# Goal/Experiment:
Normalisation and Pooling of DNA Libraries for Illumina Whole Genome Sequencing
## C-SOP-501: Normalisation and Pooling of DNA Libraries for Illumina Whole Genome Sequencing
### Authors
Ben, Mihir Kekre¹, Pascoe¹
¹The Centre for Genomic Pathogen Surveillance, Oxford, United Kingdom
---
### Abstract
Normalisation in next-generation sequencing (NGS) is the process of equalising the concentration of multiple DNA libraries for the purpose of multiplexing. Multiplexing helps maximize the use of expensive NGS technology, enabling parallel sequencing of hundreds to thousands of libraries on a single flowcell, thereby driving down per-sample costs.
Uneven library concentrations from different types and qualities of samples can lead to inconsistencies in data quality. Overrepresented libraries on the flowcell waste capacity while underrepresented ones may lead to poor read depth and unreliable data. Normalisation ensures each library is equally represented and sequenced to sufficient depth.
### Quantitation Options
- **Quick but less accurate methods**: Spectrophotometry-based quantitation.
- **Accurate methods**: Quantitative PCR (qPCR), dependent on fragment size knowledge.
A crucial factor influencing quantitation and normalisation accuracy is whether the method specifically counts adaptor-ligated double-stranded DNA (dsDNA) molecules. Illumina's best practice suggests using fluorometric or qPCR-based quantitation.
---
### Materials
1. Quantified and size-estimated double-stranded DNA libraries
2. Nuclease-free water or 10 mM Tris-HCl (pH 8.5)
3. Single-channel pipettes (P10, P200) with compatible tips (filter-free, sterile)
4. 96-well PCR-plate, low-profile, full skirted (ThermoFisher Scientific, Cat no. AB0800)
5. 2.0 mL microcentrifuge tubes
6. **For New England BioLabs NEBNext Library Quant Assay**:
- a. NEBNext® Library Quant Kit for Illumina (NEB, Cat no. E7630)
- b. Nuclease-free water
- c. qPCR machine
- d. Compatible qPCR plates and seals
- e. PCR strip tubes or microcentrifuge tubes
- f. Conical centrifuge tubes
### Safety Warnings
- NEBNext Library Quant Kit safety sheets:
- [Dilution Buffer (10X)](link)
- [DNA Standard 1](link)
- [DNA Standard 2](link)
- [DNA Standard 3](link)
- [DNA Standard 4](link)
- [ROX (High)](link)
- [ROX (Low)](link)
### Before Starting
1. Ensure that all active workbenches are cleaned with 80% ethanol, all relevant personal protective clothing is worn, and the work area is prepared according to local GLP guidelines for molecular methods.
2. Ensure you have the following library QC metrics using 'C-SOP-401: Quality Control (QC) of DNA Libraries for Whole Genome Sequencing':
- a. Mean total library fragment size (in bp) - output from Bioanalyzer 2100 or Tapestation 4150/4200.
- b. Mean library concentration (in nM) - output from a qPCR quantification assay.
> **Note:** If mean library concentration is in ng/µl, convert using [this link](link).
---
### Library Normalisation
1. **Concentration normalisation**: Dilute an aliquot of the stock library in a diluent (nuclease-free water or 10 mM Tris-HCl pH 8.5).
2. For most Illumina sequencing platforms, 2–4 nM for each library is the preferred final concentration.
**Dilution Calculation**
\[
(C_1 \times V_1) = (C_2 \times V_2)
\]
- \(C_1 = \) concentration of stock library (nM)
- \(V_1 = \) volume of stock library to be diluted (µl)
- \(C_2 = \) desired final concentration (nM)
- \(V_2 = \) desired total volume
Alternatively, use the [normalisation and pooling calculator](link) to compute library dilutions.
> **Note:** Ensure pipetted volumes are not less than 4 µl (or at least 2 µl).
**Example Dilutions**
| Starting Concentration | Volume of Stock DNA (for 4 nM final concentration) | Volume of Diluent (for 15 µl final volume) |
|------------------------|----------------------------------------------------|-------------------------------------------|
| 15 nM | 4 µl | 11 µl |
| 20 nM | 3 µl | 12 µl |
| 50 nM* | 1.2 µl | 13.8 µl |
*Intermediate dilution required:
| Starting Concentration | Volume of Stock DNA (for intermediate 20 nM concentration) | Volume of Diluent (for 15 µl volume) | Volume of Intermediate 20 nM DNA (for 4 nM final concentration) | Volume of Diluent (for 15 µl final volume) |
|------------------------|-------------------------------------------------------------|-------------------------------------|-----------------------------------------------------------------|-------------------------------------------|
| 50 nM | 6 µl | 9 µl | 3 µl | 12 µl |
3. Mix the calculated volumes of library and diluent to obtain a normalised library solution.
---
### Library Pool Creation (for Multiplexed Libraries)
1. **Volumetric pooling**: Combine equal volumes of each normalised library in a 2.0 mL microcentrifuge tube.
2. Gently pipette the contents up and down 15 times or vortex to mix thoroughly.
> **Note:** Steps 4-8 can be combined using the [norm/pool calculator](link).
### Library Pool Confirmatory QC
1. Quantify the library pool using a robust qPCR assay as described in 'C-SOP-401: Quality Control (QC) of DNA Libraries for Whole Genome Sequencing' (NEBNext® Library Quant Kit for Illumina®) and compute the pool concentration in nM.
> **Note:**
> - Ensure final pool concentration matches the expected 2-4 nM range.
> - Step 9 can be skipped under extreme budget constraints, but downstream results like clustering densities may vary.
2. The normalised pool is ready to be denatured and sequenced.
---
### Additional Information & Troubleshooting
Refer to the following resources for detailed denaturation and dilution procedures:
- [iSeq™ 100 System Guide](link)
- [MiniSeq™ System Denature and Dilute Libraries Guide](link)
- [MiSeq™ System Denature and Dilute Libraries Guide](link)
- [NextSeq™ 500/550 System Denature and Dilute Libraries Guide](link)
- [NextSeq 1000/2000 Sequencing System Guide](link)
- [HiSeq™ Systems Denature and Dilute Libraries Guide](link)
- [cBot™ System Guide](link) for HiSeq 3000/4000
- [NovaSeq™ System Guide](link)
This protocol is distributed under the terms of the [Creative Commons Attribution-NonCommercial-ShareAlike](link) license.
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