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  • #16
    When I contacted Sage they replied, "Blue Pippin users who use TruSeq kits, perform size selections at 500-600bp to achieve the desired 450 peak." Yet Sage later made this sound like an approx. range (hence my post).

    The Blue Pippin gels we're using (1.5% cassettes) have internal standards, and do Not have ethidium bromide. A marker is loaded with ea. sample.

    Another user just tried the 500-600 bp setting (different DNA source, but again Blue Pippin / TruSeq DNA), and that shifted the peak from the prior median of ~300 to ~350 bp. It's close to good enough, but a 30 bp insert (100 bp, PE, + ~120 bp for the adapters) seems a tad short. He has a lot of undesirable products at higher sizes as well -- presumably PCR artifacts with smaller/lower quantity peaks around 1.2-1.4 kb. (We have less input DNA available and cut the reagent volumes in 1/2 , so last time I didn't have the odd bubble peaks; this time we increased from 150 ng to 300 ng and I've been wondering about cutting the # of PCR cycles...).

    In talking about this the lab is concerned with potential issues/biases with the PCR (eg amplifying adapters pre-PCR) so we may see if we can just offset the Blue Pippin settings by trial and error. Given we have low input DNA to start I hesitate to go to the manual gels per Illumina's protocol, as that can (I gather) give lower yield, and do want to avoid adapters dimerizing or annealing to the product etc. I'm also not sure if we'd have to use 2X the PCR reagents if we do some cycles before and others after the size selection step; at present we're working with the TruSeq kits and have 28+ libraries so that could get expensive. Trial & error optimization to guess the apparent size of Y-ligated adapter+library DNA seems rather inefficient ... but we might be able to make it work with the 2 extra libraries I made this time for troubleshooting.

    Thank you again for all of your insight! This is my first time with the DNA TruSeq kits (and the cantankerous Blue Pippin; I love the precision of the instrument ... but adapting it for use with TruSeq is frustrating).

    Comment


    • #17
      Hi,
      Does anyone have final libraries which show shoulder peaks on their larger insert libraries?

      Thanks,
      Anna.

      Comment


      • #18
        Originally posted by hawaii454-0 View Post
        Hi,
        Does anyone have final libraries which show shoulder peaks on their larger insert libraries?

        Thanks,
        Anna.
        How large do you mean? We have only made a handful of libraries with >1000 bp inserts. I don't recall seeing a bubble peak on any of them. But the molar ratio of primers/template is higher for those, so that may explain why.

        --
        Phillip

        Comment


        • #19
          Hi Phillip,
          No not as large as that. I mean 500-600bp insert libraries. See attached Agilent trace. How do you size select your libraries? The libraries on the attached trace were size selected on an Invitrogen 2% size select gel. Prior to that, I have tried Caliper XT and a standard ethidium gel which both gave similar/larger shoulder peaks. We've been told that shoulder peaks like this can cause problems for analyses later on.

          Any advice you could give would be much appreciated.

          Thanks,
          Anna.
          Attached Files

          Comment


          • #20
            Hi Anna,

            We just use Ampure for 90% of the libraries we make.
            How many cycles of amplification did you do?

            Phillip

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            • #21
              Hi Phillip,
              Do you mean you do ampure clean up for size selection? What ratio do you use? 0.6 beads:1 library?

              At the moment, we are doing 12 cycles for the PCR.

              Thanks for your help,
              Anna.

              Comment


              • #22
                Hi Anna,
                There's also a thread (a few pages; eg I think an initial suggestion for ramping was discarded on a later page) that I found informative at: http://seqanswers.com/forums/showthread.php?t=9124

                Comment


                • #23
                  The link that Hilary posted is to a thread that includes a post where I waxed rhapsodic about bubble products, what the common wisdom was as to what they were, etc.

                  I now accept the common wisdom that "bubble products" are just that, 2 strands annealed at their TruSeq adapter ends but not in the middle where their sequences are divergent. Then these "ectopically annealed" molecules run as about 2x their true length. Also that they originate as a result of the concentration of template exceeding some threshold in the PCR reaction where a substantial fraction of the library molecules reanneal to other library molecules before a primer can anneal to them.

                  If this is what you have, Anna, the recommended course is to ignore the shoulder. If that doesn't work for you, you can re-amplify (just a couple of cycles) with fresh reagents. That should denature all the ectopically annealed strands and synthesize complements for them.

                  But, if we accept the bubble product hypothesis there is the issue that your library probably has a smaller average insert size than what you were shooting for. (The lower end looks to be 300 bp amplicon, or 180 bp insert -- about half what you said you wanted.

                  So the secondary issue likely derives from the second bugaboo of DNA TruSeq libraries -- if you do a gel-based size selection prior to enrichment PCR, you get gel migration artifacts cause either by the Y-adapters and/or ligase attachment persisting. The solution to this is said to be either:

                  (1) Do the size selection after enrichment PCR. I think this should be fine unless you are starting out with quite a bit of adapter dimers.

