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  • RRBS NGS library prep

    I am trying to prepare RRBS sequencing libraries with the NEBNext® DNA Library Prep Master Mix Set for Illumina® kit and NEBNext® Multiplex Oligos for Illumina® (Methylated Adaptor) kit. I used 5 ug starting DNA for each sample. However, I encounter several problems and I hope to find answers here.
    1. I used AMPure XP beads for sample clean up as suggested by the NEB protocol. Generally, the recovery rate for each clean up was above 85% except for the step after dA-tailing of end-repair product. The recovery rate was always below 45%. I repeated this step several times and the yield was the same. Is there something in the buffer or dA-tailing enzyme that inhibits bead binding efficiency? Can I improve the yield by adding more beads than 1.8X as suggested by the NEB DNA Library Prep protocol?

    2. The RRBS protocol that comes with the NEBNext® Multiplex Oligos for Illumina does not select proper size of DNA after adaptor ligation. I tried the dual bead-based size selection as suggested in the NEB DNA Library Prep protocol, but I am not able to recover any DNA with insert size of 100 or 200 bp. Does anyone have a working bead-based size selection protocol for RRBS? Does gel selection (3% Nusieve 3-1 agarose gel) or bead selection work better?

    Thank you and I look forward to your answers!

  • #2
    I meet the same issue, have you figured out how to solve it, please?

    Comment


    • #3
      I don't know what is happening with your post A-tailing cleanup, but when size selecting for RRBS you need to keep in mind that only ~1% of your sample will be in the 220-320bp (100-200bp insert) range so a very low recovery rate is expected.

      In my experience using beads produces a better yield than gel selecting, but both will work.
      Josh Kinman

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      • #4
        Originally posted by jdk787 View Post
        I don't know what is happening with your post A-tailing cleanup, but when size selecting for RRBS you need to keep in mind that only ~1% of your sample will be in the 220-320bp (100-200bp insert) range so a very low recovery rate is expected.

        In my experience using beads produces a better yield than gel selecting, but both will work.
        I don' t understand how do you use beads instead of gel selecting, do you mean to use beads to purify the selected gel fragment but not use the column in the kit?

        Comment


        • #5
          Originally posted by reprogrammer View Post
          I don' t understand how do you use beads instead of gel selecting, do you mean to use beads to purify the selected gel fragment but not use the column in the kit?
          I mean using only beads to perform the size-selection instead of using a gel at all.

          This blog post explains the process of size selection with beads pretty well.
          Someone recently asked me, “how do SPRI beads work” and I realized I was not completely sure so I went to find out. My lab uses kits. Lots...
          Josh Kinman

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