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

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
• 4,090 views • link
updated by Ram Ram • written by mjoyraj mjoyraj

1 answer

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

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".

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written by mjoyraj mjoyraj

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

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written by Devon Ryan Devon Ryan

Thanks.... Ryan

• 0 views • link
written by mjoyraj mjoyraj

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?

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updated by Ram Ram • written by mjoyraj mjoyraj

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