                  (2) Size select without dye in your agarose (stain after the run).

                  --
                  Phillip

                  Comment


                  • #24
                    Originally posted by hawaii454-0 View Post
                    Hi Phillip,
                    Do you mean you do ampure clean up for size selection? What ratio do you use? 0.6 beads:1 library?
                    Depends on the size we are shooting for. We always calibrate our ampure by precipitating a size standard marker with various ampure cuts. Then we run them on a high sensitivity chip.

                    Recently we have added an upper ampure cut as well because I think the very high molecular weight portion of the library was interfere with our ability to obtain an accurate titration using a KAPA qPCR kit.

                    --
                    Phillip

                    Comment


                    • #25
                      Hurrah. So we're a bit off the target 450 bp for 100 bp PE HiSeq sequencing (per the TruSeq protocol). Yet here's the result of programming a 650 bp "tight" cut (Blue Pippin 1.5% cassettes, R2 marker; loaded 30 uL of sample in "Resuspension Buffer" + 10 uL marker. Previously another user tried Illumina's Res. Buffer and I used TE as the solvent; this did not seem to affect migration ... from one trial run).

                      Yet at 490 bp, it's pretty close to the target -- perhaps good enough or if not, at least within the range that one more trial should let us nail the target. We ran 10 PCR cycles, but used 1/2 reagent volume in the TruSeq kits and 300 ng input DNA ...
                      ~Hilary
                      Attached Files
                      Last edited by Hilary April Smith; 10-29-2012, 12:18 PM.

                      Comment


                      • #26
                        Seems we have some slightly large products too (dwarfed in comparison to the 490 bp peak; we anticipated lower yield so we ran a High Sensitivity Chip but seems we should've done a regular DNA chip).

                        Anyway the attached pdf is something I found online in case that helps you Anna. I queried Illumina and here's their reply:
                        "Thank you for sending in this trace. 490bp should be fine, it just means there's a slightly longer insert for this library and it should not have an effect on this next run. I noticed that the markers for this trace are very very small however, meaning that the sample has been overloaded. As such, I can't really tell if the high molecular weight hump is significantly large or not. This can be checked by further diluting the library (so that it falls within range of the height of the marker peaks) if you're very concerned. The most common causes of high molecuar weight humps are either AMPure bead carryover or PCR artifact. Neither of which should be a problem for sequencing but can be good to double check if you are worried. Beads don't interfere with clustering at all and artifacts are usually perfectly good library that have re-annealed incorrectly. Upon denaturation of the library, this issue is resolved."
                        Attached Files

                        Comment


                        • #27
                          Hi all,
                          A few posts in this thread mentioned the possibility of doing Pippin Prep size-selection post-PCR, which means the size-selected DNA is in the Pippin Prep elution buffer when it goes into the Illumina cluster formation procedure. Is this a problem? Or is the unstated assumption that the size-selected DNA is changed into a different buffer using beads or a column clean-up step before cluster formation?

                          Thanks!

                          Comment


                          • #28
                            If you're concerned about the product being in TE buffer from the Pippin... when I contacted Illumina they once noted concern that the EDTA could interfere with subsequent steps. Thus when I do BluePippin preps BEFORE PCR, I usually run an Ampure Cleanup and elute in Illumina's Resuspension buffer after the Pippin/before the PCR. It may not be necessary, but I just didn't want to take any chances with PCR failure.

                            For running the Pippin as the final step, I'd recommend contacting the genomics core to which you'll send your samples for sequencing. We don't have a HiSeq here and have to send the samples out; if memory serves me right the center where we sent our samples would've taken them in either Illumina's Resp. Buffer or in TE. If the TE isn't accepted, just doing one more Ampure cleanup after the Pippin should let you elute in water or Illumina's Resp. Buffer (or TE). I've not done cluster formation/the actual sequencing myself.

                            Comment


                            • #29
                              Hilary,
                              Thanks for the quick reply. We have successfully used the PippinPrep eluates directly as template in PCR reactions (without changing buffer), as others in the forum have also noted. We don't have our own HiSeq either, but hoped that someone on the forum might have direct experience with the use of PippinPrep eluates in cluster formation. If TE is acceptable (with 1 mM EDTA), then the PippinPrep eluate may work fine, because the user manual for the device indicates that EDTA concentration in the eluate is in the range of 1 - 2 mM.

                              Best regards

                              Comment


                              • #30
                                Best of luck; perhaps one of the other posters from a Genomics Core can help. Also while I'm unsure if he discusses cluster formation, the webpage "http://ethanomics.wordpress.com/" has some generally helpful advice -- maybe something in there will help.
                                ~Hilary

                                Comment

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