Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Library yield problem

    Hi everyone,

    I have a specific problem for a few months now with our in-house library preparation for Illumina. We have lower than expected yields by a significant margin - in extreme cases we get 20 ng total out of 10ng of starting material after 18 cycles of PCR, which is ridiculous.

    We know that from the very same samples we get great yields with the Illumina kit when done by the core facility (800 ng final).

    What steps are the most susceptible to be the cause of the problem?

    We use End-it kit for end-repair, followed by A addition with Klenow exo-, ligation of adapters (PAGE purified with a thio bond at the last base) with Quick ligation kit from NEB, gel size selection and PCR with KAPA (Phusion gives the same, so its probably not the PCR step). So everything is pretty standard.

    Between every step we cleanup with Qiagen and at the last one with Ampure to get rid of adapter dimers, which we seem to get a LOT of when things are not working right.

    To be clear, the protocol gave great results in the past, but at some point yields decreased significantly.

    Is it possible that some reagents are more sensitive to freeze / thaw? What would you troubleshoot first? I know the End-it kit isn't directly in cause as results were the same when we got a new one.

    I'll try to reanneal adapters from the 100 uM stock to 15 uM working but aside from that I have no idea why it would fail that bad. Please help if you can

  • #2
    How much material are you starting with? Expect 80% recovery on EACH cleanup step (on a bad day). Are you eluting off of your columns with warm water/basic Tris?

    Enzymatic reagents are absolutely sensitive to freeze thaw. The likely culprit is an ATP-containing reagent, probably in your end repair mix or ligation mix.

    Also, have you looked at the electropherograms of ampure supernatant? If sample is "bleeding through" you might want to get another lot of beads.

    Comment


    • #3
      The last one was 10 ng but sometimes its lower - doesn't really matter as no matter the starting material we seem to end up with 20-100ng final, which is much lower than before.

      I'll try a new ligase and end repair mix, but I doubt it's gonna fix the problem as those are few reactions and changed pretty often - do you usually aliquot the buffer of those, thaw on ice?

      Good tip on the ampure - I noticed that all protocols recommend letting them warm to room temp, and I think lately I'm using it straight from the 4 degree, might be related.

      Comment


      • #4
        How about cleaning up with beads between your end-repair and adenylation steps? Sounds like you are using beads after ligation. If you are using 60nt adapters, then I highly recommend the beads over columns.

        Comment


        • #5
          Hi All

          We are preparing library using TruSeq kit from Illumina (further planning to do exome sequencing), and facing the same problem of lower library yield after PCR (~50ng).

          We are using gel based method instead of gel free method for size selection. (tried both with Qiagen and MDI gel extraction kits). Can this be a problem??

          Comment


          • #6
            What's your starting DNA input amount? Gel-free bead based size selection will certainly result in higher library yields.

            Comment


            • #7
              Thanks for the reply.
              We are starting with 2microgms of DNA.
              Any idea what can be other possible reasons for such low yield?

              Comment


              • #8
                Other reasons could be related to your starting input material. Have you checked your DNA quality? There may be PCR or other enzyme inhibitors in your prep. How many cycles of PCR are you using?

                Comment


                • #9
                  10 PCR cycles. Can improper adapter ligation be one of the reasons. If yes, How do we check for it?

                  Comment


                  • #10
                    You can use the bioanalyzer post ligation to check that you have adapters on both ends of your insert. Can you upload a gel image or some specs of your starting DNA input?

                    Comment


                    • #11
                      In the past when I had issues I would do a library prep with a single PCR product and then using a bioanalyzer I could easily see what percentage of the product made it to the adapter ligation step and quantify what percentage of the products had adapters ligated on each end. You can even run it on a gel if you don't want to use a chip. Just a thought.

                      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, 03-27-2024, 06:37 PM
                      0 responses
                      12 views
                      0 likes
                      Last Post seqadmin  
                      Started by seqadmin, 03-27-2024, 06:07 PM
                      0 responses
                      11 views
                      0 likes
                      Last Post seqadmin  
                      Started by seqadmin, 03-22-2024, 10:03 AM
                      0 responses
                      52 views
                      0 likes
                      Last Post seqadmin  
                      Started by seqadmin, 03-21-2024, 07:32 AM
                      0 responses
                      68 views
                      0 likes
                      Last Post seqadmin  
                      Working...
                      X