How to use (3 steps)
- Choose an example or enter library concentration (ng/µL or nM) and fragment length (bp).
- Select a target nM and final volume to see a dilution recipe (stock + water).
- Choose a pooling method and review per-library volumes and pooled nM (guide).
Conversion to nM depends on length, MW factors, and quantification method. Treat results as a guide and follow your protocol for final decisions.
Inputs (library table)
Results (conversion, dilution, pooling)
| Name | Stock (nM) | Dilution: stock (µL) | Dilution: water (µL) | Pool volume (µL) |
|---|
—
How it's calculated
- Conversion (guide): nM = (ng/µL) × 1e6 / (length × MW_per_bp)
- Dilution: C1V1 = C2V2 (V_stock = (C_target/C_stock) × V_final)
- Direct equal-molar pooling: allocate volumes so C_i × V_i is equal (given total volume)
Conversion depends on length, MW factors, and quantification method. Use these results as a guide.
How to use this calculator effectively
This calculator is designed to make scenario checks fast. Use a repeatable workflow: baseline first, one variable change at a time, then compare output direction and magnitude.
How it works
Run your first scenario with defaults. Then, change exactly one assumption and observe which result changes most. That is the fastest way to identify sensitivity and explain what drives the outcome.
When to use
Use this page when you need practical planning support, side-by-side alternatives, or a clean baseline for further discussion.
Common mistakes to avoid
- Changing multiple assumptions simultaneously.
- Confusing percent and decimal inputs.
- Mixing unit systems across scenarios.
- Relying only on rounded display output for final conclusions.
Worked example
Prepare a base case and one alternative case, then compare outputs and validate the direction, scale, and interpretation with the same assumptions across both cases.
See also
FAQ
What is ng/µL → nM conversion used for?
Use it to normalize libraries by molecule counts for equal-molar pooling (different from equal mass).
Where do I get fragment length (bp)?
Use the average size from a Bioanalyzer/TapeStation report. Keep adapter inclusion consistent with the report definition.
What is the difference between equal-mass and equal-molar pooling?
Equal mass matches ng values, while equal molar matches molecule counts (nM). If lengths differ, equal mass will not equalize molecule counts.
What is normalization?
Normalize by diluting all libraries to the same nM, then pool equal volumes. This tool provides that recommended flow.
The dilution volume is too small (e.g., 0.2 µL).
Very small volumes are hard to pipette. Consider intermediate dilution or adjusting final volume.
Can I use nM (or pM) values from qPCR?
Yes. Use the direct nM input mode. Note that quantification definitions can differ by method.
Will the pooled concentration be exact?
It is a guide. Quantification and pipetting errors and fragment length variation can shift the final value.
How to use NGS library concentration: ng/µL → nM, dilution, pooling effectively
What this calculator does
This page is for estimating outcomes by changing inputs in one controlled workflow. The model keeps your focus on variables, not output shape. Start with stable assumptions, then test sensitivity by changing one key input at a time to observe directional impact.
Input meaning and unit policy
Each input has an expected unit and a typical range. For reliable interpretation, check whether you are using the same unit system, period, and base assumptions across all runs. Unit mismatch is the most common source of unexpected drift in numeric results.
Use-case sequence
A practical sequence is: first run with defaults, then create a baseline log, then run one alternative scenario, and finally compare only the changed output metric. This sequence reduces cognitive load and prevents false pattern recognition in early experiments.
Common mistakes to avoid
Avoid changing too many variables at once, mixing incompatible data sources, and interpreting a one-time output without checking robustness. A single contradictory input can flip conclusions, so keep each experiment minimal and document assumptions as part of your note.
Interpretation guidance
Review both magnitude and direction. Direction tells you whether a strategy moves outcomes in the desired direction, while magnitude helps you judge practicality. If both agree, you can proceed; if not, rebuild the baseline and verify constraints before deciding.
Operational checkpoint 1
Record the exact values and intent before you finalize any comparison. Confirm the unit system, date context, and business constraints. Compare outputs side by side and check whether differences are explained by one changed variable or by hidden assumptions. This checkpoint often reveals the single factor that changed everything.
Operational checkpoint 2
Record the exact values and intent before you finalize any comparison. Confirm the unit system, date context, and business constraints. Compare outputs side by side and check whether differences are explained by one changed variable or by hidden assumptions. This checkpoint often reveals the single factor that changed everything.
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