Equimolar vs fraction mode?

Equimolar gives every sample equal moles (equal expected reads). Fraction mode weights each sample by the read share you enter, for uneven multiplexing.

Do the sample volumes include diluent?

No. The volumes are the amounts of each library to combine; together they make up the target pool volume. Add buffer separately if you need a lower final concentration.

Library pooling calculator

Pool several libraries to equimolar (or read-share weighted) ratios. Enter each sample’s concentration and fragment length to get the volume of each to add for a target pool volume.

Pooling mode

Target pool volume (µL)

#	Conc (ng/µL)	Length (bp)	Read share	Remove

How it works

Formula

Convert each sample to molarity with nM = (ng/µL × 10⁶) ÷ (length × 650). Volume to add is proportional to weight ÷ molarity (weight = 1 for equimolar, or the read share in fraction mode), scaled so all volumes sum to the target pool volume.

Worked example

A 10 ng/µL and a 20 ng/µL library of the same 300 bp length: the 10 ng/µL sample is half the molarity, so it needs double the volume — 66.7 µL vs 33.3 µL in a 100 µL pool — to contribute equal moles.

When to use it

When combining multiple libraries on one run. Equimolar pooling aims for equal reads per sample; fraction mode lets you deliberately over- or under-represent samples by read share.

Sensible defaults

Start with the example samples, then edit concentrations and lengths, add or remove rows, and set your target pool volume. Use the mean fragment size including adapters from your fragment-analyzer trace.

FAQ

Equimolar vs fraction mode?: Equimolar gives every sample equal moles (equal expected reads). Fraction mode weights each sample by the read share you enter, for uneven multiplexing.
Do the sample volumes include diluent?: No. The volumes are the amounts of each library to combine; together they make up the target pool volume. Add buffer separately if you need a lower final concentration.