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  • #31
    Originally posted by korostin View Post
    Hiro,

    Maybe I don't understand main Ampliseq feature correctly? Because for my mind, super-multiplex provided by step-out PCR based on universal identical 5'-tail on all primers. So, primer cleavage means 5'-tail cutting. Also, universal and specific parts of every primer separated by uracil. After PCR all products would be like:

    5' universal-tail-U-NNNNNNNNNNNN......................... 3'
    3' .......................NNNNNNNNNNNN-U-5'universal-tail 5'

    [dots mean nothing – only for visualisation]
    Because classic DNA polymerases can't read uracil and stop synthesis.
    If so, uracil DNA glycosylase and endonuclease IV treatment would produce blind ends. If PCR products have no 5' overhangs, uracil DNA glycosylase and endonuclease IV would create nicks only.

    Could you explain, please?
    In our protocol, we performed AmpliSeq multiplex PCR using the following materials:
    1) original AmpliSeq primers: containing target-specific sequences with uracil nucleobases but not containing universal sequences
    2) KAPA2G FAST Multiplex Kits: uracil-tolerant polymerase and regular dNTPs (A, C, G and T)
    3) genomic DNA

    The uracil DNA glycosylase and endonuclease IV treatment for the amplicons followed by the AMPure XP cleaning obtained blunt-ended uracil-less fragments being ready for Illumina library construction.

    I hope that answers your question.

    Hiro.

    Comment


    • #32
      Currently I'm looking at buying the components for a DIY Ampliseq protocol using ampliseq primers. I'm thinking the following should work:

      1. NEB OneTaq Hot Start Quick-Load 2X Master Mix with GC Buffer - for initial multiplex PCR with uracil-containing AmpliSeq primers. 10 ul master mix, 4 ul 5X primers, 6 ul gDNA + H2O. I'll modify the PCR program to run similar to the Ampliseq program with something like a 4-8 minute annealing/extension time per cycle.
      2. UDG + Endo IV - to be added directly to the PCR product (another thread suggested using USER + Exo I however it was never tested). 1 ul of each enzyme added directly to the PCR product. 37C for 1 hour.
      2.1. It would be nice to add 3'-dA tailing for convenient off-the-shelf Illumina adapter ligation in the next step rather than needing blunt-ended Illumina Y-adapters.
      3. Blunt/TA Ligase Master Mix + AmpliSeq P1 + X adapter mix (Ion Xpress barcodes or blunt-end Illumina adapters). This reaction has to be scaled up from NEB's recommended 20 ul reaction to a ~50ul reaction but I'll keep adapter concentrations the same as what Thermo recommends. I"m not sure if I'll need to do an ampure wash before this or if the PCR product with all the extra primers, UDG, and Endo IV won't interfere with the ligation. 25C for 15 minutes.
      4. Ampure wash following the AmpliSeq protocol but scaled up.
      5. Library amp of ligated fragments using NEB Q5 Hifi 2X master mix and custom P1/X primers (not sure what primer sequence to use for the X end. For Illumina libraries I would just use P5/P7 primers).
      6. Size selection and cleanup per AmpliSeq protocol by Ampure beads.

      The cost of these NEB enzymes as of now comes out to $7.67 per sample.

      The Ion library amp primers can be custom ordered from IDT or ordered off-the-shelf from IDT as the xGen library amp primer mix (P5/P7 primers) at $0.50 per reaction at a 500 pM final primer concentration or 250 pM per primer.

      Let me know if I'm missing anything. I'm trying to order everything from NEB for convenience but all of these components are generic for the most part.

      Comment


      • #33
        Currently I'm looking at buying the components for a DIY Ampliseq protocol using ampliseq primers. I'm thinking the following should work:

        1. NEB OneTaq Hot Start Quick-Load 2X Master Mix with GC Buffer - for initial multiplex PCR with uracil-containing AmpliSeq primers. 10 ul master mix, 4 ul 5X primers, 6 ul gDNA + H2O. I'll modify the PCR program to run similar to the Ampliseq program with something like a 4-8 minute annealing/extension time per cycle.
        2. UDG + Endo IV - to be added directly to the PCR product (another thread suggested using USER + Exo I however it was never tested). 1 ul of each enzyme added directly to the PCR product. 37C for 1 hour.
        2.1. It would be nice to add 3'-dA tailing for convenient off-the-shelf Illumina adapter ligation in the next step rather than needing blunt-ended Illumina Y-adapters.
        3. Blunt/TA Ligase Master Mix + AmpliSeq P1 + X adapter mix (Ion Xpress barcodes or blunt-end Illumina adapters). This reaction has to be scaled up from NEB's recommended 20 ul reaction to a ~50ul reaction but I'll keep adapter concentrations the same as what Thermo recommends. I"m not sure if I'll need to do an ampure wash before this or if the PCR product with all the extra primers, UDG, and Endo IV won't interfere with the ligation. 25C for 15 minutes.
        4. Ampure wash following the AmpliSeq protocol but scaled up.
        5. Library amp of ligated fragments using NEB Q5 Hifi 2X master mix and custom P1/X primers (not sure what primer sequence to use for the X end. For Illumina libraries I would just use P5/P7 primers).
        6. Size selection and cleanup per AmpliSeq protocol by Ampure beads.

