Droplet Digital™ PCR Provides Accurate Quantification of Next-Generation Sequencing Libraries
A study published recently found that Droplet Digital PCR (ddPCR™) quantification is a more accurate and precise method for quality control of next-generation sequencing (NGS) libraries than conventional real-time PCR methods. NGS library QC is essential for optimizing sequencing data yield, thereby increasing efficiency and throughput while lowering cost. This study appears in the in the August issue of BioTechniques.
“While real-time PCR has traditionally been used to quantify libraries, we determined that the only truly accurate way to reproducibly quantify our NGS libraries is with ddPCR,” said lead author Dr. Jason Bielas of the Public Health Sciences Division at Fred Hutchinson Cancer Research Center in Seattle, WA.
Why NGS Library Quantification Matters
Various commercial NGS technologies require users to load a precise number of viable DNA library molecules onto the instrument to optimize data yield. Performing a sequencing run with either too many or too few library molecules results in compromised data and sometimes no data at all, wasting sample, expensive reagents, user time, and instrument time.
Moreover, fewer bases can be sequenced when library molecules are not the appropriate length to fully utilize the sequencing platform, thus limiting throughput. Therefore, quantifying library molecules and determining the fragment size range have become crucial steps in library preparation.
NGS instrument manufacturers recommend quantifying libraries using real-time quantitative PCR (qPCR) and determining their size range using gel or capillary electrophoresis. However, these techniques both have limitations, and the steps recommended to address the problems can be time-consuming and expensive.
Advantages of ddPCR for Quantifying NGS Libraries
To simultaneously quantify and determine the size distribution of target DNA with a single ddPCR assay, Dr. Bielas and his team exploited a relationship between droplet fluorescence and amplicon size. They confirmed the accuracy and precision of this method by applying it to NGS library preparation.
The ddPCR assay they designed — known as QuantiSize — was developed using Bio-Rad’s QX100™ Droplet Digital™ PCR System. QuantiSize offers the ability to determine the absolute quantity of target DNA and a detailed amplicon size distribution in a single ddPCR reaction well, thus avoiding the drawbacks of other independent quantification and size determination methods.
“Now that we have discovered this new correlation, we can also use ddPCR to extract more information on the characteristics of DNA based on the range of fluorescence that can occur within each droplet,” said Bielas.
Having demonstrated the efficacy of this technique, Dr. Bielas is now planning to leverage the relationship between ddPCR fluorescence and amplicon size to explore mutagenic deletion events in both the human nuclear and mitochondrial genomes.