American Biotechnologist

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.

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Professor Alec Morley, a pioneer in digital PCR, has chosen Bio-Rad Laboratories’ QX100 Droplet Digital PCR system to develop a diagnostic test for chronic myeloid leukemia (CML).

In 1992, Prof. Morley and his lab at Flinders University and Medical Center in Adelaide, South Australia, published a general method called “limiting dilution PCR” for quantifying PCR targets. As a proof of concept, they used this method for the quantification of marker mutations in acute leukemia. By diluting DNA samples so that only one or two copies per well were present and then amplifying those copies with PCR, Morley’s team was able to detect two copies of leukemic DNA against a background of 160,000 normal genomes.

They subsequently reported in The Lancet that the outcome of acute leukemia can be predicted by measuring the response to treatment using limiting dilution PCR to quantify the leukemic cells at high sensitivity. In later work, the Morley Lab used real-time quantitative PCR (qPCR) to develop a highly sensitive method for isolating and quantifying the chromosomal translocation that is typically associated with CML.

Using droplet digital PCR to diagnose leukemia
Because the translocation point for each patient is different in CML, real-time PCR conditions may vary from patient to patient and may therefore produce different results. The lab has now returned to digital PCR.

“Advancements in digital PCR have given us the ability to overcome variations in real-time PCR amplification efficiency and have also enabled us to do away with using a standard curve,” Prof. Morley said.

Monoquant, a company associated with Flinders University, recently used Bio-Rad’s QX100 system to refine the new clinical test for CML. Not only does the instrument offer high sensitivity, it also removes variability in amplification efficiency that results from using patient-specific PCR primers, a traditional sticking point for the FDA. Monoquant hopes the results from the QX100 system will fast-track the FDA approval process for its test.

“It’s a great feeling knowing that something we helped create is propelling our work today,” Prof. Morley said. “We are hoping that this new test we’re developing will offer a better degree of monitoring and better disease management for patients by tracking the progression or remission of CML.”

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The Ji Research Group at Stanford University, in collaboration with Bio-Rad’s Digital Biology Center, demonstrated that the QX100™ Droplet Digital PCR (ddPCR™) system enables accurate and precise measurements of cancer genome amplifications in archival cancer tissue samples. The results of their study were published online in Translational Medicine.

“The cancer research community is greatly interested in accurately identifying and characterizing genome amplifications and other copy number variations because they are a critical component for understanding and treating human cancers,” said Dr. Hanlee Ji, MD, director of the Ji Research Group. “Using ddPCR, we demonstrated the superiority of this method for copy number analysis of DNA in archival material.”

Certain copy number variations (CNVs) known as genomic amplifications may lead to overexpression of specific oncogenes that drive cancer development. Targeting amplified oncogenes could move us closer to long-sought personalized therapies for cancer treatment.

Detecting amplifications in cancer tissue is technically challenging for two reasons. One is that normal tissue is known to dilute the presence of genomic amplifications. The other is that clinical samples are typically of poor quality because they are traditionally processed as formalin-fixed, paraffin-embedded (FFPE) tissues. This preservation method leads to irreversible damage to the genomic DNA. More sensitive methods of evaluation are thus needed to overcome the poor DNA quality found in FFPE samples and to detect small fractions of tumor DNA.

Bio-Rad’s QX100 system partitions samples into 20,000 droplets for individual PCR reactions. This partitioning reduces background interference and provides a more reliable measurement of a target sequence within a complex sample. As a result, measurement precision is less affected by suboptimal PCR conditions in FFPE samples than when less sensitive methods are used.

Research Findings
Dr. Ji and Digital Biology Center researchers tested the QX100 system to determine its effectiveness in detecting cancer gene amplifications in FFPE cancer tissue samples. They diluted gastric cancer genomic DNA containing an FGFR2 gene amplification in decreasing ratios with DNA from a normal genome sample. Their analysis confirmed the accuracy, reproducibility, and sensitivity of ddPCR in quantifying the FGFR2-amplified gene even when there was a 1,000-fold dilution with normal genome DNA.

The researchers then compared qPCR and ddPCR methods for measuring FGFR2 gene amplifications. They confirmed the presence of an approximately sevenfold amplification of the FGFR2 locus in an FFPE-processed gastric tumor using ddPCR. That amplification was very similar to the value determined using a microarray analysis on a “matched” flash-frozen sample. In contrast, qPCR analysis of the same FFPE tumor sample found a copy number estimate of 35, demonstrating that ddPCR is more accurate than qPCR for determining copy number variants in these FFPE-derived samples.

“We were able to demonstrate that ddPCR provides the sensitivity needed to detect genomic amplifications in archival material,” said Dr. Ji. “Now we can conduct a variety of genomic studies using the QX100 system that could not have been done using traditional methods, such as real-time PCR.”

For further details about the QX100, please visit http://bit.ly/ddPCR_QX100.

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