Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Using alternative normalization method in DESeq analysis of gene enrichment

    I'm using DESeq to identify differentially expressed genes in a next-gen sequencing dataset. (DESeq: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3218662/). In my experiment, the normalization of read counts as implemented by DESeq may not perform as well as anticipated. DESeq uses a 'size factor' to achieve a common scale of count values across samples. The size factor for a given library is defined as the median of the ratios of observed counts to the geometric mean of each corresponding target over all samples.

    For my dataset, upper-quartile scaling (using the 75th percentile of data, which often has low read counts, for linear scaling) may improve performance. In my specific case it may even be possible to define a set of genes as an internal scaling standard.

    In order for the statistical test for differential gene expression to work correctly, am I allowed to use an alternative method for data normalization?

  • #2
    Sure, see the CQN package for an example of a different normalization method that can be used with DESeq2, which you should be using rather than DESeq.

    Comment


    • #3
      Thanks a lot for your quick answer!

      May I ask, what are the formal constraints when changing normalization methods, such that the basic concept of DESeq(2) still works? Ie. assumption of a negative binomial distribution, Fisher's exact testing? As long as I transform the data linearly and get count values out, I'm fine to transform the data?

      Comment


      • #4
        And to extend my question, would I be fine to apply a LOESS transformation? It appears to me that in some of my conditions, there are very active targets, which then cause a bias in reads. Thus, a LOESS transformation may be relevant. Here are some plots of my data, MA plots given on the right side of the diagonal.

        (Here's the link in case the picture doesn't show: https://www.dropbox.com/s/qssqov0hzr...2_v01.png?dl=0 )
        Last edited by simon_seq; 08-27-2015, 11:00 AM. Reason: Link doesn't work.

        Comment


        • #5
          The trick is to not transform the data at all. Leave the data untouched and supply offsets and such to produce the needed normalization. Again, see how the CQN package and DESeq2 interact.

          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
          8 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, Yesterday, 06:07 PM
          0 responses
          8 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-22-2024, 10:03 AM
          0 responses
          49 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 03-21-2024, 07:32 AM
          0 responses
          67 views
          0 likes
          Last Post seqadmin  
          Working...
          X