Library normalization is the process of diluting libraries of variable concentration to the same concentration before volumetric pooling, ensuring an even read distribution for all samples. Normalization best practices can be used for any Illumina library preparation requiring a manual normalization. Steps for normalization are:

1. Determine your library size
2. Quantify your libraries
3. Plan your dilution calculations
4. Pool the normalized libraries

Some library preparation kits, such as Nextera XT and Illumina DNA Prep, (M) Tagmentation (formerly known as Nextera DNA Flex), offer a bead-based normalization approach. Manual normalization is not needed when bead-based normalization is performed. However, when the input amount is lower than 100 ng for Illumina DNA Prep, (M) Tagmentation or the final library yield is less than 10–15 nM for Nextera XT, the internal normalization is not compatible. In these situations, it is necessary to perform manual normalization

Note: the bead normalization chemistries are different in the Illumina DNA Prep, (M) Tagmentation and Nextera XT workflows; because of this difference, the library denature and dilution procedures prior to sequencing are different. Contact Illumina Technical Support (techsupport@illumina.com) for additional information.

Determine your library size

Validate the library size by running the samples on a Bioanalyzer or Fragment Analyzer. This step also provides visibility into possible library issues, such as adapter dimers or unexpected library sizes.

Quantify your libraries

Quantify the library using the recommended quantification method as specified in the library preparation guide, summarized in the Library Quantification and Quality Control Quick Reference Guide bulletin.

Convert this value from ng/µl to nM using the average library size obtained from the Bioanalyzer or Fragment Analyzer, and the Converting ng/µl to nM When Calculating dsDNA Library Concentration bulletin.

Plan your dilution calculations

The concentration normalization occurs in this step. Dilution can be done using molecular grade water or 10 mM Tris-HCl pH 8.5. The Pooling calculator can also be used to calculate library dilutions, and can be used if the libraries have the same or different concentration.

1. Determine the common concentration to dilute the libraries for subsequent applications. For most Illumina sequencing platforms, 2–4 nM for each library is the preferred starting concentration for the denaturation and dilution guidelines; consult the respective instrument user guides for more information.
2. Calculate the dilution of the libraries using the following equation:



For the most even representation of samples and most reliable cluster density, make sure that all pipetted volumes are at least 2 µl. Pipetting less than 2 µl can introduce significant concentration errors. Adjust calculations to make sure that appropriate volumes are used. For highly concentrated libraries, use one or more intermediate concentrations.

For example, the following calculations demonstrate how to dilute 3 libraries (with different starting concentrations of 15 nM, 20 nM, and 50 nM) to a final concentration of 4 nM in a final volume of 15 µl for each library.

Starting Concentration	Volume of stock DNA (for 4 nM concentration)	Volume of diluent (for 15 ul final volume)
15 nM	4 ul	11 ul
20 nM	3 ul	12 ul
50 nM*	1.2 ul	13.8 ul

* The 50 nM library example only uses 1.2 µl of stock DNA to dilute down to 4 nM library. For the most accurate results, an intermediate dilution of 20 nM is necessary to achieve at least a 2 µl pipet volume.

Intermediate Dilution			Final Dilution
Starting concentration	Volume of stock DNA (for intermediate 20 nM concentration)	Volume of diluent (for 15 ul volume)	Volume of intermediate 20 nM DNA (for 4 nM final concentration	Volume of diluent (for 15 ul volume)
50 nM	6 ul	9 ul	3 ul	12 ul

 

3. Dilute the libraries according to the calculations above. The libraries are now normalized.

Pool the normalized libraries

Volumetric pooling: Combine equal volumes of each normalized library into a microcentrifuge tube and gently pipette contents up and down 10 times to mix thoroughly.

The normalized pool is now ready to be denatured and sequenced. For detailed instructions on denaturation procedures, use the appropriate instrument denaturation and dilution guide:

• iSeq 100 System Guide for dilution recommendations
• MiniSeq System Denature and Dilute Libraries Guide
• MiSeq System Denature and Dilute Libraries Guide
• NextSeq 500/550 System Denature and Dilute Libraries Guide
• HiSeq Systems Denature and Dilute Libraries Guide
• cBot System Guide for denaturing and diluting libraries for the HiSeq 3000/4000 
• NextSeq 1000/2000 Sequencing System Guide for denaturing and diluting libraries on the NextSeq 1000/2000
• NovaSeq System Guide for denaturing and diluting libraries on the NovaSeq