Posts Tagged ‘droplet digital pcr’

Droplet Digital™ PCR provides accurate quantification of next-generation sequencing libraries

 :: Posted by American Biotechnologist on 08-19-2013

A study published today found that Droplet Digital PCR (ddPCR™) can be used as an accurate and precise method for quality control of next-generation sequencing (NGS) libraries. NGS library QC is essential to optimizing sequencing data yield, thereby increasing efficiency and throughput while lowering cost. The research was published 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 Dr. Jason Bielas, lead author and Assistant Member in the Public Health Sciences Division at Fred Hutchinson Cancer Research Center in Seattle, Wash.

Quantifying NGS Libraries and Why It 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 might be sequenced if library molecules are not the appropriate length to fully utilize the sequencing platform, thus limiting throughput. Given this, quantifying library molecules and determining 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. Each of these has its limitations, though, and the steps recommended to address them, 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 the QX100 ddPCR system from Bio-Rad Laboratories. QuantiSize offers the ability to determine the absolute quantity and the detailed size distribution of target DNA 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.

Bio-Rad Introduces New Digital PCR Assays for Human Copy Number Variation and Mutation Detection

 :: Posted by American Biotechnologist on 08-01-2013

Bio-Rad Laboratories, Inc. announced the launch of its PrimePCR™ assays for Droplet Digital™ PCR. These new predesigned assays for mutation detection and copy number variation (CNV) have been experimentally validated to provide single-copy PCR resolution without a standard curve.

Droplet Digital PCR (ddPCR™) technology provides an absolute measure of target DNA molecules. Researchers can use ddPCR assays to accelerate discovery and enable new research strategies for inherited disorders, cancer, and infectious diseases.

“We are excited because these are the first assays that have been made available as fully validated for digital PCR,” said Richard Kurtz, marketing manager of the Gene Expression Division at Bio-Rad. “These predesigned assays remove the burden of assay design and make digital PCR even easier for our customers.”

In this first release, Bio-Rad offers its customers 14 mutation detection assays with matching wild-type assays for biologically relevant and highly prevalent cancer mutation targets chosen from the COSMIC database. Commonly studied cancer gene mutations such as BRAF V600E and EGFR T790M are among the first set of assays available.

The assays, combined with the sensitivity of Bio-Rad’s ddPCR system, are capable of detecting a single mutant copy in a background of 2,000 or more wild-type molecules (0.05% mutation frequency). The precision of ddPCR also enables cancer researchers to discriminate small-fold copy number changes with the 62 assays targeting common cancer genes and two reference target assays now available.

Bio-Rad has partnered with Biogazelle, a qPCR data analysis and services company, and Integrated DNA Technologies (IDT), a world-class manufacturer of oligonucleotides, to design, optimize, and experimentally validate the assays. The PrimePCR ddPCR assays employ universal cycling conditions and do not require optimization. Primer specificity has been validated by next-generation sequencing, and all assays have been validated on Bio-Rad’s QX100™ Droplet Digital PCR system.

PrimePCR ddPCR assays are available in multiple reaction sizes.

For more information on Bio-Rad’s PrimePCR products, please visit www.bio-rad.com/PrimePCR.

Bio-Rad Launches New ddPCR Library Quantification Kit to Optimize Performance of Illumina NGS Platforms

 :: Posted by American Biotechnologist on 04-17-2013

Bio-Rad Laboratories, Inc., today announced the availability of its new ddPCR library quantification kit for Illumina TruSeq sample preparation protocols. Used with Bio-Rad’s QX100™ Droplet Digital™ PCR system, the new kit offers researchers a way to precisely and directly measure amplifiable library concentrations.

The TruSeq sample preparation method is the technique behind Illumina’s MiSeq and HiSeq next-generation sequencing (NGS)platforms. Using the ddPCR library quantification kit to quantify TruSeq DNA libraries maximizes the number of useable reads, enables consistent loading, and optimizes the utilization of every sequencing run. The resulting data provide additional measures of library quality not provided by other methods, including the percentage of nonamplifiable species such as adaptor dimers as well as the size range of library inserts.

Additional key benefits of the ddPCR library quantification kit include:

  • Superior performance — reduces PCR bias due to sample partitioning during quantification
  • Simple workflow — easily incorporated into the TruSeq library construction workflow
  • Efficient utilization of sequencing runs — provides fluorescence amplitude data, a metric for library quality

Kits for other NGS platforms are also in development. For more information on the ddPCR library quantification kit, please visit http://bit.ly/ddPCR_QKL.

News: Droplet Digital PCR Improves Detection Sensitivity of Telomerase Activity Assay

 :: Posted by American Biotechnologist on 04-10-2013

Researchers from the University of California, San Francisco, and Bio-Rad have demonstrated that Bio-Rad’s Droplet Digital PCR (ddPCR) technology can dramatically improve the sensitivity, precision, and throughput of a popular assay for telomerase activity.

Currently, researchers are investigating telomerase activity as a biomarker for cancer diagnosis and as a target for anticancer drugs. Measuring it’s activity more sensitivity may enhance our understanding of it’s role in diseases.

This research was presented at the American Association of Cancer Research (AACR) Annual Meeting on Tuesday, April 9.

You can view the press release here: http://bit.ly/12KJyUk

Droplet Digital PCR and the HIV Baby

 :: Posted by American Biotechnologist on 03-28-2013

In a recent BioTechniques podcast, Drs. Matthew Strain and Daniel Kuritzkes talk about how droplet digital PCR was used to verify that an baby born with HIV had indeed been cured. Listen to the podcast below: