Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • CuffDiff: genes with highly abundant reads have FPKM 0.

    I just started learning rna-seq data anlaysis following Nature Protocols 7, 562–578 (2012).
    I used CuffDiff after tophat, but the genes with highly abundant reads have FPKM 0.
    I tried all the different normalization options in CuffDiff, and --max-bundle-frags 2000000, but still FPKM 0 for the genes with the largest number of mapped reads. Is there any upper bound like Microarray excludes the saturated spots ?

    I added example below,
    In sample A, it has FPKM 0, because it has the most number of reads, but the same gene in sample B has FPKM 3789, because it's in the confidence zone in sample B ? Please, help me to get FPKM for those genes with highly abundant reads. How can I get FPKM for PM_0405A60 from the example below?
    Thanks,


    Both examples below were sorted by the # reads in the descending order.
    I counted the number of reads using 'htseq-count'

    Sample A

    Gene | # reads | FPKM
    PYYM_0405060 | 1447079 | 0
    PYYM_1351050 | 675926 | 0
    PYYM_1007060 | 559162 | 0
    PYYM_1209040 | 421148 | 29315.7

    Sample: B
    Gene | # reads | FPKM
    PYYM_0501060 | 1633262 | 0
    PYYM_1351050 | 757339 | 0
    PYYM_0405060 | 552553 | 3789.85

    CuffDiff –verbose printed the below; PM_0405A60’s loci is PyYM_04_v1:214849-216007


    Inspecting bundle PyYM_04_v1:206968-210685 with 5956 reads
    Inspecting bundle PyYM_04_v1:211950-212458 with 368 reads
    Inspecting bundle PyYM_04_v1:213051-213787 with 2864 reads

    Inspecting bundle PyYM_04_v1:214849-216007 with 943663 reads

    Inspecting bundle PyYM_04_v1:218737-223186 with 746 reads
    Inspecting bundle PyYM_04_v1:223748-224267 with 889 reads
    Inspecting bundle PyYM_04_v1:225620-226957 with 399 reads
    Inspecting bundle PyYM_04_v1:227958-238759 with 10900 reads
    Last edited by uniportdb; 02-18-2013, 04:50 PM.

  • #2
    I encountered something like this last year, where I was getting transcripts flagged as HIDATA in Cufflinks output.

    You might try increasing the --max-bundle-frags setting in from its default setting to get around this issue. See:

    --max-bundle-frags <int> Sets the maximum number of fragments a locus may have before being skipped. Skipped loci are listed in skipped.gtf. Default: 1000000

    Paul Krzyzanowski
    Blog: www.checkmatescientist.net

    Comment


    • #3
      I'm having the same problem but in my case there are no genes in the skipped.gtf file, so it's not because of HIDATA flag but there is another problem that I couldnt figure out yet... Any help would be really appreciated!!

      Comment


      • #4
        Thank you, PaulK.

        I tried --max-bundle-frags 2000000, but still 0 FPKM.
        And then I tried --max-bundle-frags 3000000, at last I have none-zero FPKM 107219.
        I guess it's paired-end, so the max should be > 2 * No. reads.
        Last edited by uniportdb; 02-18-2013, 04:59 PM.

        Comment

        Latest Articles

        Collapse

        • seqadmin
          Strategies for Sequencing Challenging Samples
          by seqadmin


          Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
          03-22-2024, 06:39 AM
        • seqadmin
          Techniques and Challenges in Conservation Genomics
          by seqadmin



          The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

          Avian Conservation
          Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
          03-08-2024, 10:41 AM

        ad_right_rmr

        Collapse

        News

        Collapse

        Topics Statistics Last Post
        Started by seqadmin, Yesterday, 06:37 PM
        0 responses
        12 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, Yesterday, 06:07 PM
        0 responses
        10 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 03-22-2024, 10:03 AM
        0 responses
        52 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 03-21-2024, 07:32 AM
        0 responses
        68 views
        0 likes
        Last Post seqadmin  
        Working...
        X