Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Difference between FPKM and raw reads?

    hey,

    we sent 3 Affected and 3 Control Mice Samples for Sequencing to compare Gene Expression Levels and we got back a bunch of files that were generated with cufflinks. So theoretically we would now compare the Gene expression Levels between Affected and Control to see where they vary and what might cause the phenotype.

    The problem is that I dont know which files to use.
    basically there are 3 excel Files.
    isoform_exp.diff.with.fpkm_denovo
    isoform_exp.diff.with.fpkm_known
    dge_results
    (Differential Gene Expression)

    so the first 2 are the fpkm values(de novo and known) and the last one are basically raw read counts from what I can understand.
    Theroetically it shouldnt matter which file I use because all we need is the ratio from control group to affected to find out which genes are less/more expressed in the affected group.
    But when I try that with the different files I get huge differences that should not be there(if I understand it correctly)
    For example Errc2.
    In dge results it's a 1:1 ratio between control and affected, in fpkm_known it's 1:10 and in fpkm_denovo the gene isnt even listed.

    also I would like to know how they get their FPKM values.
    FPKM means something like Fragments per kilobase of exom per million reads.
    But some Genes have like [S1:40, S2:34, S3:54] in raw reads, but in FPKM it looks like [S1:1.6, S2:0.00005, S3: 1.3]

    I also dont understand how cufflinks calculates p-values.
    There are some genes where FPKM is zero for all the samples, but they still got totally different p values how can that be?

  • #2
    Hi Big Cheese,
    I am new to the sequencing field, but from what you write I might give you some advices:
    first, I understood a lot by reading the manual on cufflinks web site http://cufflinks.cbcb.umd.edu/.

    Have a go and you will see you will find your answers.

    Also there is a recent paper from Trapnell where they did what you are doing basically and you might want to have a go there as well: "Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks"

    Also, as you will read, you might want to run cuffdiff on you samples. Cufflinks it's only assembling your aligned reads, what you want is the difference between you two group samples after assembling them.

    Best,
    ib

    Comment


    • #3
      hm,
      the files I got were already generated with cuffdiff.

      And I read the manual, but it hasn't really helped me
      Last edited by Big_Cheese; 07-30-2012, 11:09 AM.

      Comment


      • #4
        Hi Big_Cheese,

        Call whomever did the sequencing and analysis for you and ask the questions. That's fastest way to have your questions answered.

        Best regards,
        Douglas

        Comment

        Latest Articles

        Collapse

        • 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
        • 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

        ad_right_rmr

        Collapse

        News

        Collapse

        Topics Statistics Last Post
        Started by seqadmin, 04-11-2024, 12:08 PM
        0 responses
        11 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 04-10-2024, 10:19 PM
        0 responses
        17 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 04-10-2024, 09:21 AM
        0 responses
        14 views
        0 likes
        Last Post seqadmin  
        Started by seqadmin, 04-04-2024, 09:00 AM
        0 responses
        43 views
        0 likes
        Last Post seqadmin  
        Working...
        X