        The cost of these NEB enzymes as of now comes out to $7.67 per sample.

        The Ion library amp primers can be custom ordered from IDT or ordered off-the-shelf from IDT as the xGen library amp primer mix (P5/P7 primers) at $0.50 per reaction at a 500 pM final primer concentration or 250 pM per primer.

        Let me know if I'm missing anything. I'm trying to order everything from NEB for convenience but all of these components are generic for the most part.

        Ivan

        Comment


        • #34
          Originally posted by korostin View Post
          We aren't looking for easy ways)) Price per 1 library more 100$ – it's too expensive
          Yeah I've been looking at alternatives too but it's hard to beat the sequence quality and workflow simplicity.

          I'll try out a DIY ampliseq approach using NEB enzymes with some modifications:
          1. OneTaq Hot Start Master Mix with GC buffer should be sufficient for the multiplex PCR reaction with uracil-containing ampliseq primers. I would scale the reaction down to 20 ul and run a 4/8/16 minute anneal extend. NEB recommends a 68C extension however such a long annealing should provide enough extension activity. Also annoying that Thermo calls their PCR enzyme "AmpliSeq Hifi" when it probably isn't even a high fidelity enzyme.
          2. There was a post suggesting using USER and Exonuclease I for the FuPa digest however it was never tested and I'm not sure what the difference is between USER and UDG. I would simply add 10 units of UDG and Endonuclease IV directly to each PCR reaction and incubate at 37C for 1 hour. Adding 3' dA tailing would be convenient for Illumina adapter ligation
          3. Blunt/TA Ligase master mix, scaled up to ~ 50 ul though it might be possible to find a 5x mix to keep the ratios as similar to Thermo's as possible. This would work with Ion Xpress adapters or off-the-shelf Illumina barcoded adapters if dA-tailing was performed.
          4. Ampure wash, same as Thermo's recommendation but scaled up.
          5. Library amp with Q5 Hot start HiFi 2x master mix and P1/X primers or P5/P7 primers.
          5. Final size selection and ampure wash as per Thermo's guidelines.

          The total cost of the NEB enzymes came out to around $8 per sample. I would order custom P1/X primers from IDT as well as off-the-shelf P5/P7 primers off IDT. Most expensive components would be the AmpliSeq primers followed by the sequencing adapters. The total cost per sample would still be far less than the $100/sample Thermo and Illumina are charging for without the AmpliSeq primers or barcoded sequencing adapters.

          Let me know if I'm missing anything.

          Ivan

          Comment


          • #35
            Originally posted by idedios View Post
            Yeah I've been looking at alternatives too but it's hard to beat the sequence quality and workflow simplicity.

            I'll try out a DIY ampliseq approach using NEB enzymes with some modifications:
            1. OneTaq Hot Start Master Mix with GC buffer should be sufficient for the multiplex PCR reaction with uracil-containing ampliseq primers. I would scale the reaction down to 20 ul and run a 4/8/16 minute anneal extend. NEB recommends a 68C extension however such a long annealing should provide enough extension activity. Also annoying that Thermo calls their PCR enzyme "AmpliSeq Hifi" when it probably isn't even a high fidelity enzyme.
            2. There was a post suggesting using USER and Exonuclease I for the FuPa digest however it was never tested and I'm not sure what the difference is between USER and UDG. I would simply add 10 units of UDG and Endonuclease IV directly to each PCR reaction and incubate at 37C for 1 hour. Adding 3' dA tailing would be convenient for Illumina adapter ligation
            3. Blunt/TA Ligase master mix, scaled up to ~ 50 ul though it might be possible to find a 5x mix to keep the ratios as similar to Thermo's as possible. This would work with Ion Xpress adapters or off-the-shelf Illumina barcoded adapters if dA-tailing was performed.
            4. Ampure wash, same as Thermo's recommendation but scaled up.
            5. Library amp with Q5 Hot start HiFi 2x master mix and P1/X primers or P5/P7 primers.
            5. Final size selection and ampure wash as per Thermo's guidelines.

            The total cost of the NEB enzymes came out to around $8 per sample. I would order custom P1/X primers from IDT as well as off-the-shelf P5/P7 primers off IDT. Most expensive components would be the AmpliSeq primers followed by the sequencing adapters. The total cost per sample would still be far less than the $100/sample Thermo and Illumina are charging for without the AmpliSeq primers or barcoded sequencing adapters.

            Let me know if I'm missing anything.

