Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • RNA-seq library prep without poly-A selection

    Hello. I'm working on a project to examine gene expression in a system where RNA-processing has gone awry. I expect to see aberrant splicing, alternative transcription start site usage, aberrant poly-adenylation and capping, etc. RNA-seq is the perfect tool for looking at both differential expression and strange RNA-structural events.

    But I don't want to do a poly-A selection because I'll probably lose all these odd transcripts that I'm most interested in that might not be properly polyadenylated, spliced, capped, etc. Yet I don't want to sequence a ton of rRNAs and tRNAs.

    What kind of library preparation techniques should I investigate that don't require a poly-A selection?

    Many thanks.

  • #2
    Yet I don't want to sequence a ton of rRNAs and tRNAs.

    What kind of library preparation techniques should I investigate that don't require a poly-A selection?
    Given that RNA selection can be biased and will not necessarily remove all rRNAs, and current NGS sequencers give you oodles of data to play around with, I don't see a problem with sequencing the rRNA as well as other RNA.

    Let's say you have 80% rRNA in your sample, and 80% mappable reads. The amount of mappable non-rRNA sample will be about 16% of the original reads, so if you've got 100M single-end reads then that's 16M non-rRNA reads. With 50bp reads, that's a mean coverage of ~7x for a 120Mb transcriptome (vs ~33x for a "perfect" rRNA extraction).

    Comment


    • #3
      One way to do it is two extract RNA in two steps. 1.) Capture Poly-A tail 2.) Perform ribosomal depletion on the leftover sample and the leftover should contain Poly A-.

      When we do this we still see about 70-80% of the #2 sample still contain rRNA/tRNA.

      I would be interested in knowing if others can get a cleaner sample and methods used.

      Thanks!
      -Abhi

      Comment


      • #4
        Consider using duplex-specific nuclease (DSN):

        Comment


        • #5
          If you don't get rid of the rRNA, you might miss some of the rare/odd transcripts you mentioned you wanted to look for. Size selection is enough to get rid of tRNAs, which are generally under 100bp long. For rRNAs, I would use a ribosome depletion kit. Getting rid of those reads would be very useful in getting the depth you might need to see aberrant transcripts.

          Comment


          • #6
            You can just do two rounds of rRNA removal. I've used invitrogen's ribominus, cleared more than 90% of rRNA in first round (confirmed by real time PCR of 18s).

            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
            10 views
            0 likes
            Last Post seqadmin  
            Started by seqadmin, Yesterday, 06:07 PM
            0 responses
            9 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