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Digital PCR

Posted in Clinical & Translational, Clinical Diagnostics, Genetics & Innovations in Treatment, Infectious Disease & New Antibiotic Targets, Innovations, Microbiologial genetics, Molecular Genetics & Pharmaceutical, tagged diagnostics, Digital pathology, Digital polymerase chain reaction, medical devices and digital health, PCR on March 24, 2016| Leave a Comment »

Digital PCR

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

GEN Roundup: Digital PCR Advances Partition by Partition  

By Partitioning Samples Digital PCR Is Lowering Detection Limits and Enabling New Applications

GEN  Mar 1, 2016 (Vol. 36, No. 5)       http://www.genengnews.com/gen-articles/gen-roundup-digital-pcr-advances-partition-by-partition/5697

 

• Digital PCR (dPCR) has generated intense interest because it is showing potential as a clinical diagnostics tool. It has already proven to be a useful technique for any application where extreme sensitivity or precise quantification is essential, such as identifying mutations or copy number variations in tumor cells, or examining gene expression at the single-cell level.

  GEN interviewed several dPCR experts to find out specifically why the technique is increasing in popularity. GEN also asked the experts to envision dPCR’s future capabilities.

• GEN: What makes dPCR technology such a superior tool for discovery and diagnostic applications?

  Dr. Shelton The high levels of sensitivity, precision, and reproducibility in DNA and quantification are the major strengths of dPCR. The technology is robust where differences in primer efficiency or the presence of sample-specific PCR inhibitors are trivial to the final quantification through an end-point amplification reaction.

  This provides value to discovery as a trusted tool for validating potential biomarkers and hypotheses generated by broad profiling techniques such as microarrays or next-generation sequencing (NGS). In diagnostics applications, the reproducibility and rapid results of dPCR are critical for labs around the world to quickly compare and share data, especially for ultra-low detection of DNA where variability is high.

  Dr. Garner Digital PCR provides a precise direct counting approach for single molecule detection, thereby providing a straightforward process for the absolute quantification of nucleic acids in samples. One of the biggest advantages of using a system such as ours is its ability to do real-time reads on digital samples. When samples go through PCR, their results are recorded after each cycle.

  These results build a curve, and customers can analyze the data if something went wrong. If it isn’t a clean read—from either a contamination issue, primer-dimer issue, or off-target issue—the curve isn’t the classic PCR curve.

  Dr. Menezes Digital PCR allows absolute quantification of target concentration in samples without the need for standard curves. Obtaining consistent, precise, and absolute quantification with regular qPCR is dependent on standard curve generation and amplification efficiency calculations, which can introduce errors.

  Ms. Hibbs At MilliporeSigma Cell Design Studio, the implementation of dPCR has improved and accelerated the custom cell engineering workflow. After the application of zinc finger nuclease or CRISPR/Cas to create precise genetic modifications in mammalian cell lines, dPCR is used to characterize the expected frequency of homologous recombination and develop a screening strategy based on this expected frequency.

  In some cell lines, homologous recombination occurs at a low frequency. In such cases, dPCR is used to screen cell pools and subsequently identify rare clones having the desired mutation. Digital PCR is also used to accurately and expeditiously measure target gene copy number. It is used this way, for example, in polyploid cell lines.

  Dr. Price The ability to partition genomic samples to a level that enables robust detection of single target molecules is what sets dPCR apart as an innovative tool. Each partition (droplet in the case of the RainDrop System) operates as an individual PCR reaction, allowing for sensitive, reproducible, and precise quantification of nucleic acid molecules without the need for reference standards or endogenous controls.

  Partitioning also provides greater tolerance to PCR inhibitors compared to quantitative PCR (qPCR). In doing so, dPCR can remedy many shortcomings of qPCR by transforming the analog, exponential nature of PCR into a digital signal.

  Mr. Wakida Digital PCR is an ideal technology for detecting rare targets at concentrations of 0.1% or lower. By partitioning samples prior to PCR, exceptionally rare targets can be isolated into individual partitions and amplified.

