Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Should one combine normalization methods in RNA-seq?

    Does anyone know if RPKM normalization and quantile normalization of RNA-seq read counts can or should be combined?

    For normalization of paired-end reads in RNA-seq, is it accurate to normalize the raw read counts using the quantile method, then following that calculate the RPKM value for each transcript using the quantile normalized values?

    It seems that RPKM normalization using the total millions of mapped reads is somewhat redundant with an initial quantile normalization of read counts. Are these two methods mutually exclusive or should they be combined? Any recommendations?

    thanks!

  • #2
    It's a bit late for this answer but maybe future users will benefit from that:
    When you use FPKM / RPKM as a measure unit you already have a normalized unit, which accounts for the gene length. However, when you have many samples sometimes you have different sequencing depths and thus different expression values that are the same thing but at two different depths. To account for that, using a global scaling or a full quantile normalization will help. In fact, when doing it you will end up with all the different "lanes" having the same statistical properties and thus remove the problem of the sequencing depth.

    So, in conclusion, use RPKM/FPKM and then do a full quantile on all the samples, it works and is a good practice.

    Comment


    • #3
      A better solution would be to never use RPKM/FPKM, since it tends to kill your statistical power, which is related to the raw counts and gets normalized away by the RPKM transformation.

      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
      11 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
      51 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