Library Quantification

Introduction

Next-generation sequencing, or NGS, uses large-scale DNA sequencing technology to determine the sequence of DNA or RNA. This allows for researchers to study and identify genetic variations associated with diseases or other inherited conditions, to discover novel pathogens, and study genetic modifications that arise in tumor subclones. Beyond the research lab, NGS is also used in the clinical setting to diagnose various disorders.

A sequencing library is needed to perform NGS. To produce a sequencing library, the genetic material from a sample is randomly fragmented and adapter sequences are added to both the 3’ and 5’ ends. These ligated adapters serve to create the primer annealing site and allow for specific enrichment of only these products during subsequent PCR. These adapters serve as a way to anchor the fragment to a surface, such as a bead, for sequencing down the line. This collection of genetic fragments is referred to as the NGS library.

To ensure equal representation of indexed libraries for use in NGS, libraries must be quantified prior to pooling. Library quantification is also essential to ensure optimal dilution of library pools prior to sequencing. It is a key step in the NGS workflow because it ensures the recommended amount of DNA is loaded into the flowcell, preventing saturation and increasing coverage and depth.

Real-time PCR and Library quantification

There are multiple ways to perform library quantification, but qPCR is the gold standard because it produces more precise results due to the amplification of only the fragments with the adapter sequences added and disregards other DNA fragments, primer dimers, etc. These sequences with the adapter sequences on them will then serve as templates during the amplification process. Another bonus of using qPCR to do library quantification is it uses a minimal amount of material and can be automated. This allows for more high-throughput runs to be performed.

How does the Cielo improve on the library quantification process?

Having an efficient, reliable qPCR machine is important for optimizing library quantification. The Azure Cielo is a qPCR system with advanced and high-performance optical technology allowing for high-quality data generation.

Boasting broad-spectrum detection capability, exceptional specificity and precision, and faster run times, the Cielo consistently provides reproducible qPCR data which makes it a superior choice for use in library quantification.

The Azure Cielo qPCR systems are designed with innovative optical technology with two sets of 16 optical fibers. This allows 16 individual wells to be imaged simultaneously, meaning an entire 96-well plate can be imaged in up to  six fluorescent channels in just nine seconds saving time and getting results quicker. This would ultimately increase high-throughput and allow for more libraries to be quantified and faster.

Additionally, Cielo uses fiber optics to directly deliver light to each individual well with precision, which reduces background excitation. The data obtained is reliable due to the minimal crosstalk.

REFERENCES

  1. “Best practices for library quantification.” Illumina, 18 November 2020, https://emea.support.illumina.com/bulletins/2020/11/best-practices-for-library-quantification.html. Accessed 24 March 2022.

  2. Gurson, Natalie. “Next Generation Sequencing Library Preparation – Seq It Out #10.” Thermo Fisher, 22 October 2015, https://www.thermofisher.com/blog/behindthebench/next-generation-sequencing-library-preparation-seq-it-out-10/. Accessed 18 March 2022.

  3. “An Overview of Next-Generation Sequencing.” Technology Networks, 17 March 2021, https://www.technologynetworks.com/genomics/articles/an-overview-of-next-generation-sequencing-346532#D1. Accessed 18 March 2022.

Related Products

Smart protocols, high-quality data, and robust durability

Looking for a full list of applications?

Related blog posts…

Multiplexing

Multiplexing Real-time PCR (qPCR) is a widely-used method of detecting specific DNA sequences.  Using target-specific fluorescent probes, sequences are amplified over multiple cycles. As the

Read More
Purple pipette

Genotyping

Genotyping Genotyping looks at the genetic information of a sample. This application allows researchers to make correlations with an observable phenotype. Genotyping is often used

Read More

Pathogen Detection

Pathogen Detection What is pathogen detection? A pathogen, or infectious agent,  is an organism which causes disease to its host. Pathogens include viruses, bacteria, fungi,

Read More
Shopping cart
There are no products in the cart!
Continue shopping