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  • AmpliSeq on the MiSeq

    I'm trying to hijack the AmpliSeq protocol and get it onto the MiSeq instead of the PGM. Has anyone figured out a good way to do this? The best way I think will be to just substitute an Illumina Adapter (blunted ended) into the AmpliSeq ligation step and then use multiplexed PCR primers in the enrichment PCR step (the 2nd round of PCR). If I go about this way, I need to know the molarity of the Adapter being added during the AmpliSeq ligation and whats the molarity of the PCR primers in the PCR step. Has anyone figured this out or have a guess?

    In addition, has anyone tried using another polymerase to amplify the AmpliSeq amplicons? I was looking into Platinum® PCR SuperMix High Fidelity to use, as that is what LifeTech says to use for the Amplicon Fusion method (amplicons with the PGM sequencing adapter added to the PCR primers, so once done the amplicon PCR the resulting library is ready for sequencing without any other modifications). If I use this polymerase, I won't even need to do end repair or A-Tailing, because most of the product has an A-overhang and thus I can just ligate the Illumina adapter directly to the PCR products and then do PCR to enrich for those libraries.

    Any suggestions or advice will be very helpful.

  • #2
    I am about to undertake a similar endeavor and was wondering if you'd had any luck in the last couple weeks and were willing to share what has worked so far (or what hasn't).

    Comment


    • #3
      Me too, I was hoping to be able to do the Ampliseq reaction, treat with USER to remove the dUTP bases and then do the standard blunt end ligation prep putting on Illumina adapters. Ampliseq looks useful but we don't plan to buy a PGM to run it.

      Comment


      • #4
        Also curious if this has worked for anyone. Is it not possible to simply add Illumina adapters as tails onto the designed gene-specific sequences?

        Comment


        • #5
          It would be cheaper and easier to add a universal sequence and then do a short second PCR adding on the adapters (and indexing) using home made oligos or Illumina's Indexing kit. Same way TruSeq Custom Amplicon panels work.

          Full on bivalent primers quickly get expensive and inflexible.

          Comment


          • #6
            Here's my update so far...

            I got the molarities of AmpliSeq's oligos using the Qubit. The adapters are added at 2.5uM and the same with the primers. If I do one adapter (like a Y-shaped Illumina adapter), I should add 5uM because you add two adapters for AmpliSeq both at 2.5uM.

            I'm still working out logistics, but here are all the options (ranked easiest to hardest in my book) to achieve the desired results. I'm currently trying all the options, but option 2. Despite being difficult to pull off, we are aiming for Option 4 currently.

            1) Make custom dual-indexed primers for the 2nd PCR that prime off the AmpliSeq universal adapters (non-multiplexed) that contain the P5 and P7 sequences for Illumina clustering/seq. Similar to what epistatic said above. Then just order custom sequencing primers for the MiSeq. Will need to order 20 different (similar to TSCA, 12 index 1's and 8 index 2's) and 3 sequencing primers (read 1, index 1, read 2).
            Pros:
            -Easy to do, since only changing the primers at one step
            Cons:
            -Always need to do the 2nd PCR (which could add basis) to get a library
            -Need to custom sequencing on the MiSeq

            2) Add some Illumina sequence to the tails of the initial primers used in AmpliSeq. I can't do this because using the Cancer Panel V2 and my boss didn't do this to our custom panel. Thus just need to do the initial PCR, cleanup, and then use Illumina primers to do a second round of PCR to make a library.
            Pros:
            -Easy and less steps
            Cons:
            -Expensive, since have to buy the whole AmpliSeq kit just for the first PCR step (or could try the Platinum SuperMix HiFi that I think is just the DNA Pol they use).

            3) Ligate on an Illumina Universal Sequence adapters instead of the AmpliSeq adapters and then use TSCA primers in the 2nd round of PCR. The TSCA primers' molarity is at 2.5uM, so that's nice. This is also similar to what epistatic said above.
            Pros:
            -Don't need custom sequencing primers.
            -Don't need to make custom PCR primers (just purchase through Illumina).
            Cons:
            -Always need to do the 2nd PCR (which could add basis) to get a library
            -The TSCA oligo sequences aren't published (and we think we have them) so it makes it tricky.
            -Have to deal with ligation step

            4) Ligate on custom made TruSeq gDNA HT adapters and do 2nd round of PCR with universal PCR primers (like the P5 and P7 sequences that can amplify any Illumina library).
            Pros:
            -Have a library after ligation, so don't need to do 2nd round of PCR if have a QPCR machine to quantify.
            -Don't need custom sequencing primers.
            Cons:
            -Really hard to do.
            -Need to buy custom oligos for the adapters and anneal them myself (they have to be blunt ended Illumina adapters, so just add the A to the adapter that goes on the 3' end).

