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  • cement_head
    replied
    Basically one would design PCR primers such that the 3' portion is complementary to the target, and the 5' half contains the barcode and the Illumina adaptor sequences for the instrument you are using. You can get the Illumina sequences from the sequence letter. Just use Google to find online resources to help design the primers.

    Try this first: https://www.google.com/url?sa=t&rct=...NSQ0L-KGPUg4QQ

    Leave a comment:


  • Anne zhao
    replied
    Where I can find the sequence of Ampliseq primers, so I can design the second round pcr fusion primers. Does the modification in the Ampliseq primers affect this second pcr? Thanks a lot!

    Leave a comment:


  • cement_head
    replied
    Originally posted by Zaag View Post
    I will be trying this in a week or two, so, did you make any progress? ;-)

    From what I understand it should be possible to remove the primers from ampliseq and subsequently ligate adapters, so I was planning on ligating MiSeq adapters instead of PGM adapters.
    I wouldn't do ligation, just do another round of PCR with primers for Illumina flow cells (get them from IDT).

    Leave a comment:


  • cement_head
    replied
    Originally posted by [email protected] View Post
    Hi I have made an Ampliseq panel for use on the PGM, it has 280 targets of sizes between 250-150bp.
    We would like to also sequence these same targets on the MiSeq (we are testing to see how the two platforms compare on our types of targets and may possibly buy the one we like best).
    Has anyone before used the Ampliseq primer panel to prepare a library for the MiSeq?
    Or have any suggestions about which library prep method would be best?

    We have looked at the truseq custom amplicon designer but we would prefer to use the ampliseq primer panel we already have. This is due to the cost of purchasing the full truseq amplicon kits (they only coming in 96sample minimums when we really only want to try on around 10 samples to begin with).
    I think bunce is correct. I would probably buy 10 primers that JUST had your target sequence, perform your PCR. Then take the amplicons (seperately) and do five cycles of PCR using barcoded Illumina adaptor primers and then continue on with the MiSeq sequencing. Essentially doing this (Berry et al. (2011)) but with Illumina primers.

    Leave a comment:


  • Zaag
    replied
    I will be trying this in a week or two, so, did you make any progress? ;-)

    From what I understand it should be possible to remove the primers from ampliseq and subsequently ligate adapters, so I was planning on ligating MiSeq adapters instead of PGM adapters.

    Leave a comment:


  • bunce
    replied
    Hi Ryan,
    A very real option for you to -'readapt' the A/P1 Ampliseq products (via PCR) with the illumina p5/p7. You would typically do 12-20 cycles of PCR with the fusion Primers. This would be a cheap and easy way for doing the reaction. It is not, technically, a valid comparison as the MiSeq library would have gone through subsequent PCR (=more error) but.....If you are sequencing microsats you will find the MiSeq data much (MUCH) cleaner. The story is much the same for SNPs. The homopolymer error in the PGM is difficult to cope with when scoring alleles. Happy to discuss this with you 'offline' - drop me a line (michael.bunce 'at' curtin.edu.au). Cheers.

    Leave a comment:


  • ryan.england@esr.cri.nz
    started a topic Ampliseq panel on MiSeq

    Ampliseq panel on MiSeq

    Hi I have made an Ampliseq panel for use on the PGM, it has 280 targets of sizes between 250-150bp.
    We would like to also sequence these same targets on the MiSeq (we are testing to see how the two platforms compare on our types of targets and may possibly buy the one we like best).
    Has anyone before used the Ampliseq primer panel to prepare a library for the MiSeq?
    Or have any suggestions about which library prep method would be best?

    We have looked at the truseq custom amplicon designer but we would prefer to use the ampliseq primer panel we already have. This is due to the cost of purchasing the full truseq amplicon kits (they only coming in 96sample minimums when we really only want to try on around 10 samples to begin with).

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