Posts Tagged ‘droplet digital pcr’

What is droplet digital PCR?

 :: Posted by American Biotechnologist on 09-04-2013

Yesterday we told you how scientists at the Fred Hutchinson Cancer Research Center were using droplet digital PCR to accurately quantify microRNA biomarkers in an accurate and reproducible fashion.

As a courtesy to those readers who may have missed it previously, we are reposting a video that was produced a couple of years ago with a comprehensive technical introduction to digital PCR. Please let us know if you have any questions regarding this video or droplet digital PCR in general.

Droplet Digital™ PCR Enables Reproducible Quantification of microRNA Biomarkers

 :: Posted by American Biotechnologist on 09-03-2013

Muneesh Tewari

A study published online in Nature Methods today demonstrated that Droplet Digital PCR (ddPCR™) technology can be used to precisely and reproducibly quantify microRNA (miRNA) in plasma and serum across different days, paving the way for further development of miRNA and other nucleic acids as circulating biomarkers.

“In the field of circulating microRNA diagnostics, Droplet Digital PCR enables us to finally perform biomarker studies in which the measurements are directly comparable across days within a laboratory and even among different laboratories,” said Dr. Muneesh Tewari, associate member in the Human Biology Division at the Fred Hutchinson Cancer Research Center and lead author of the study.

Challenges in miRNA Quantification
miRNAs are small regulatory RNA molecules with diverse cellular functions. The human genome may encode over 1,000 miRNAs, which could target about 60 percent of mammalian genes. Because they are abundant in many cell types, exist in highly stable extracellular forms, and may provide direct information about disease processes, they are being actively studied as blood-based biomarkers for cancer and other diseases.

Quantitative real-time PCR (qPCR) has been used for the analytical measurement of miRNAs in blood samples; however, researchers have found that qPCR measurements of miRNAs in serum or plasma display unacceptably high interday variability, undermining the use of miRNAs as reliable blood-based biomarkers. An approach that yields more dependable results has therefore been sought by researchers in this field.

Advantages of ddPCR for miRNA Detection
Digital PCR has many advantages over qPCR including the ability to provide absolute quantification without a standard curve and robustness to variations in PCR efficiency across different samples and assays. These and other advantages are embodied by Bio-Rad Laboratories’ QX100™ Droplet Digital PCR (ddPCR) system, which was introduced in 2011.

“We chose to use Bio-Rad’s QX100 Droplet Digital PCR system because it was the first system on the market that could make digital PCR practical from a cost and throughput standpoint for routine use in the lab,” said Dr. Tewari.

To assess the imprecision introduced by each workflow step — serial dilution, reverse transcription (RT), and the preparation of PCR technical replicates — Dr. Tewari and his team conducted nested analyses of ddPCR vs. qPCR on cDNA from a dilution series of six different synthetic miRNAs in both water and plasma on three separate days. In comparison to qPCR, the researchers found that ddPCR demonstrated greater precision (48–72% lower coefficients of variation) with respect to PCR-specific variation

Next, the team performed a side-by-side comparison of qPCR to ddPCR for detecting miRNAs in serum. They collected sera samples from 20 patients with advanced prostate cancer and 20 age-matched male controls and measured the abundance of miR-141, which has been shown to be a biomarker for advanced prostate cancer. Samples were analyzed by qPCR and ddPCR with individual dilution series replicates prepared on three different days. The team found that ddPCR improved day-to-day reproducibility sevenfold relative to qPCR. It was also able to demonstrate differences between case vs. control specimens with much higher confidence than qPCR (P=0.0036 vs. P=0.1199).

“Droplet Digital PCR will allow us to accurately follow serum microRNA biomarker concentrations over time during a patient’s treatment course, something that has been nearly impossible to achieve with real-time PCR,” Dr. Tewari said.

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.