            5) Just try Qiagen's GeneRead DNAseq Gene Panels and then can go into any library prep kit.
            Pros:
            -Can use any library kit after the initial PCR.
            Cons:
            -Might get costly, because they are charging $100 a sample for just the PCR. Then have to add cost of the library kit in.
            -Not known for being in the NGS field.

            Comment


            • #7
              Hi foxaj,
              I have been following this thread. How's your trial?
              I read the product insert of AmpliSeq Kit. It seems that the primers were modified and a "FuPa reagent" is needed to digest the primers before constructing the library. So I am not sure whether you can use the PCR product from AmpliSeq and then construct the library using QIAGEN GeneRead Kit.
              On the other hand, the AmpliSeq Library Prep kit is very very expensive (>$100 per sample just the sample prep) so it may not be feasible to use AmpliSeq Library Kit and then QIAGEN/NEB library kit for adaptor ligation.

              Comment


              • #8
                Here are my best two options that worked, but you need to optimize for your needs...

                1) Use the AmpliSeq kit and make your own short BLUNT-ENDED Illumina Adapter and PCR Primers. It's a decent amount of work, because you have to vary your Adapter in the ligation (0.5uM-1.5uM is a good starting point) to avoid adapter junk in the final PCR (because Illumina's adapter all together is 120bp, where as LifeTech's ~90bp so their's is lost by the Ampure Bead cleanups whereas Illumina's is not). Don't forget the adapter needs the phosphothioate bonds (*) on the 3' ends and you need the Phosphate on the 5' end that ligates to the 3' end of the insert. Ensure the final molarity of the Multiplex PCR Primers are 200-300nM in the final PCR, and you need to do between 7-10 PCR cycles. For help with ordering the oligos ...http://www.med.upenn.edu/kaestnerlab...on_Apr2012.pdf

                2) Just take the AmpliSeq PCR product and go into the NEBNext or NEB Ultra library prep kit. Pretty much any Illumina library kit, just skip the end repair because the products are already blunt. This is the most expensive option. Again keep the adapter molarity low, like around 1uM.

                Good Luck

                Comment


                • #9
                  Thanks foxaj,

                  Very useful info. What method do you clean up the initial AmpliSeq products? AMPure beads? In what ratio?

                  Thanks

                  Comment


                  • #10
                    I used 1.5x Ampure Beads.

                    Comment


                    • #11
                      Ampliseq and AMPure

                      Hi foxaj,
                      I would like to use the NEBnext DNA library kit on ampliseq PCR product. I have one question on the selection of the adaptor ligated DNA using AMPure. Which are the conditions that you use considering the different sizes of the library (insert + adaptor) ? Thanks for your help
                      Emanuele

                      Comment


                      • #12
                        Originally posted by foxaj View Post
                        2) Just take the AmpliSeq PCR product and go into the NEBNext or NEB Ultra library prep kit. Pretty much any Illumina library kit, just skip the end repair because the products are already blunt. This is the most expensive option. Again keep the adapter molarity low, like around 1uM.

                        Good Luck
                        You say the Amplicon PCR product, is that before or after digestion with the infamous "FuPa"?

                        Comment


                        • #13
                          Hi foxaj,
                          I am about to undertake your same trial (sequencinq Ampliseq products on Illumina) and I was wondering if one of your option worked fine. Which is the best approach I should try?

                          Thanks

                          Comment


                          • #14
                            Hi guys,

                            instead of going through the trouble i would seriously consider to trial Illumina's new low input custom amplicon kit. That way you at least are using a standard optimised protocol with less risk. It starts from 16 sample size, 1-10ng for non FFPE and 10-50ng for FFPE samples.

                            Results should be at least equivalent if not better due to the higher accuracy of the chemistry.

                            Comment


                            • #15
                              Hi, has anyone already tried to sequence the AmpliSeq exome library on a NextSeq500? Do you think for this, you could also use the NEBNext kit after the AmpliSeq PCR?

                              Thanks!

                              Comment

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