How do I pool sequencing libraries? Can you pool them for us?

If you have access to fluorometric DNA quantification and a Bioanalyzer library pooling is not difficult. We offer the pooling of sequencing libraries for a small fee.  Prerequisites for the pooling of customer libraries are:

  • all libraries were generated using the same protocol and are PCR amplified
  • their fragment size distribution is similar (and within the Illumina specs) as demonstrated by Bioanalyzer traces
  • uniquely indexed adapters
  • all libraries need to have DNA concentrations in about the same range

Library pooling requires precise pipetting of very small volumes and we can’t work magic with wildly variable samples.  PCR-free libraries are best quantified by qPCR.  Other libraries can be quantified by fluorometry (e.g. Qubit).  For sequencing libraries generated by the core labs, the pooling is included in the service.

We suggest the following procedure:

  • verify that Bioanalyzer traces of your libraries show the same fragment size distribution
  • quantify each library by fluorometry (Qubit or the plate reader)
  • if necessary dilute some of the highly concentrated libraries (to bring them in line with the others)
  • re-quantify the newly diluted libraries (Qubit)
  • pool the same amounts for each library:  e.g. the same number of femtomoles for each library (the femtomole amount is calculated by multiplying the concentration (in nM) by volume (in ul).
  • quantify the resulting pool by Qubit

Please note that the combined library concentration of the pool should be 5 nM or higher; this means that the concentration of individual libraries in the pool can and will be considerably lower.


Category: 04 Library Preparation and QC

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