Equimolar library pooling, explained

When you multiplex several libraries on one run, each one competes for reads. To share reads evenly you pool them to equal molarity — equal numbers of molecules — not equal mass or equal volume.

Why molarity, not mass

A sequencer counts molecules: every fragment that clusters gets sequenced, regardless of how heavy it is. Two libraries with the same mass concentration (ng/µL) but different fragment sizes contain different numbers of molecules — the one with shorter fragments has more of them. Pool by mass and the shorter-fragment library hogs the reads. Pooling by molarity fixes that.

Mass becomes molarity via length

The bridge is the ng/µL → nM conversion, which needs each library’s mean fragment length: nM = (ng/µL × 10⁶) ÷ (length × 650). Once every sample is in nM, the volume to add is proportional to 1 ÷ molarity — the lower the molarity, the more you add — scaled so the volumes sum to your target pool size.

A worked example

Two libraries of the same 300 bp length, one at 10 ng/µL and one at 20 ng/µL, pooled into 100 µL. Converting: the 10 ng/µL sample is 51.3 nM and the 20 ng/µL sample is 102.6 nM — the second has double the molarity. To contribute equal moles the first needs double the volume:66.7 µL of the dilute library and 33.3 µL of the concentrated one, giving a ≈68 nM pool.

Deliberately uneven pools

Sometimes you want unequal reads — say a deep sample alongside several shallow ones. Then you weight each sample by its target read share instead of equal moles, but the principle is the same: work in molarity, then scale the volumes to your desired proportions.