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  • question about paired-end libraries

    I haven't yet done any paired-end libraries, but I'm thinking of adding to a project that's currently in the planning stage. I understand the concept behind them and how they work, but some of the technical details of the protocols don't yet make sense. As I've looked through the protocols, some parts of them seem needlessly complicated. Why can one not just follow the standard Rapid Library procedure from the point of nebulization and add on the amplification at the end? The fill-in reaction, immobilization, single-stranded library isolation seem pointless to me. Can anyone explain why the Rapid protocol followed by amplification wouldn't work as well or better?

  • #2
    After nebulization the standard paired end library protocol calls for end polishing and immobilization. The later one is for separating only pared fragments. I have another question: have anybody tried to use MIDs adaptors for pared end library construction?
    Thanks

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    • #3
      The PE protocol pre-dates the rapid library protocol. So it is a bit antiquated. If you don't mind most of your reads being non-paired end, then you can just take all the DNA after circularization/nuclease treatment, fragment it, and construct a rapid library from that. You will want to adenylate after blunting, of course (that is part of the rapid library protocol).

      Alternatively, you could do the immobilization step, blunt+a-tail on the bead. Then ligate rapid adapters on. Then PCR. This is the way we used to construct Illumina mate end libraries using TruSeq adapters. So it should work the same for rapid adapters.

      vlee2: Yes we used MID adapters during the construction of a paired end library. Worked okay.

      --
      Phillip

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      • #4
        Interesting: in which step do you add MIDs? How does the software processes it? RL and PE libraries have different keys.

        Comment


        • #5
          Same as normal. The MIDs, whether GSMIDs or RLMIDs are part of the adapters.

          Yes, you are right about the keys. RL and the older "standard" libraries must not be mixed in the same region because their keys are not compatible. Other than that, I don't think it makes any difference which you choose -- other than end polishing, of course. (Blunt for standard, A-tailed for RL).

          The MID PE libraries we made were of the old type (standard). I don't think gsAssembler would choke on the combination of RLMIDs and PE libs, but I have not tried it myself.

          --
          Phillip

          Comment


          • #6
            Do they still sell GSMIDs? We never used those.

            Comment


            • #7
              Originally posted by pmiguel View Post
              f you don't mind most of your reads being non-paired end, then you can just take all the DNA after circularization/nuclease treatment, fragment it, and construct a rapid library from that.
              Will the software work with mixed data like this would produce, i.e. with mixed PE and non PE reads? Or will it just get confused?

              Comment


              • #8
                Originally posted by MissDNA View Post
                Do they still sell GSMIDs? We never used those.
                Not sure. We are still working off the first batch we ever bought. You could always order them from an oligo company, if needed.

                --
                Phillip

                Comment


                • #9
                  Originally posted by ajthomas View Post
                  Will the software work with mixed data like this would produce, i.e. with mixed PE and non PE reads? Or will it just get confused?
                  Mixed PE and non PE reads always happens, even if you do the immobilization step. So it is not an issue per se. The software handles this gracefully -- treating the read as a normal shotgun (non-PE) read unless it finds the center adapter sequence.

                  The issue would be if you do not get a high enough PE:non-PE ratio, your assembly might not be as good.

                  --
                  Phillip

                  Comment


                  • #10
                    Originally posted by pmiguel View Post
                    Not sure. We are still working off the first batch we ever bought. You could always order them from an oligo company, if needed.

                    --
                    Phillip
                    Is not expired yet? If so, how often do you guys use expired kits in your preps? We already have used most of the kits expired with no problems.

                    I don´t know the sequences but I am sure in case of need would be easy to find out. It is good to know that works though. Did you use it for 3, 8 or 20kb PE? Also, this GSMID adaptor substitutes the library adaptor, correct?

                    Comment


                    • #11
                      Originally posted by MissDNA View Post
                      Is not expired yet? If so, how often do you guys use expired kits in your preps? We already have used most of the kits expired with no problems.

                      I don´t know the sequences but I am sure in case of need would be easy to find out. It is good to know that works though. Did you use it for 3, 8 or 20kb PE? Also, this GSMID adaptor substitutes the library adaptor, correct?
                      Yes, most library construction kits I would use past their expiration dates because if some component is failing, I can pick that up via QC.

