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  • #31
    I did several PCR reactions to test (had enough pre-pcr sample to spare). I tried 0.5, 1, 2, and 4 ul of my 10 nm working soln primer with both 15 and 18 cycles, and only using 4 ul got rid of the smears (which would be 8x more than I usually use for ChIP). Perhaps I had more sample than usual, or my primer soln was too dilute.

    Can't answer your question about the ramp step but sounds like something worth trying.

    Comment


    • #32
      Originally posted by jlove View Post
      Hi Phillip,
      I'm curious to know if you tried this slow ramp step and if it worked.

      -Jen
      Hi Jen,
      I never tried it. If I did, I think I would try the same one used in ETHANol's protocol for annealing Y-adapters:
      (from:
      Any non-primary sequence heritable modification of genetic material. ChIP-SEQ, DNA methylation (Bisulfite-SEQ), chromatin modifications (methylation, acetylation, etc), non coding RNA.

      )


      Anneal oligos an a thermal cycler with the following program:
      a. 2 min at 95 oC
      b. 140 steps of
      30 sec at 95 oC (decrease temp 0.5. C every step)
      c. Hold at 4. C

      If that does not work, I do not know what would.

      --
      Phillip

      Comment


      • #33
        Hi Phillip, I tried this yesterday but did not do such a slow ramp (I did 0.1C/ sec from 98-4C) and got a very strange result..... some of my "500 bp" library split into two humps, one right at 300 bp where it belongs and the other way, way higher. I guess this does suggest that there is some bubble and/or chimera foming. I decided to do another ramp where I simply put the sample on a heat block at 98 and took it off the heater and let it go down slowly overnight at RT. Maybe I'll try this setting as well. Can't hurt. I'm also doing some PCR using 10X the amount of PCR primer. I'll keep you all posted.

        Comment


        • #34
          Hi Jen,
          Similarly, when we denatured the sample and then allowed to reanneal at room temp we saw more of the sample end up in the slow peak (whatever that is), not less.

          I will be interested to see what your 10X PCR primer result will be.

          --
          Phillip

          Comment


          • #35
            We tried this suggestion last week (slow ramp speed on the last cycle). Our bubble product was also greatly increased, but our concentrations determined by KAPA qPCR were the same as our previous preps that used the TruSeq protocol as written.

            Next time I'd like to try increasing the amount of primer. Has anyone done this yet, and what were the results?

            Originally posted by pmiguel View Post
            How about for the last cycle doing a very slow ramp from 98 oC to 60 oC? That way the full length products will have lots of time to find each other.

            Comment


            • #36
              In our lab, we've either re-PCRd with 4x primer, or take the bubble'd product and perform an additional 2 cycles of PCR with extra primer. Both of these methods worked to remove the extra HMW product.

              Comment


              • #37
                You can also just ignore the larger peak and go with your qPCR titration.

                We usually go with fewer cycles of enrichment PCR now and rarely see the multimer peaks caused by ectopic annealing.

                --
                Phillip

                Comment


                • #38
                  Using ssDNA and Agilen RNA Chip to solve longer than expected library sizes

                  Find below an example of what we thought was a library overamplification problem. When the same libraries are denatured at 97 C for 2 minutes and run with RNA chip, the larger fragments disappear. So this supports the bubble and daisy-chain explanation. My conclusion is that if we use qPCR to quantify, these shouls be OK for sequencing and not form e.g. chimeric sequences. Also, reducing number of PCR cycles or adding primers might be a solution. We tried size based selection with AMPure, but it did not work out, which makes sense if this is caused by ssDNA annealing e.g. as heteroduplexes/bubble products.

                  Click image for larger version

Name:	ssDNABioanalyzer.jpg
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ID:	305172

                  Comment


                  • #39
                    Originally posted by PyhaTanja View Post
                    Find below an example of what we thought was a library overamplification problem. When the same libraries are denatured at 97 C for 2 minutes and run with RNA chip, the larger fragments disappear. So this supports the bubble and daisy-chain explanation. My conclusion is that if we use qPCR to quantify, these shouls be OK for sequencing and not form e.g. chimeric sequences. Also, reducing number of PCR cycles or adding primers might be a solution. We tried size based selection with AMPure, but it did not work out, which makes sense if this is caused by ssDNA annealing e.g. as heteroduplexes/bubble products.

                    [ATTACH]4555[/ATTACH]
                    Yes, exactly.

                    Although I've always wondered what a sample would look like after treating it with a single-strand-specific nuclease. That could confirm that there was single-stranded + double stranded regions in the amplicons.

                    But I've been too lazy to actually do the experiment...
                    --
                    Phillip

                    Comment


                    • #40
                      Hello folks,

                      Interesting thread! I got a lot of high molecular weight smear in my last run as well (following EMP protocols for soil samples).

                      I'm trying to figure out the details of the daisy-chain hypothesis:

                      From what I see the Illumina adapters on either end aren't complementary. So I guess the thought is that a few annealing bps are enough to lead to 3' extension. However, why would this lead to preferential extension to form multimers, thus starting at the adapter sequences? If it is just a few bps duplexing, surely this could happen anywhere within the amplicon.

                      Is my thinking off. Am I missing a point?

