Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • Is anyone having trouble with tophat's SAM file -> cufflinks?

    I am having trouble running cufflinks on my own SAM file generated from tophat. I used many SAM files generated from real data as well as simulated data, but I can't get cufflinks to assemble the transcripts using any of my SAM files.

    I tried downloading the file: test_data.sam and it works fine. It identifies three exons and notes that they are part of one transcript. When I try this on my SAM file, it identifies all the exons as their own transcripts, even though I see many reads that span the exons.

    Any help would be appreciated. Thanks

  • #2
    Did you run Tophat on more than one sequence reads at the same time? If so you might want to run it one at a time. If not, then well, at the moment I can't think of an answer.

    Siva

    Comment


    • #3
      Would this matter if I specify a different ouuput file for each run?

      I am trying to figure out if the SAM format is a problem or if the actual reads do not cover the junction enough for the transcript to be called.

      Comment


      • #4
        Actually it is really strange. I cannot get any of my tophat-generated SAM outputs to successfully run using cufflinks (it runs but it lists each exon as its own transcript). I've done this with real data and simulated data.

        Comment


        • #5
          Originally posted by peterlchang View Post
          Actually it is really strange. I cannot get any of my tophat-generated SAM outputs to successfully run using cufflinks (it runs but it lists each exon as its own transcript). I've done this with real data and simulated data.
          Hi are u working with an organism whose typical intron size is very large? If so you can change Tophat's default parameters. Cufflinks typically gives you a GTF file detailing FPKM etc, a comma delimited transcript.expr file and a list of gene coordinates (genes.expr).

          Comment


          • #6
            The gene I am testing is 34K long (ie. the reference sequence is 34K long). The default intron length for tophat is 500K and I changed that parameter to 50K. The default intron length for tophat is 300K, and that was left unchanged.

            In addition, tophat is able to find reads that span the junction. I don't know what the issue could be. I already emailed Cole so I hope he can get back to me asap.

            Thanks

            Comment

            Latest Articles

            Collapse

            • seqadmin
              Essential Discoveries and Tools in Epitranscriptomics
              by seqadmin




              The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist...
              04-22-2024, 07:01 AM
            • seqadmin
              Current Approaches to Protein Sequencing
              by seqadmin


              Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...
              04-04-2024, 04:25 PM

            ad_right_rmr

            Collapse

            News

            Collapse

            Topics Statistics Last Post
            Started by seqadmin, Yesterday, 08:47 AM
            0 responses
            12 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 04-11-2024, 12:08 PM
            0 responses
            60 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 04-10-2024, 10:19 PM
            0 responses
            60 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, 04-10-2024, 09:21 AM
            0 responses
            54 views
            0 likes
            Last Post seqadmin  
            Working...
            X