region.

ADD COMMENT • link 9.9 years ago by Devon Ryan 104k

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Yes, I observed the skipped region are mostly my target genes which are highly expressed (as can be seen from mapped reads). So I am trying again cufflinks-assembly with "--max-bundle-frags 1000000000000".

ADD REPLY • link 9.9 years ago by mjoyraj ▴ 80

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Something like that, I always have to look up cufflinks parameters.

ADD REPLY • link 9.9 years ago by Devon Ryan 104k

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Thanks.... Ryan

ADD REPLY • link 9.9 years ago by mjoyraj ▴ 80

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Dear Ryan, when I used --max-bundle-frags 1000000000000 in cufflinks, the run continues for a long time and it is still running showing waiting for two threads to complete. Is it okay?. Do I need to increase the number of threads?

ADD REPLY • link updated 2.7 years ago by Ram 44k • written 9.9 years ago by mjoyraj ▴ 80