Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Questions about RNA-seq experiment design

    I try to sequence the RNAs from 2 conditions, 5 replicates per condition, using multiplex Illumina Hiseq 2000. I would like to consult about experiment design. There are 3 plans. Any suggestions are greatly appreciated, especially for the third one.

    First, 5 samples are put in one sequencing lane (2 for condition A, 3 for condition B, and vice versa). This will be asymmetric.

    Second, randomly choose 4 samples in one lane (2 for condition A, 2 for condition B). This reduce the replicates but increase the reads number for each sample.

    Third, keep 5 reps each and pool all 10 and then run half a pool each on two lanes. Each lane would generate 10 separate fastq files and one could then concatenate the 2 fastq files (from the 2 lanes) for the same samples to get more reads for each sample, ending up with a total of 10 fastq files. We are not sure if there are other considerations in this scenario.

  • #2
    We do all our RNA-Seq through option 3.

    Obviously this is not always possible when you have hundreds of samples, and then the randomised/mixed approach is fine.

    But with 10 samples, just index them all separately, pool and sequence down two lanes and combine the Fastq.

    Comment


    • #3
      Option 3 every time.

      You can multiplex up to 96 samples in one pool on HiSeq. I always recommend pooling the whole experiment if possible, and if not consulting with a statistician beforehand to get a controlled randomisation.

      A MiSeq QC can help chekc your pool is balanced. However for your experiment I'd suggest getting the seqeuncing done without MiSeq as a single-end 50bp read (what we'd use for DGE) is cheaper thana MISeq QC run in many labs!

      Comment


      • #4
        A problem we have is balancing the number of reads between samples. As James suggested using a MiSeq is a good way to do figure out the balance before running the HiSeq. But even a better method (if you have the time) is to run a pooled sample in a single HiSeq lane and use the information from that lane to balance a second run. You'll get much more data that way.

        Comment


        • #5
          Thank you very much. I really appreciate your help.

          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
          66 views
          0 likes
          Last Post seqadmin  
          Working...
          X