            Ivan
            1. are you sure, OneTaq Mix is tolerant to U?
            2. I'd add size-select step after 1st PCR to solve primer dimers problem (especially if starting with small amounts of DNA)

            Comment


            • #36
              Originally posted by korostin View Post
              1. are you sure, OneTaq Mix is tolerant to U?
              2. I'd add size-select step after 1st PCR to solve primer dimers problem (especially if starting with small amounts of DNA)
              I checked and OneTaq is compatible with uracil templates. They use it for their bisulfite protocol.

              Too bad OneTaq is not a HiFi enzyme. For any liquid biopsy assay where I would want to make calls around 0.1% I would definitely need a HiFi enzyme. Too bad I couldn't find one that is compatible with uracil templates. On the upside OneTaq claims to have 2x higher fidelity than regular Taq.

              Size selection after 1st PCR should not be necessary with the FuPa digestion which should digest all primer sequences including template incorporated ones and dimers.
              Last edited by idedios; 05-22-2018, 09:24 AM.

              Comment


              • #37
                Originally posted by HiroMishima View Post
                In our protocol, we performed AmpliSeq multiplex PCR using the following materials:
                1) original AmpliSeq primers: containing target-specific sequences with uracil nucleobases but not containing universal sequences
                2) KAPA2G FAST Multiplex Kits: uracil-tolerant polymerase and regular dNTPs (A, C, G and T)
                3) genomic DNA

                The uracil DNA glycosylase and endonuclease IV treatment for the amplicons followed by the AMPure XP cleaning obtained blunt-ended uracil-less fragments being ready for Illumina library construction.

                I hope that answers your question.

                Hiro.
                Hi Hiro,

                This seems like an interesting approach. Perhaps you could comment on a couple of things to help make me understand the logic behind your approach.

                Only the primers contain uracil and in the subsequent amplicons, the opposite strand would be a non-uracil nucleotide. What makes you think the products are blunt after your treatment with UDG & Endo IV? As opposed to being blunted/having the 3' overhangs being chewed away during the first step of the Kapa library prep protocol? I think this is important for those of us trying to put together a homemade protocol since your paper is the first published evidence of getting AmpliSeq on the MiSeq.

                Also, what made you decide to go with Endonuclease IV? One of the modifications I've considered for my own protocol is using Endonuclease VIII & UDG together to create 5'-P moieties that allow direct ligation of the amplicons, but I think I would likely need to treat with DNA Pol 1 or T4 DNA Polymerase to actually chew away the 3' overhangs which would be complementary to the AmpliSeq primer sequences and be uracil-less.

                Comment


                • #38
                  Thank you all the above guys, you provided many informations.

                  I'm also trying to make my own home-made ampliseq libray kit (for Ion torrent and Illumina)

                  Based on posts above, I think that the most import thing blongs to :
                  1) a HIFI DNA polumerase that could tolerrent U in primer( template), there are several candidate: OneTaq (not so HIFI),Phusion U Multiplex PCR Master Mix, KAPA2G FAST Multiplex Kits. I wonder which works best.
                  2) enzymes that digest primer region and make blunt-end (or single A-tailed) amplicons。 There are many options. UDG & Endo IV might be good choice, because UDG has 100% activity in Endo IV buffer, and Endo IV have both endonuclease and 3'-5' exonuclease activity. However, UDG & Endo IV treated end might be still not able to ligate adaptor. T4 PNK might be needed in this case.


                  Back to the erly beginning, Do we really need to digest primers? Especially in the conditon that so many tricky details underlines.

                  Comment


                  • #39
                    Originally posted by Buckethead84 View Post
                    Hi Hiro,

                    This seems like an interesting approach. Perhaps you could comment on a couple of things to help make me understand the logic behind your approach.

                    Only the primers contain uracil and in the subsequent amplicons, the opposite strand would be a non-uracil nucleotide. What makes you think the products are blunt after your treatment with UDG & Endo IV? As opposed to being blunted/having the 3' overhangs being chewed away during the first step of the Kapa library prep protocol? I think this is important for those of us trying to put together a homemade protocol since your paper is the first published evidence of getting AmpliSeq on the MiSeq.

                    Also, what made you decide to go with Endonuclease IV? One of the modifications I've considered for my own protocol is using Endonuclease VIII & UDG together to create 5'-P moieties that allow direct ligation of the amplicons, but I think I would likely need to treat with DNA Pol 1 or T4 DNA Polymerase to actually chew away the 3' overhangs which would be complementary to the AmpliSeq primer sequences and be uracil-less.


                    I think you are right, when you use Endonuclease VIII, you might need T4 pol(or DNA pol1).When you use Endonuclease IV, you might need T4 PNK.

                    Then the next question is: How to determine concentration of these enzymes?

                    Comment


                    • #40
                      I didn't get information that KAPA 2G could tolerate uracil.

                      But this one is claimed capable:

                      KAPA HiFi HotStart Uracil+ ReadyMix (1 x 1.25 mL)
                      Last edited by zany; 06-06-2018, 06:41 PM.

                      Comment

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