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  • miseq custom primers

    Hello,

    Recently I had a sequencing run on Miseq with custom Read1 and Read2 primers and I’m not very happy with quality of basecalls.
    Miseq generated ~700K cluster/mm^2 but Read1 >=Q30 was 88.2% and Read2 >=Q30 71.5%

    Could it be the case of not efficient annealing of primers?
    Tm of my custom primers:
    Read1 77.6°C;
    Read2 79.3°C.

    Native illumina’s primers have following Tm’s:
    Multiplexing Read 1 Sequencing Primer
    5' ACACTCTTTCCCTACACGACGCTCTTCCGATCT 77.5°C
    Multiplexing Read 2 Sequencing Primer
    5' GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC 79.2°C
    Multiplexing Index Read Sequencing Primer
    5' GATCGGAAGAGCACACGTCTGAACTCCAGTCAC 79.2°C


    My custom primers have almost the same temp. properties.

    Do you guys have any ideas or recommendations how custom primers should be designed?


    Thank you very much!

  • #2
    I wrote a blog on it here, when I was looking at doing this.

    If you do a search on this forum you should find several threads talking about just this.

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