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Droplet Digital PCR enables measurement of potential cancer survival biomarker

 :: Posted by American Biotechnologist on 01-09-2014

Researchers at Fred Hutchinson Cancer Research Center have used Droplet Digital PCR (ddPCR™) to demonstrate for the first time the quantification of a special class of tumor-attacking immune cell known to improve cancer survival, a subpopulation of T-cells called tumor-infiltrating T-lymphocytes or TILs. The study, led by Dr. Jason Bielas, Associate Member of the Public Health Sciences Division at Fred Hutch, paves the way for further study of the role of TIL quantification in immunotherapy and as a cancer survival predictor.

“Now that we have the sensitivity and ability to reproducibly count TILs in tumors, we may be able to stratify and more effectively treat patients based on tumor TIL count, especially with immunotherapeutics coming to market,” said Dr. Bielas, one of the lead authors of a paper reporting the TIL quantification results in Science Translational Medicine.

Quantifying TILs Using ddPCR

TILs directly attack tumor cells in a variety of cancer types. While the presence and quantity of TILs strongly correlate with increased patient survival, current tests are semiquantitative at best. As a result, TILs cannot be used for clinical decision making.

According to Dr. Bielas, TILs have a “genomic signature that can be digitally exploited.” This signature, which exhibits a vast amount of diversity, determines the genetic identity, or clonality, of the T-cell receptors (TCR) expressed on the surface of each TIL. With the advent of digital PCR – and the generation of tens of thousands of data points produced by Droplet Digital PCR – it is now possible to quantify these signatures, enabling the determination of the number of TILs.

“There’s no way you could do this with any method other than digital PCR because of the numerous primer pairs and probes that we have (45 forward primers, 13 reverse primers, and 30 probes),” said Dr. Bielas. “Digital PCR partitions all the reactions so you can amplify these targets independently of PCR efficiency without any competing side reactions.”

Fred Hutch researchers developed the Droplet Digital PCR-based “QuanTILfy” assay using Bio-Rad Laboratories’ QX100 ddPCR system. They then used QuanTILfy to count TILs, determine their frequency, and develop a grouping system to classify “clonality,” which might be a marker of druggable targets.

Study Results

Fred Hutch researchers performed the QuanTILfy assay on primary tumors from 30 ovarian carcinoma patients with known survival outcomes, ranging from 1 to 122 months. TIL frequency was approximately threefold higher in patients with a survival rate of more than five years compared with patients with survival rates of less than two years. These results show that higher TIL levels correlate positively with patient survival, consistent with the hypothesis that TILs play an active role in suppressing tumor formation.

The researchers also demonstrated that QuanTILfy can be used to accurately and reproducibly characterize T-cell clonality in patients with T-cell acute lymphoblastic leukemia. In each case, they saw a single QuanTILfy assay subgroup, indicative of clonal T-cell expansion. This finding was confirmed by deep sequencing.

The QuanTILfy assay proved to be both sensitive and accurate. In a mixture of human T-cells purified from blood and normal human lung fibroblasts, the assay demonstrated the ability to detect a single TCR rearrangement among 10,000 tumor cells. Importantly, it also demonstrated the ability of ddPCR technology to quantify a large number of markers simultaneously in a single reaction through multiplexing.

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.

Protein Purification Parody

 :: Posted by American Biotechnologist on 03-11-2013

Chromatography Comic

 :: Posted by American Biotechnologist on 12-18-2012

Protein Purification Blues

 :: Posted by American Biotechnologist on 12-10-2012