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.
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)
- Under the precondition, that all libraries show very similar fragment size distributions there are in principle two options:
#1 Dilute all libraries to the same concentration, re-quantify the libraries, then pool the same volumes. This option is more laborious than #2, but allows more precise re-pooling if needed (since all libraries have similar concentrations at least).
#2 Pool the same a amounts of each library based on the first round of quantifications. This means pooling varying volumes. As long as the libraries are consistent in length this could mean pooling the same amounts in ng. If the length is a bit variable one should pool the same number of femtomoles for each library; the femtomole amount is calculated by multiplying the concentration (in nM) by volume (in ul). The molarity calculation will consider the fragment lengths and compensate for varying length. But see tip 3) below !!! - 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 (Illumina sequencers) or 16 nM or higher (AVITI); 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/
Three more tips for library pooling:
1) Best pipette volume ranges that allow the pipetting to be reproducible and accurate while still saving some library. For example aiming for a volume range from 3 ul to 10 ul.
Likely you will pool much more library than is needed in the end for sequencing.
2) In case you chose option #2 above and your library concentrations vary more than +- 60%, it is often helpful to work with two sub-pools: One for the higher concentration samples and one pool for the lower concentration samples.
The example numbers here are arbitrary. Under the precondition, that all libraries show very similar fragment size distribution, one would for example pipette 100 ng of each high-concentration library into one tube (Pool A) and 20 ng of each low-concentration library into another tube (Pool B). Vortex mix and spin down each of the tubes twice.
For the final combined pool one could then use the complete volume of Pool B and add one fifth of the total volume of Pool A. Vortex and spin down the combined pool twice. The final combined pool thus should contain 20 ng for each of the libraries aiming for balanced sequencing data. (Please note that the library amounts and pooling ratios mentioned here are only examples and that you have to choose appropriate ratios based on the library concentrations of your experiment).
3) If the fragment length distribution of the libraries is variable, one should try to adjust for these length differences (convert the library concentrations into molarities for this purpose). Further consider that shorter library molecules will be faster and cluster preferentially. To generate similar read numbers, one has to pool more library molecules for longer library molecules. Obviously more guessing and a bit of gambling will be involved in this case and pooling will be less accurate.
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