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  • Problem with sequencing short reads 454Jr

    We use 454 Jr system and we have to sequence short reads (about 100-150 bp), do we have to change quality filtering parameters?
    I read that something should be done in nedit, but i didnĀ“t quite understand, what exactly.

  • #2
    If you are sequencing such short reads then you are going to want to decrease the amount of primer you use in the emulsion PCR. When you are making up the Live Amp Mix use just 20uL of primer rather than the 80uL stated in the manual and make up the rest of the volume with H2O.
    This should reduce any over-amplification and will minimise cross-talk between wells on the chip.
    Personally, I would also reduce the number of thermal cycles to 35 (rather than 50) for the same reason.
    When sequencing short fragments a slight underload of the chip may be beneficial also. On the Junior I aim to load just below the 500 000 mark on the bead counter.
    Hope that helps

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