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  • Can I use default sequencing primers for Miseq?

    I use below PCR primers to prepare libraries for Miseq amplicon sequencing:

    i7-INDEX2-Spacer- gene_specific_Primer

    CAAGCAGAAGACGGCATACGAGAT TCGCCTTA GTCTCGTGGGCTCGG +gene_specific_Primer

    i5-INDEX2-Spacer+gene_specific_Primer
    AATGATACGGCGACCACCGAGATCTACAC-CTCTCTAT-TCGTCGGCAGCGTC+gene_specific_Primer

    Compared to Nextera adapters, my primers do not include the tagmentation sequence. I tried to look up Miseq sequencing primers, but still not sure whether my adapters are compatible with Miseq sequencing primers.

    Can I use default sequencing primers in Miseq cartridge for sequencing? Does anybody know any website or document to verify that?

  • #2
    My guess is that you cannot use the "default" sequencing primers with your scheme.

    You appear to have directly attached your gene-specific primer sequence directly to the i5 and i7 illumina indexing primers and therefore lack the sequencing primer binding site, which is actually contained within the Nextera transposase sequence.

    Pages 3 and 4 of the following Illumina document might help you:

    Comment


    • #3
      Originally posted by ATϟGC View Post
      My guess is that you cannot use the "default" sequencing primers with your scheme.

      You appear to have directly attached your gene-specific primer sequence directly to the i5 and i7 illumina indexing primers and therefore lack the sequencing primer binding site, which is actually contained within the Nextera transposase sequence.

      Pages 3 and 4 of the following Illumina document might help you:

      https://support.illumina.com/content...0002694-11.pdf
      Thanks a lot for your reply.
      Are there any document/post(s) mention that "the sequencing primer binding site is actually contained within the Nextera transposase sequence"?

      Comment


      • #4
        Originally posted by ATϟGC View Post
        My guess is that you cannot use the "default" sequencing primers with your scheme.

        You appear to have directly attached your gene-specific primer sequence directly to the i5 and i7 illumina indexing primers and therefore lack the sequencing primer binding site, which is actually contained within the Nextera transposase sequence.

        Pages 3 and 4 of the following Illumina document might help you:

        https://support.illumina.com/content...0002694-11.pdf
        You are right!

        Comment

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