                      Comment


                      • #41
                        Originally posted by Cistron View Post
                        Hello folks,

                        Interesting thread! I got a lot of high molecular weight smear in my last run as well (following EMP protocols for soil samples).

                        I'm trying to figure out the details of the daisy-chain hypothesis:

                        From what I see the Illumina adapters on either end aren't complementary. So I guess the thought is that a few annealing bps are enough to lead to 3' extension. However, why would this lead to preferential extension to form multimers, thus starting at the adapter sequences? If it is just a few bps duplexing, surely this could happen anywhere within the amplicon.

                        Is my thinking off. Am I missing a point?
                        No, I think you are right. I just hadn't thought carefully enough about it previously. It is very likely that the bubble product hypothesis is correct.

                        Wait, I just re-read your post. The bubble product hypothesis explains the presence of an extra peak with higher apparently molecular weight. Molecules comprising the "high MW" peak are not actually higher MW, they are just largely "di-monoplex" rather than duplex. That is, the ends are duplex, the middle comprises two non-complementary strands.

                        This doesn't directly explain a "smear" extending up into much higher MWs. Unless one posits that the bubble products are chain-linking together into larger structures. Not "daisy-chaining", but creating loops that have actually interlocked. (I'm sure there is a term in topology for this, but I don't know what it would be.)

                        To test this I guess you could massively dilute your sample, and subject it to denaturing temperatures. Then cool it diluted and re-concentrate it. If true, one would expect to get just 1 or 2 peaks afterwards, not the smear.

                        That said, if you just want libraries to sequence, the answer is generally to ignore the issue, use qPCR to titrate and move ahead. Or just construct libraries with fewer cycles of amplification.

                        --
                        Phillip

                        Comment


                        • #42
                          Originally posted by pmiguel View Post
                          No, I think you are right. I just hadn't thought carefully enough about it previously. It is very likely that the bubble product hypothesis is correct.

                          Wait, I just re-read your post. The bubble product hypothesis explains the presence of an extra peak with higher apparently molecular weight. Molecules comprising the "high MW" peak are not actually higher MW, they are just largely "di-monoplex" rather than duplex. That is, the ends are duplex, the middle comprises two non-complementary strands.

                          This doesn't directly explain a "smear" extending up into much higher MWs. Unless one posits that the bubble products are chain-linking together into larger structures. Not "daisy-chaining", but creating loops that have actually interlocked. (I'm sure there is a term in topology for this, but I don't know what it would be.)

                          To test this I guess you could massively dilute your sample, and subject it to denaturing temperatures. Then cool it diluted and re-concentrate it. If true, one would expect to get just 1 or 2 peaks afterwards, not the smear.
                          Hi Philip,

                          Thanks for your answer!

                          Right, so "daisy-chains" aren't really extended much, but entangled products. That makes more sense.

                          The bubble products made sense when I first heard about it, however, the appearance of seemingly multiples of the original product on the gel led me off track.

                          That said, if you just want libraries to sequence, the answer is generally to ignore the issue, use qPCR to titrate and move ahead. Or just construct libraries with fewer cycles of amplification.

                          --
                          Phillip
                          The stuff I do for my students

                          Comment


                          • #43
                            Originally posted by Cistron View Post
                            Hi Philip,

                            Thanks for your answer!

                            Right, so "daisy-chains" aren't really extended much, but entangled products. That makes more sense.
                            If you like. I had envisioned daisy-chains as linear arrays of products, each linked by duplex ends.

                            But I was clearly wrong, as you point out.

                            --
                            Phillip

                            Comment


                            • #44
                              Originally posted by pmiguel View Post
                              If you like. I had envisioned daisy-chains as linear arrays of products, each linked by duplex ends.

                              But I was clearly wrong, as you point out.

                              --
                              Phillip
                              Wait, no!! I was Jedi mind-tricked. No reason my original vision of linear daisy chains wouldn't happen.

                              Each double stranded library amplicon after it is created by the polymerase from a single-stranded template molecule will comprise 2 strands.

                              Code:
                              [FONT="Courier New"]
                              5e-A--M-B'-3e
                                 |  | |
                              3e-A'-M-B--5e
                              [/FONT]
                              Where "5e" denotes "5 prime end", "A" is the entire top strand of the left (forward) adapter, "A'" is the reverse complement, or bottom strand of the left (forward) adapter. "M" should be considered the insert, either strand. "B" and "B'" are just the right (reverse) adapter strand.

                              During denaturation the two strand melt apart and during annealing could form, for example:

                              Code:
                              [FONT="Courier New"]
                              5e-A--M-B'-3e
                                      |
                                   5e-B-M-A'-3e
                                          |
                                       5e-A--M-B'-3e
                              [/FONT]
                              Except this is a linear diagram and you would need to imagine the 5' end fo B annealing to the 3' endo fo B'.

                              For any number of links of these type to form, the intermolecular kinetics must be faster than the intramolecular formation of a bubble product. Which would require very high DNA concentration and/or longer inserts.

                              --
                              Phillip

                              Comment


                              • #45
                                Ah, yes - do you mean like this?

                                Code:
                                     3'5'      3'5'
                                     | |       | |
                                5'__/   \_____/   \___3'
                                I guess this could still form a complex bubble.

                                Comment

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