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GS Junior pair-end sequencing trouble



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  • GS Junior pair-end sequencing trouble

    We just did yesterday a run with paired-end libraries on the GS Junior and the results are very strange.
    We had no problem to prepare the libraries. Good concentration and good profile on Agilent Bioanalyzer.

    We did the emulsion as usual and get 5% of enrich beads.
    Usually we 8-9% but since we had 5% we decided to go on.

    Today we get the results and it was very bad.

    We obtain a normal range of raw wells and sample key pass but we had a lot of mix and dots and very few passed filter wells.

    I would to understand if my libraries are good or not.

    Can you tell me were is the sample key? Before or after adaptors?
    With such results I wonder if adaptors ligation went well.

    I don't understand why I have a lot of mixed and dot beads but a good beads enrichment...

    If someone can help me to understand!

    I hope I post in the appropriate category!

    Thank you in advance!
    Last edited by LuLuNe; 10-31-2013, 12:45 AM.

  • #2
    I don't see there is problem with your library. key is located just after sequencing primer. The template can't be amplified onto the beads without adaptor.

    High dot and mix usually indict the emPCR has problem. You just need repeat emPCR with higher DNA input. I think 5% enrichment is too low, aim to 15%, but below 20% is good beads. and make sure you wash the beads enough before final melting.


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