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  • Equimolar pooling of hundreds of libraries for sequencing

    Recently Ilumina released additional sets of barcoded Nextera primers which enable multiplexing 384 samples for sequencing. High level of multiplexing for sequencing on NextSeq 500 system High output kit sometimes is essential as lanes can not be clustered separately. Equimolar pooling of that many libraries by current gold standard (QPCR+ average library sizing by Bioanalyzer or TapeStation) is a daunting task. A possible work around to avoid hundreds of QPCR is to use beads for normalisation when pooling libraries of similar size distribution in which the equal mass (volume) pooling is equimolar pooling followed by QPCR quantification of pool. Currently there are two products for normalisation. They work by binding (switching charge by pH) or precipitating DNA (PEG+salt) onto beads with low capacity for interaction with DNA. AMPure beads have high capacity and cannot be saturated by concentrations of most NGS libraries. It will be good if anyone with experience using this normalisation products or homebrew versions to share their experience with Seqanswers community.
    Last edited by nucacidhunter; 05-08-2014, 06:24 AM.

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