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Pooling of amplicon libraries for MiSeq

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  • Pooling of amplicon libraries for MiSeq

    Hi folks,

    I have limited experience in pooling libraries but now I am trying to pool (fungal ITS1) amplicon libraries for MiSeq V3 600 cycles. First, I sent some samples for fragment analyzer to check for their size distribution. Surprisingly some of them have double peaks at different sizes (please see attached figures; e.g. 289 bp and 431 bp in library no.1, and 387 bp and 455 bp in library no.2). I am using a pooling calculator from Illumina, which asks for library size. My question is that (1) which size I should bring to the calculation since I have two values? and (2) I think that peaks below 200 bp are potentially primer-dimer since no fungal ITS is that small and I shall remove them (which I can do it after pooling). Is this correct?

    Thank you very much for your help in advance and looking forward to have your advice and suggestions.

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