  Digital PCR produces absolute quantitative results, so in some respects, it is easier than qPCR because it doesn’t require a standard curve, with the added advantages of being highly tolerant of inhibitors and being able to detect more minute fold changes. Absolute quantification is useful for generating reference standards, detecting viral load, and preparing NGS libraries.

• GEN: In what field do you think dPCR will have the greatest impact in the future?

  Dr. Shelton dPCR will have a great impact on precision medicine, especially in liquid biopsy analysis. Cell-free DNA from bodily fluids such as urine or blood plasma can be analyzed quickly and cost-effectively using dPCR. For example, a rapid dPCR test can be performed to determine mutations present in a patient’s tumor and help drive treatment decisions.

  Iterative monitoring of disease states can also be achieved due to the relatively low cost of dPCR, providing faster response times when medications are failing. Gene editing will also be greatly impacted by dPCR. Digital PCR enables refinement and optimization of gene-editing tools and conditions. Digital PCR also serves as quality control of therapeutically modified cells and viral transfer vectors used in gene-therapy efforts.

  Dr. Garner The BioMark™ HD system combines dPCR with simultaneous real-time data for counting and validation. This capability is important for applications such as rare mutation detection, GMO quantitation, and aneuploidy detection—where false positives are intolerable and precision is paramount.

  Any field that requires precision and the ability to detect false positives is a likely target for Fluidigm’s dPCR. Suitable applications include detecting and quantifying cancer-causing genes in patients’ cells, viral RNA that infects bacteria, or fetal DNA in an expectant mother’s plasma.

  Dr. Menezes This technology is particularly useful for samples with low frequency sequences as, for example, those containing rare alleles, low levels of pathogen, or low levels of target gene expression. Teasing out fine differences in copy number variants is another area where this technology delivers more precise data.

  Ms. Hibbs Digital PCR overcomes limitations associated with low-abundance template material and quantification of rare mutations in a high background of wild-type DNA sequence. For this reason, dPCR is poised to have significant impacts in diverse clinical applications such as detection and quantification of rare mutations in liquid biopsies, detection of viral pathogens, and detection of copy number variation and mosaicism.

  Dr. Price Due to its high sensitivity, precision, and absolute quantification, the RainDrop dPCR has the potential to extend the range of nucleic acid analysis beyond the reach of other methods in a number of applications that could lend themselves to diagnostic, prognostic, and predictive applications. The precision of dPCR can be extremely useful in applications that require finer measures of fold change and rare variant detection.

  Digital PCR is suitable for addressing varied research and clinical challenges. These include the early detection of cancer, pathogen/viral detection and quantitation, copy number variation, rare mutation detection, fetal genetic screening, and predicting transplant rejection. Additional applications include gene expression analysis, microRNA analysis, and NGS library quantification.

  Mr. Wakida Digital PCR will have an impact on applications for detecting rare targets by enabling investigators to complement and extend their capabilities beyond traditionally employed methods. One such application is using dPCR to monitor rare targets in peripheral blood, as in liquid biopsies.

  The monitoring of peripheral blood by means of dPCR has been described in several peer-reviewed articles. In one such article, investigators considered the clinical value of Thermo’s QuantStudio™ 3D Digital PCR system for the detection of circulating DNA in metastatic colorectal cancer (Dig Liver Dis. 2015 Oct; 47(10): 884–90).

• GEN: Is there a new technology on the horizon that will increase the speed and/or efficiency of dPCR?

  Dr. Shelton High-throughput sample analysis can be an issue with some dPCR systems. However, Bio-Rad’s Automated Droplet Generator allows labs to process 96 samples simultaneously, a capability that eliminates user-to-user variability and minimizes hands-on time.

  We also want users to get the most information from one sample. Therefore, we are focused on expanding the multiplexing capabilities of our system. In development at Bio-Rad are new technologies that increase the multiplexing capabilities without loss of specificity or accuracy in the downstream workflow.

