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  • mdubs
    Junior Member
    • May 2013
    • 2

    miRNA precipitation prior to Illumina TruSeq Small RNA lib prep

    Hi, I have isolated small RNA using the miRvana kit and would like to concentrate my product for input into the TrueSeq Small RNA library prep kit and minimize loss (no use of columns). My question is, has anyone used glycogen (with NaOAC, EtOH) in a precipitation just prior to the library step? My concern is that glycogen may interfere with the the adaptor ligations to come.

    I spoke with Illumina and they did not have a clear answer to this. However, I did find an earlier version of their TruSeq Small RNA manual that included glycogen in a concentration step just prior to ligation processes. Although, in the current manual, there is no mention of it. Any info would be much appreciated.

    Thanks in advance.
  • Genohub
    Registered Vendor
    • Mar 2013
    • 210

    #2
    glycogen or linear acrylamide

    You can certainly use glycogen prior to the library step. In my experience, glycogen does not interfere with ligation. You may want to also try out linear acrylamide, its inert and has less of a chance of being contaminated with host nucleic acid.


    Originally posted by mdubs View Post
    Hi, I have isolated small RNA using the miRvana kit and would like to concentrate my product for input into the TrueSeq Small RNA library prep kit and minimize loss (no use of columns). My question is, has anyone used glycogen (with NaOAC, EtOH) in a precipitation just prior to the library step? My concern is that glycogen may interfere with the the adaptor ligations to come.

    I spoke with Illumina and they did not have a clear answer to this. However, I did find an earlier version of their TruSeq Small RNA manual that included glycogen in a concentration step just prior to ligation processes. Although, in the current manual, there is no mention of it. Any info would be much appreciated.

    Thanks in advance.

    Comment

    • NextGenSeq
      Senior Member
      • Apr 2009
      • 482

      #3
      We anyone uses the Illumina Small RNA kit is beyond me. It's been published for two years that the ligation bias using that kit gives ENTIRELY WRONG results.

      Background The use of nucleic acid-modifying enzymes has driven the rapid advancement in molecular biology. Understanding their function is important for modifying or improving their activity. However, functional analysis usually relies upon low-throughput experiments. Here we present a method for functional analysis of nucleic acid-modifying enzymes using next generation sequencing. Findings We demonstrate that sequencing data of libraries generated by RNA ligases can reveal novel secondary structure preferences of these enzymes, which are used in small RNA cloning and library preparation for NGS. Using this knowledge we demonstrate that the cloning bias in small RNA libraries is RNA ligase-dependent. We developed a high definition (HD) protocol that reduces the RNA ligase-dependent cloning bias. The HD protocol doubled read coverage, is quantitative and found previously unidentified microRNAs. In addition, we show that microRNAs in miRBase are those preferred by the adapters of the main sequencing platform. Conclusions Sequencing bias of small RNAs partially influenced which microRNAs have been studied in depth; therefore most previous small RNA profiling experiments should be re-evaluated. New microRNAs are likely to be found, which were selected against by existing adapters. Preference of currently used adapters towards known microRNAs suggests that the annotation of all existing small RNAs, including miRNAs, siRNAs and piRNAs, has been biased.

      Comment

      • Genohub
        Registered Vendor
        • Mar 2013
        • 210

        #4
        Here is the original paper that identified the bias:

        Identification and remediation of biases in the activity of RNA ligases in small-RNA deep sequencing

        To solve the problem they used adapters with randomized ends.

        Comment

        • peterawe
          Member
          • Mar 2013
          • 14

          #5
          Hi. Thanks for the link! The ligation bias is unfortunate and difference in read counts between different miRNAs are not readily interpreted as a measure of their biological expression. However, the profiles generated from these protocols could still be used to access e.g. relative expression between groups.

          A question: In the Sorefan et al. Silence Journal paper they used the older Illumina kit, not the current Truseq small RNA. Did Illumina adress some of these issues with the new version?

          Comment

          • Genohub
            Registered Vendor
            • Mar 2013
            • 210

            #6
            No, unfortunately the ligation bias has not been solved with Illumina's latest TruSeq Small RNA Kit. Relative comparisons between groups can be made with the same microRNAs, but different microRNAs cannot accurately be compared to each other.

            Comment

            • NextGenSeq
              Senior Member
              • Apr 2009
              • 482

              #7
              There are a couple small companies releasing kits with the modified adapters which supposedly reduce or eliminate the ligation bias. We are testing them now and comparing the results to QPCR and microarray expression profiling.

              Comment

              • peterawe
                Member
                • Mar 2013
                • 14

                #8
                Thanks for the answer. On the positive side, the Truseq small RNA kit gives in our hand excellent technical reproducibility during library prep - which is also a very important point.

                Do you have link to any of the kits you are testing and when do you think your results would be out? I am really interested if you have any experience/data/papers on which kits would perform better. We are about to start profiling a large series and are investigating different options.

                Comment

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