Equimolar Library Pooling Calculator
Enter the measured concentration and average fragment size for four double-stranded DNA libraries. The calculator converts each result to nM, assigns equal molecular contributions, and shows the library and diluent volumes for the requested pool.
Diluent volume (µL)14.06
- Library molarities (nM)
- Library 1: 6.734 nM; Library 2: 6.734 nM; Library 3: 6.734 nM; Library 4: 6.734 nM
- Library 1 volume (µL)
- 1.48
- Library 2 volume (µL)
- 1.48
- Library 3 volume (µL)
- 1.48
- Library 4 volume (µL)
- 1.48
- Molecules contributed per library (fmol)
- 10
- Maximum achievable pool molarity (nM)
- 6.73
- Feasibility and pipetting check
- Feasible without concentrating the entered libraries. All calculated library transfers are at least 1 µL.
Assumes double-stranded DNA with an average molecular mass of 660 g/mol per base pair.
Volumes are unrounded; rounding transfers changes the achieved pool molarity and equal contribution slightly.
How to use this calculator
- Enter the pool molarity and total volume you need before the platform-specific denaturation or loading dilution.
- For each library, enter its measured concentration in ng/µL and average fragment size including adapters.
- Use the four calculated library volumes and the diluent volume to prepare the pool.
- Review the feasibility and pipetting messages before transferring very small volumes.
How equimolar library pooling is calculated
Mass concentration alone does not tell you how many library molecules are present. A shorter DNA library has more molecules per ng/µL than a longer library at the same mass concentration. For equal sequencing representation, first convert each double-stranded DNA library from mass concentration to molarity.
For each library, the calculator uses M = (C × 1,000,000) ÷ (660 × L), where M is molarity in nM, C is concentration in ng/µL, and L is average fragment length in base pairs. The factor 660 g/mol per base pair is an approximate average molecular mass for double-stranded DNA.
With four libraries, a target concentration T in a final volume V requires Q = (T × V) ÷ 4 fmol from each library. The volume of library j is vᵢ = Q ÷ Mᵢ. The remaining volume is diluent: D = V − Σvᵢ.
Feasibility and transfer limits
There is a concentration ceiling when you only mix the entered libraries and do not concentrate them. The calculator reports Mmax = 4 ÷ Σ(1/Mᵢ), the harmonic mean of the four library molarities. A target above that value needs more molecules than can fit in the requested volume, so the library set must be concentrated or the target reduced.
The least molar library often controls this limit and needs the largest transfer. In contrast, a highly concentrated library can require a very small transfer. Transfers below 1 µL deserve a pipetting check; below 0.2 µL, prepare a working dilution or make a larger pool rather than relying on an imprecise direct transfer.
What the calculation leaves out
This tool assumes that the reported average fragment size and mass concentration are representative. It does not correct for fragment-size distributions, quantification bias, damaged or adapter-dimer molecules, index balance, cluster-density targets, or differences in library behavior on a particular platform. It also does not prescribe denaturation, PhiX addition, loading concentration, or quality-control steps; follow the instructions for the sequencing platform, library kit, and facility.
The 660 g/mol per base-pair conversion is for double-stranded DNA. Do not use this calculation unchanged for single-stranded libraries, where molecular mass and workflow requirements can differ. After practical rounding, record the actual volumes and requantify or validate the completed pool when the workflow calls for it.
Worked example
Suppose all four libraries measure 2 ng/µL and have an average fragment size of 450 bp. Each library is (2 × 1,000,000) ÷ (660 × 450) = 6.734 nM. For a 20 µL pool at 2 nM, the pool needs 40 fmol total, or 10 fmol from each library.
Each transfer is 10 ÷ 6.734 = 1.485 µL. The four libraries use 5.94 µL, leaving 14.06 µL diluent. Because all four molarities are the same, the maximum pool molarity without concentration is also 6.734 nM, so the 2 nM target is feasible.
Common questions
Why should sequencing libraries be pooled by molarity instead of mass?
Equal masses of DNA do not contain equal numbers of molecules when fragment lengths differ. Pooling by molarity is intended to give each library an equal molecular contribution, which is the relevant starting point for cluster generation and sequencing representation.
How is ng/µL converted to nM for double-stranded DNA?
Divide the concentration in ng/µL times 1,000,000 by 660 times the average fragment length in base pairs. The 660 factor approximates the molecular mass of one base pair of double-stranded DNA, so the result is an estimate that depends on the accuracy of the fragment-size measurement.
Should adapter length be included in the average fragment size?
Yes. Molarity depends on the mass of the complete sequencing molecule, so use the average length including adapters. Use the fragment-size value reported for the final library rather than the insert size alone.
What should I do when a calculated transfer is too small to pipette?
For a transfer below your validated pipetting range, make a working dilution of that library or scale up the total pool volume. A volume below 0.2 µL should not be treated as a reliable direct transfer in a routine manual workflow.
Why is my requested pool concentration infeasible?
The requested pool would require more total library volume than the final pool can hold. Lower the target molarity, concentrate one or more libraries, or prepare a pool at the maximum achievable molarity shown by the calculator.
Can this calculation be used for single-stranded libraries?
Not without changing the molecular-mass conversion and checking the protocol for that library type. This calculator explicitly uses the double-stranded DNA approximation of 660 g/mol per base pair.