  Dr. Garner Much of the industry direction seems to be in offering ever-higher resolution, or the ability to run more samples at the same resolution. Thus far, however, customers haven’t found commercial uses for these tools. Also, with increasing resolution and the search for even rarer mutations, the challenge of detecting false positives becomes an even bigger issue.

  Dr. Menezes Use of ZEN™ Double-Quenched Probes by IDT in digital PCR provides increased sensitivity and a lower limit of detection. Due to the second quencher, ZEN probes provide even lower background than traditional single-quenched probes. And this lower background enables increased sensitivity when analyzing samples with low copy number targets, where every droplet matters.

  Ms. Hibbs Quantification relies upon counting the number of positive partitions at the end point of the reaction. Accordingly, precision and resolution can be increased by increasing the number of partitions. We are now capable of analyzing on the order of millions of partitions per run, further extending the lower limit of detection. Additionally, the workflow is amenable to the integration of automation in order to increase throughput and standardize reaction set up.

  Dr. Price Although dPCR is still an emerging technology, there is tremendous interest in its potential clinical diagnostics applications. Enabling adoption of dPCR in the clinical lab requires addressing current gaps in workflow, cost, throughput, and turnaround time.

  Digital PCR technology has the potential for being improved significantly in two dimensions. First, one can address the problem of serially detecting positive versus negative partitions by leveraging lower-cost imaging detection technologies. Alternatively, one may capitalize on the small partition volumes to dramatically reduce the time to perform PCR. Ideally, the future will bring both capabilities to bear.

  Mr. Wakida Compared to qPCR, dPCR currently requires more hands-on time to set up experiments. We are investigating methods to address this.

 

PCR Shows Off Its Clinical Chops   

Thanks to Advances in Genomics, PCR Is Becoming More Common in Clinical Applications

• MaryAnn Labant

• Last May, Roche Molecular Systems announced that its cobas Liat Strep A assay received a CLIA waiver. This clinic-ready assay can detect Streptococcus pyogenes (group A ß-hemolytic streptococcus) DNA in throat swabs by targeting a segment of the S. pyogenes genome.

  Since its invention by Kary B. Mullis in 1985, the polymerase chain reaction (PCR) has become well established, even routine, in research laboratories. And now PCR is becoming more common in clinical applications, thanks to advances in genomics and the evolution of more sensitive quantitative PCR methodologies.

  Examples of clinical applications of PCR include point-of-care (POC) molecular tests for bacterial and viral detection, as well as mutation detection in liquid or tumor biopsies for patient stratification and treatment monitoring.

  Industry leaders recently participated in a CHI conference that was held in San Francisco. This conference—PCR for Molecular Medicine—encompassed research and clinical perspectives and emphasized advanced techniques and tools for effective disease diagnosis.

  To kick off the event, speakers shared their views on POC molecular tests. These tests, the speakers insisted, can provide significant value to healthcare only if they support timely decision making.

  Clinic-ready PCR platforms need to combine speed, ease of use, and accuracy. One such platform, the cobas Liat (“laboratory in a tube”), is manufactured by Roche Molecular Systems. The system employs nucleic acid purification and state-of-art PCR-based assay chemistry to enable POC sites to rapidly provide lab-quality results.

  The cobas Liat Strep A Assay detects Streptococcus pyogenes (group A β-hemolytic streptococcus) DNA by targeting a segment of the S. pyogenes genome. The operator transfers an aliquot of a throat swab sample in Amies medium into a cobas Liat Strep A Assay tube, scans the relevant tube and sample identification barcodes, and then inserts the tube into the analyzer for automated processing and result interpretation. No other operator intervention or interpretation is required. Results are ready in approximately 15 minutes.

  According to Shuqi Chen, Ph.D., vp of Point-of-Care R&D at Roche Molecular Systems, clinical studies of the cobas Liat Strep A Assay demonstrated 97.7% sensitivity when the test was used at CLIA-waived, intended-use sites, such as physicians’ offices. In comparison, rapid antigen tests and diagnostic culture have sensitivities of 70% and 81%, respectively (according to a 2009 study Tanz et al. in Pediatrics).

