Need help? Call us: +1 416-549-8100 or customerservice@mbpinc.net

Quick Order Contact Us About Us Blogs Sign In

Product Filters

DNA Normalization and Concentration for NGS Library Pooling

DNA normalization and Concentration covers the reagents and methods used to equalize DNA library concentrations and volumes after quantitation, before pooling multiple indexed libraries into a single multiplexed sequencing run. Magnetic SPRI bead-based cleanup methods are most commonly used for both size selection and normalization of sequencing libraries. Accurate normalization ensures each library contributes equal sequencing depth in a multiplexed run. Academic and core laboratories performing multiplexed sequencing can benefit from guidance when selecting normalization strategies and bead-based cleanup workflows for consistent library pooling.

Looking for a DNA normalization workflow? Contact customerservice@mbpinc.net for a quotation and expert guidance on selecting the right normalization method and SPRI-based cleanup system for your sequencing libraries.

DNA normalization and Concentration

Per Page:

Sort By:

MQP-50000

Magbio MagQuant Plus DNA Kit V2

List Price:

USD42.89 - USD17,514.44

Online Price:

USD30.64 - USD12,510.31

Your Price:

Showing 1 to 1 of 1 results

What is DNA normalization and concentration for NGS?

DNA normalization for NGS is the process of adjusting all individual indexed sequencing libraries to the same molar concentration before pooling them into a single multiplexed sequencing run, so that each library contributes approximately the same number of sequencing reads to the total run output. Concentration may also be needed when a library is too dilute after cleanup or elution. SPRI (solid-phase reversible immobilization) magnetic bead-based cleanup, most commonly using AMPure XP beads, is the standard method for post-library-prep cleanup, size selection, and concentration in NGS workflows.

What you will find:

Magbio MagQuant Plus DNA Kit: for automated, high-throughput nucleic acid concentration and normalization workflows

How to normalize and concentrate NGS libraries

Quantify each library by qPCR to calculate molar concentration accurately

Kapa Library Quantification qPCR kits or equivalent measure the amplifiable, adapter-ligated molecules in a library, providing a molar concentration based on insert size that can be used to pool libraries at equal molar ratios. Fluorometric quantitation gives mass per volume but not molar concentration, and libraries with different insert sizes have different mass-to-moles conversion factors.

Calculate library molar concentration using measured mass and average insert size

If molar quantitation by qPCR is not available, the molar concentration of a library can be estimated from its fluorometric mass concentration and the average insert size from Bioanalyzer or TapeStation: molar concentration (nM) = (mass in ng/µl × 10^6) / (insert size in bp × 660 g/mol/bp). This converts mass to moles using the average molecular weight per base pair.

Pool indexed libraries at equal molar ratios for balanced depth

After calculating the molar concentration of each library, pool volumes delivering equal moles of each library into the final pool. Even small quantitation errors translate into coverage imbalances between samples in the sequencing output.

Use SPRI bead cleanup to concentrate libraries and remove adapter dimers

SPRI bead cleanup with AMPure XP beads at the appropriate bead-to-sample ratio selects for DNA fragments above a size cutoff while removing smaller adapter dimers and free adapter molecules. The final elution in a smaller volume of buffer concentrates the library. The bead ratio determines the minimum retained fragment size, with higher ratios retaining smaller fragments.

Verify the pooled library before sequencing

After pooling and final SPRI cleanup, measure the pooled library by qPCR and Bioanalyzer to confirm molar concentration and size distribution before loading, since loading concentration determines cluster density and affects sequencing performance.

Specifications context

AMPure XP SPRI beads use polyethylene glycol and sodium chloride to precipitate DNA onto paramagnetic beads in a size-dependent manner, making the bead-to-sample volume ratio the key parameter for controlling the minimum retained fragment size. As of 2026, automated bead-based normalization kits that combine quantitation and normalization remain available for high-throughput settings, though most academic labs perform manual quantitation and normalization before pooling.

Browse through the catalog and contact the expert team to book high-quality DNA normalization and concentration products today.

FAQ

If individual libraries are pooled without normalization, libraries present at higher molar concentration will produce proportionally more clusters on the flow cell and consume more sequencing reads, resulting in unequal coverage across samples in the run output. Equal-molar pooling ensures each sample receives approximately the same number of sequencing reads, which is particularly important when comparing expression levels or variant frequencies between samples in the same run.

qPCR-based library quantitation specifically measures the amplifiable, adapter-ligated molecules that will form clusters on the flow cell, while fluorometric assays measure total DNA mass regardless of whether each molecule is adapter-ligated and amplifiable. Libraries with different insert sizes also have different mass-to-moles conversion factors, so fluorometric mass quantitation from different-sized libraries cannot be directly compared without knowing and correcting for insert size.

Molar concentration in nanomolar can be calculated as (mass concentration in ng/µl multiplied by 1,000,000), divided by (average insert size in base pairs multiplied by 660 g/mol/bp). For example, a library with a fluorometric concentration of 2.5 ng/µl and a 350 bp average insert size has a molar concentration of approximately 10.8 nM, which can be used to calculate the volume needed for equal-molar pooling.

AMPure XP beads are paramagnetic SPRI beads in polyethylene glycol and sodium chloride that bind DNA size-dependently: at higher bead-to-sample volume ratios, smaller DNA fragments bind along with larger ones, while at lower ratios only larger fragments bind and smaller ones remain in solution. The bead ratio is the primary variable controlling minimum retained fragment size, with higher ratios retaining adapter dimers and lower ratios leaving them behind.

Yes, AMPure XP beads can be used to concentrate a library by eluting the bead-bound DNA in a smaller volume than the original sample, increasing the library concentration. The beads are added in excess to bind all DNA, the supernatant is removed, beads are washed, and then the library is eluted in the desired smaller volume, effectively concentrating the library proportional to the volume reduction.

Loading a library pool at too high a concentration on an Illumina flow cell produces overclustering, where clusters are so dense they overlap and interfere with each other's optical detection, resulting in low data quality, poor cluster passing filtering, and low usable read output. Loading too low produces underclustering, where fewer clusters form than expected and sequencing output is lower than the instrument's capacity. Accurate quantitation and pooling within the instrument's recommended loading range is critical for obtaining optimal data.

Bead-based normalization kits, sometimes called beadlinking or SequalPrep-type normalization, use a solid-phase substrate that binds a fixed maximum amount of DNA per bead, so that regardless of the starting concentration, all samples elute at approximately the same DNA concentration after washing. This approach normalizes library concentrations without requiring individual quantitation of each sample, which is useful in high-throughput workflows, though it sacrifices some accuracy compared with manual quantitation-based normalization.

Yes, MBP offers academic and bulk pricing for SPRI bead-based cleanup and normalization reagents and Kapa library quantification kits for NGS workflows, with specialist support for bead ratio and pooling guidance. Orders ship from MBP's US office in Houston, Texas, with stock available in both USD and CAD.

We provide the highest quality of products and anytime customer service.

Featured Categories

Useful Links

+1 416-549-8100

customerservice@mbpinc.net

online payment

fast delivery

technical support

24/7 support