If you have access to fluorometric DNA quantification and a Bioanalyzer (or equivalent), library pooling is not difficult. We offer the pooling of sequencing libraries for a small fee. For sequencing libraries generated by the Core, pooling is included in the library preparation service.
Prerequisites for the pooling of customer libraries are:
- all libraries were generated using the same protocol and are PCR amplified
- the library fragment sizes have to be similar for all libraries * (and within Illumina specs) as demonstrated by Bioanalyzer traces (or gel images if correct balancing is not that critical)
- have uniquely indexed adapters
- all libraries have DNA concentrations in the same range
- PCR-amplified libraries can be quantified based on fluorometric measurements (e.g. Qubit), but PCR-free libraries are best quantified by qPCR.
Library pooling requires precise pipetting of very small volumes. Even with the best libraries, there will be some imbalances. However, and we can’t work magic with variably sized samples.
* The clustering efficiency of Illumina sequencing libraries varies with the fragment lengths. Shorter molecules are more mobile and will always cluster preferentially compared to longer molecules (smaller molecules will “win the race” to the flowcell surface oligos).
Thus, accurate pooling is impossible when combining libraries of varying lengths and we can’t vouch for the results. In some cases, it is advisable to size-select the libraries stringently before quantification and pooling.
PCR-amplified libraries can be quantified by fluorometry (e.g. Qubit), but PCR-free libraries are best quantified by qPCR. For sequencing libraries generated by the Core, pooling is included in the library preparation service.
We suggest the following procedure when pooling libraries yourself:
- verify that Bioanalyzer traces of your libraries show the same fragment size distribution
- quantify each library by fluorometry (Qubit or 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 i.e., 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 to verify that it has the expected concentration (we will quantify once more by qPCR before sequencing)
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.
You can get trained and use a Qubit in our lab: http://dnatech.genomecenter.ucdavis.edu/qubit-fluorometer/