                      That said, I am not recommending it as "good practice". But the further away from the $0.5 million instrument the process is, the farther from Roche dogma I am willing to stray. So I pay $200 for the Roche wash kits and instrument runs are done "by the book". But a library is a library.

                      So, I don't think there is much difference between the 3 and 8 kb protocols. We would usually shear to something in the 3-8kb range, then go with the 3 kb protocol. There was one time where MIDs looked useful, so we used those. I would have to actually look at the protocol to answer your question...

                      Okay, yes use the GSMID adapters instead of the library adapters. Actually, I think we did order the GSMID adapter oligos ourselves and those would be the ones we used. Not sure if using a biotinylated "B" adapter would work as well--since that might interact with the streptavidin beads. But since you are going to do PCR anyway, you might be alright either way.

                      --
                      Phillip

                      Comment


                      • #12
                        Originally posted by pmiguel View Post
                        Yes, most library construction kits I would use past their expiration dates because if some component is failing, I can pick that up via QC.
                        That's usually true, but not always. We had an issue a few months ago where we couldn't make a functional library for several months. The libraries were of a good concentration and looked beautiful on the BioAnalyzer, but they always failed completely in emPCR. We had the same issue with a few different library prep kits, all from the same lot. It wasn't until we switched out the enzymes in the kit with enzymes from another manufacturer that it worked. We then got replacement library prep kits from Roche and everything was fine again. I don't know for sure, but I think the problem was probably the PNK. If that is bad, then the fragments won't be phosphorylated, and the adapters will be ligated on, but only on one strand. The result will be fragments that will quantify just fine because the fluorescent tag is attached, but once the duplex is melted there will be only one adapter on each molecule and the emPCR will fail.

                        Comment


                        • #13
                          We have used expired XLR70 kits, and it worked. Of course is not the ideal thing to do but is hard to throw away reagents that cost a lot. We always use the wash kits, however I have to say we´ve used those expired too! Sometimes Roche deliveries reagents to us that are almost expiring.

                          I´ve constructed plenty of 3kb libraries but never an 8 kb. The protocols are different, being 8kb way more trick although not as much as 20kb.

                          Another question: you talked about blunt and A-tailed adapter. I didn´t actually know there was a difference. Is that difference due to the fact one library is single stranded and the other double?

                          Comment


                          • #14
                            Originally posted by ajthomas View Post
                            That's usually true, but not always. We had an issue a few months ago where we couldn't make a functional library for several months. The libraries were of a good concentration and looked beautiful on the BioAnalyzer, but they always failed completely in emPCR. We had the same issue with a few different library prep kits, all from the same lot. It wasn't until we switched out the enzymes in the kit with enzymes from another manufacturer that it worked. We then got replacement library prep kits from Roche and everything was fine again. I don't know for sure, but I think the problem was probably the PNK. If that is bad, then the fragments won't be phosphorylated, and the adapters will be ligated on, but only on one strand. The result will be fragments that will quantify just fine because the fluorescent tag is attached, but once the duplex is melted there will be only one adapter on each molecule and the emPCR will fail.
                            Were your kit expired or was a bad batch? Because we once had a problem with an emPCR reagent lot, which was not expired, but didn´t work properly. Once we switched lots everything went back to normal. Roche replaced kits, however they never admitted the lot was bad.

                            Comment


                            • #15
                              No, the kits were not expired. They were getting close, but we still had a few months before the expiration date. We thought for a while that the problem was the emPCR kits because all of the failed emPCRs were also using kits from the same lot. We finally decided it was the library prep kits for sure after three pieces of evidence: we sent some DNA to another facility and had them make a library for us that we could sequence fine, I sequenced some cDNA libraries I had made using a different method, and we could sequence Lib-L amplicon libraries just fine. To be sure, we swapped the enzymes from the library prep kits with enzymes that we bought separately and those libraries were just fine, although the titer was lower than expected. Roche said anyone else had a problem with the batch, so I still don't understand why we had a problem with several kits from that lot (ordered at different times, but same lot).

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