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  • #16
    All I can say is (some) DNA oligos must run not true to the markers on the RNA chips. It just goes against everything I know about PCR. Sometimes you have to believe the data, but sometimes the data is misleading. Someone needs to mass spec them. We have a mass spec but not the PPC oligos. Weird stuff!!!!!

    And if the oligos are so long, why does Illumina say to use a 60˚ C anneal temp for the PCR? Is there something that I am missing completely?
    --------------
    Ethan

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    • #17
      To be honest, I am mystified as well.

      Tm for the Universal Adapter is 75 oC. About the same for the Index adapter oligo. 60 oC would be a full 15 oC below the Tm.

      --
      Phillip

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      • #18
        Has there been any problems with just using the p5 and p7 primers? It seems it would be easier and make more sense in terms of Tm compared to these longer primers.

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        • #19
          It seemed to work with the primers I designed which are a couple nucleotides longer then the p5 and p7 primers as far as generating a nice smear on the gel. But I'll have to wait until my samples are sequenced to be sure. I'm not sure how long the queue is. I've searched extensively and haven't seen anything.
          --------------
          Ethan

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          • #20
            Probably would work better with p5 and p7, or something similar, like the primers ETHANol designed. Still one can't help being curious as to what the Illumina guys are doing...

            --
            Phillip

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            • #21
              Just to follow up and say the protocol worked great. I've posted the details on my new blog at:
              I got the data back from the ChIP-seq samples I sent out using my TruSeq library preparation protocol. I can definitely say the library preparation protocol works great, clearly better than what Il…
              --------------
              Ethan

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              • #22
                Great protocol! Question about adapter concentration. Is 0.25 uM stock the concentration of the combined adapters or each one? Is it basically the Illumina tube of adapter at 25 uM diluted 1:100?

                Thanks!

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                • #23
                  I was thinking about this again. How about if the PPC oligos have ~60 nt 5' "heads" on them. For example 60 A's, then the actual priming site oligo. The result, after a couple of cycles, would be each product amplicon as a single stranded molecule would have a poly A tail on one end and a poly T tail on the other. (Those are just examples, could be other sequences.)

                  What for? This would allow a stem structure to form. The stem would be less available for primer annealing, once it formed. Because the formation of this stem would be unimolecular, it might be faster than the bimolecular primer annealing step. The stability of the stem loop structure would, in part, be determined by how large the loop (the insert) was. This is the basis of the the "PCR suppression effect" or sometimes called "pan-handle PCR". It can be used to compensate for PCR's short product bias.

                  --
                  Phillip

                  Originally posted by pmiguel View Post
                  Here is some evidence that the TruSeq PCR primers are just the entire TruSeq adapter oligos:
                  We ran an Agilent RNA pico chip on 0.2 ul from the "PPC" (PCR primer cocktail) from:

                  A DNA TruSeq A kit:


                  An RNA TruSeq Bkit:


                  Here is the ladder electropherogram for that chip:


                  The sizes are a little longer than expected (58 and 63 nt for the A strand and B strand oligos, respectively). But I guess that is down to mediocre accuracy for the pico chip in that size range.
                  --
                  Phillip

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                  • #24
                    Originally posted by jlove View Post
                    Great protocol! Question about adapter concentration. Is 0.25 uM stock the concentration of the combined adapters or each one? Is it basically the Illumina tube of adapter at 25 uM diluted 1:100?

                    Thanks!
                    0.25 μM means 0.25 μM for each adapter primer. You might consider diluting them a little more say to 0.125 μM. I'm not sure what the concentration of the adapters form Illumina are.

                    Phillip, I think you may be on to something. But since the goal is not to make long PCR fragments but short ones in the neighborhood of 300-500 bp, it doesn't sound exactly right. But the overhang sounds like a reasonable explanation. Maybe the p5 and p7 binding sites are repeated and that enhances attachment to the flow cell and cluster generation. Or maybe the extended length increases yield with the Agencort beads. I don't know but I am curious. There has to be someone that can mass spec them. Such a simple thing to do.
                    --------------
                    Ethan

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                    • #25
                      In a test I have to sequencing the DNA fragments as short as possible (above 40 bases).

