How to pool NGS libraries at equal molarity

Updated October 30, 2026

When several sequencing libraries share a run, each one should get a fair share of the reads. Sequencers count molecules, not nanograms, so libraries are pooled by molarity. This guide walks through the calculation with a real example.

Why pool by molarity, not mass

A nanogram of short fragments contains more molecules than a nanogram of long fragments. If you pool equal masses of a 300 bp library and a 600 bp library, the shorter one contributes about twice as many molecules and takes about twice as many reads. Converting everything to nM first removes that bias.

Step 1: Measure concentration

Use a fluorometric dsDNA assay such as Qubit. Absorbance (NanoDrop) also counts free nucleotides, primers and single-stranded DNA, so it usually overestimates library concentration.

Step 2: Measure the average fragment size

Run each library on a Bioanalyzer or TapeStation and read the average size of the library peak, including adapters. Exclude primer-dimer or adapter-dimer peaks from the region.

Step 3: Convert ng/µL to nM

Use an average mass of 660 g/mol per base pair:

nM = ng/µL × 10⁶ ÷ (660 × average size in bp)

Example: a library at 12.4 ng/µL with an average size of 420 bp is 12.4 × 10⁶ ÷ (660 × 420) = 44.7 nM.

Step 4: Work out each library's volume

Decide the final pool concentration and volume. Each of n libraries contributes an equal share of the pool's molarity, so each library's volume is:

V(library) = (pool nM ÷ n) × pool volume ÷ library nM

For a 4 nM, 50 µL pool of four libraries, each library should end up at 1 nM:

Practical tips

• Avoid sub-microlitre volumes. Pipetting under about 0.5 µL is inaccurate. Pre-dilute concentrated libraries (for example 1:10) or make a larger pool.
• Want unequal read depth? Give a library a larger share of the pool molarity. A sample that needs twice the reads gets twice the share.
• Confirm the pool by qPCR. Only molecules with both adapters form clusters. A library quantification qPCR on the final pool gives the most accurate loading concentration.
• Then follow your instrument's guide for denaturation and final loading concentration, which differ between platforms and kits.