When pooling libraries, it is critical to accurately quantify individual libraries to ensure equimolar pooling. Otherwise, you may see large variation in representation across samples, for example, sample 1 achieves 200× coverage while sample 2 achieves 50× coverage. Note that if individual products are over- or under-represented due to a multiplex PCR process, this cannot be overcome by any pooling strategy.
Other considerations when pooling libraries include the following:
• Choose libraries of identical size range for pooling.
• Choose libraries with indices that do not share sequence homology.
• Quantitate libraries using a functional assay, for example, qPCR.
• In the case that libraries of different size ranges must be pooled, employ a weighted strategy to achieve a more even representation.
Note
When planning a multiplex sequencing experiment, consider creating a single multiplex library pool and distributing the pool over the desired number of flow cell lanes to achieve the required read coverage per library. This strategy will mitigate variation in library coverage owing to differences in sequencing yield or quality across flow cell lanes.