Why pooling libraries at the same concentration does not change the final pool concentration

When pooling libraries for Illumina sequencing, the libraries are first normalized to the same concentration, then equal volumes are pooled to yield a pool of the same concentration of each of the original normalized libraries.

• When adding two solutions of the same concentration, the final concentration does not change.

  • For example, if adding 20 µl of a 4 nM solution to 20 µl of another 4 nM solution, the final solution is 4 nM.*

  • Note that the concentration of the entire solution is 4 nM, but the concentration of each library would be < 4 nM. For sequencing purposes to obtain the optimal cluster density or occupancy, it is the concentration of the entire solution that matters.

Definitions:

Molarity is the number of moles per liter of solution.

Nanomolarity is nanomoles (nmol) per liter.

*The following is an example calculation pooling the same volume of two normalized solutions from the above example. (See also the diagram below.)

In 20 µl of a 4 nM solution:

4 nM = X nmol/0.000020 liters X = 0.00008 nmol

The final number of moles will be the total sum of the nmol:

0.00008 nmol+ 0.00008 nmol= 0.00016 nmol

The total volume of solution:

0.000020 liters + 0.000020 liters = 0.0000400 liters

Nanomolarity is number of nanomoles per liter:

0.00016 nmol/0.0000400 liters = 4 nmol/liter = 4 nM

Adding different volumes

The same holds true if combining different volumes of the same concentration, eg, 60 µl of a 4 nM solution to 540 µl of a 4 nM solution.

In 540 µl of a 4 nM solution:

4 nM = X nmol/0.000540 liters X = 0.00216 nmol

In 60 µl of a 4 nM solution:

4 nM = X nmol/0.000060 liters X = 0.00024 nmol

The final number of moles will be the total sum of the nmol:

0.00216 nmol+ 0.00024 nmol= 0.00240 nmol

The total volume of solution:

0.000540 liters + 0.000060 liters = 0.000600 liters

Nanomolarity is number of nanomoles per liter:

0.00240 nmol/0.000600 liters = 4 nmol/liter = 4 nM

For any feedback or questions regarding this article (Illumina Knowledge Article #8258), contact Illumina Technical Support [e-mail].