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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.
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Last edited by nucacidhunter; 05-08-2014, 06:24 AM.
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Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...04-04-2024, 04:25 PM -
Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...03-22-2024, 06:39 AM