Loading concentration: why molarity, not mass, drives clustering

Sequencers cluster individual molecules onto a flow cell, so the loading concentration they specify is a molar one — picomolar or nanomolar. Load by mass and you will over- or under-cluster whenever the fragment size shifts.

Clustering counts molecules

Cluster density depends on how many library molecules reach the flow cell, not how much they weigh. Two libraries at the same ng/µL but different fragment lengths present different numbers of molecules, so they cluster differently. That is why loading targets are quoted in molarity, and why quantification for loading has to end up in nM (or pM).

The dilution: C1·V1 = C2·V2

Once you know your library’s molarity, diluting it to the target loading concentration is the standard dilution identity. The volume of stock is:

V₁ = (target conc × final volume) ÷ stock conc, with diluent = final volume − V₁.

A 20 nM stock down to 4 nM in 50 µL: V₁ = (4 × 50) ÷ 20 =10 µL of stock topped up with 40 µL of diluent.

When your stock is in ng/µL

If the library was quantified by mass, convert to molarity first using the mean fragment length: nM = (ng/µL × 10⁶) ÷ (length × 650). For example 10 ng/µL of a 470 bp library is 32.7 nM; diluting that to 2 nM in 25 µL needs about 1.5 µL of stock and 23.5 µL of diluent. Use the fragment size including adapters from your Bioanalyzer or TapeStation trace — the insert size alone will bias the molarity.