                      There is a question:
                      What is the bottom line of size that we can sequence with TreSeq system?
                      Particularly, If I use 75bp X 2 sequencing, can I do sequencing as short as 40 bases (inset size)? or I have to sequence >75 bp insert?

                      Your opinion will be very well appreciated.

                      Victor

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                      • #26
                        Originally posted by ETHANol View Post

                        [...]

                        Phillip, I think you may be on to something. But since the goal is not to make long PCR fragments but short ones in the neighborhood of 300-500 bp, it doesn't sound exactly right. But the overhang sounds like a reasonable explanation. Maybe the p5 and p7 binding sites are repeated and that enhances attachment to the flow cell and cluster generation. Or maybe the extended length increases yield with the Agencort beads. I don't know but I am curious. There has to be someone that can mass spec them. Such a simple thing to do.
                        I don't have a mass spec handy. (By which I mean I would have to submit it to another core on campus.) I heard that by doing a limited Snake Venom Diesterase digestion + mass spec you can determine oligo sequences.

                        Seems like cloning some PCR "enriched" TruSeq amplicons and Sanger sequencing would be one way to go. But that would be getting a little "meta" -- kind of an Easter Egg hunt, courtesy of Illumina, Inc. Maybe someone has already done this, to QC a library? If so, maybe they could slake our curiosity?

                        --
                        Phillip

                        Comment


                        • #27
                          duplicate post, sorry
                          Last edited by jlove; 08-22-2011, 06:14 AM. Reason: duplicate post

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                          • #28
                            [QUOTE=jlove;49657]
                            Originally posted by ETHANol View Post
                            0.25 μM means 0.25 μM for each adapter primer. You might consider diluting them a little more say to 0.125 μM. I'm not sure what the concentration of the adapters form Illumina are..

                            Regarding adapter - I made my own TruSeq adapters and I wanted to get them the same concentration as Illumina's...so I ran them out at a few concentrations on the smRNA chip (boiled first) alongside the Illumina ones. For what it's worth, my 8-15 uM (each) dilutions looked most similar to the Illumina ones. I had also made 25 uM, 20 uM and the peaks were much higher

                            Comment


                            • #29
                              Hi ETHANol, pmiguel, I'm glad you're both on this thread because I have questions for both.

                              I tried your chip-seq protocol (thanks for making this available by the way) with 10 ng of fragmented genomic DNA (not chip) and was able to get libraries at 220 bp however I see a higher molecular weight band between 400 and 500 bp on my agarose gel. These are the only two bands that I see on the gel. After my gel size selection I only expected to see the 220 bp band. The higher MW band is twice as intense as my expected band. Any suggestions?
                              pmiguel, I've read some of your posts about these mysterious higher MW bands and am wondering if this is the same thing.

                              Are there any magic bullets to eliminate these? Has anyone tried sequencing their sample, leaving it in there? Unfortunately because they are so close you can't really separate one from the other unless you do a gel cut. I'm just worried about that because I considered myself fortunate to even see the low input on a gel.

                              Comment


                              • #30
                                avanT, just to be sure I am understanding you correctly you see the 450 bp band after amplification. If so this is from over amplification of your library.

                                See these threads:
                                Bridged amplification & clustering followed by sequencing by synthesis. (Genome Analyzer / HiSeq / MiSeq)

                                Techniques and protocol discussions on sample preparation, library generation, methods and ideas

                                and probably more.

                                Bottom line is it doesn't matter and that big band is actually 220 bp DNA fragments annealed at the adapters but with non-homologous inserts which form after PCR primers becoming limiting. Your library is fine. Send it for sequencing as is. You could have gotten away with less PCR cycles, but it is not big deal. The DNA will be denatured for cluster generation so it's all ok.

                                If you really want to get rid of the 450 bp band do one cycle of PCR with your library (98˚ 1 min - 72˚ C 5 min with enzyme, dNTPS and PCR Primer Oligos!!) and the big band will magically disappear. But you do not need to do this and it is preferable not to because you just will get one more cycle of PCR bias.
                                Last edited by ETHANol; 10-13-2011, 08:34 AM.
                                --------------
                                Ethan

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