Next-generation sequencing (NGS) uses large-scale DNA sequencing technology to determine the sequence of DNA or RNA. Researchers use NGS to study and identify genetic variations associated with diseases and 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.
qPCR is the gold standard to performing library quantification because it produces more precise results, uses less sample, is more sensitive, and quantifies amplifiable molecules. qPCR only amplifies 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 is it uses a minimal amount of material and can be automated. These qualities allow for more high-throughput runs to be performed.
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. It consistently provides reproducible qPCR data which makes it a superior choice for use in library quantification.
The Cielo is 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 ultimately increases high-throughput and allow for more libraries to be quantified and faster.
“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.
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.
“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.
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