  The cobas Liat assay preserved the same ease-of-use and rapid turnaround as the rapid antigen tests. It addition, it provided significantly faster turnaround than the lab-based culture test, which can take 24–48 hours.

  A CLIA waiver was announced for the cobas Liat Strep A assay in May 2015. CLIA wavers have been submitted for cobas Liat flu assays, and Roche intends to extend the assay menu.

  POC tests are also moving into field applications. Coyote Bioscience has developed a novel method for one-step gene testing without nucleic acid extraction that can be as fast as 10 minutes from blood sample to result. Their portable devices for molecular diagnostics can be used as genetic biosensors to bring complex clinical testing directly to the patient.

  “Instead of sequential steps, reactions happen in parallel, significantly reducing analysis time. Buffer, enzyme, and temperature profiles are optimized to maximize sensitivity,” explained Sabrina Li, CEO, Coyote Bioscience. “Both RNA and DNA can be analyzed simultaneously from a drop of blood in the same reaction.”

  The first-generation Mini-8 system was used for Ebola detection in Africa where close to 600 samples were tested with 98.8% sensitivity. Recently in China, the Mini-8 system was applied in hospitals and small community clinics for hepatitis B and C and Bunia virus detection. The second-generation InstantGene system is currently being tested internally with clinical samples.

• Digital PCR

  Conventional real-time PCR technology, while suited to the analysis of high-quality clinical samples, may effectively conceal amplification efficiency changes when sample quality is inconsistent. A more effective alternative, Bio-Rad suggests, is its droplet-digital PCR (ddPCR) technology, which can provide absolute quantification of target DNA or RNA, a critical advantage when samples are limited, degraded, or contain PCR inhibitors. The company says that of the half-dozen clinical trials that are using digital PCR, half rely on the Bio-Rad QX200 ddPCR system.

  Personalized cancer care requires ultra-sensitive detection and monitoring of actionable mutations from patient samples. The high sensitivity and precision of droplet-digital PCR (ddPCR) from Bio-Rad Laboratories offers critical advantages when clinical samples are limited, degraded, or contain PCR inhibitors.

  Typically, formalin-fixed and paraffin-embedded (FFPE) tissue samples are processed. FFPE samples work well for immunohistochemistry and protein analysis; however, the formalin fixation can damage nucleic acids and inhibit the PCR reaction. Samples may yield 100 ng of purified nucleic acid, but the actual amplifiable material is less than 1%, or 1 ng, in most cases.

  “Current qPCR technology depends on real-time fluorescence accumulation as the PCR is occurring, which can be an effective means of detecting and quantifying DNA targets in nondegraded samples,” commented Dawne Shelton, Ph.D., staff scientist, Digital Biology Center, Applications Development Group, Bio-Rad Laboratories. “Amplification efficiency is critical; if that amplification efficiency changes because of sample quality it is hidden in the qPCR methodology.”

  “In ddPCR, that is a big red flag,” Dr. Shelton continued. “It changes the format of how the data look immediately so you know the amount of inhibition and which samples are too inhibited to use.”

  Tissue types vary and contain different degrees of fat or other content that can also act as PCR inhibitors. In blood monitoring, the small circulating fragments of DNA are extremely degraded; in addition, food, supplements, or other compounds ingested by the patient may have an inhibitory effect.

  Clinical labs test for these variabilities and clean the blood, but remnant PCR inhibitors can remain. In ddPCR, a single template is partitioned into a droplet. If the droplet contains a good template, it produces a signal; otherwise, it does not—a simple yes or no answer.

  “Even if there is no PCR inhibition, most clinical samples yield very small amounts of nucleic acid,” Dr. Shelton added. “To make a secure decision using qPCR is difficult because you are in a gray zone at the very end of its linear range. ddPCR operates best with small sample amounts and provides good statistics for confidence in your results.”

  Currently, at least a half dozen clinical trials worldwide are using digital PCR, half of them are using the Bio-Rad QX200 Droplet Digital PCR system. Examples of studies include examining BCR-ABL monitoring in patients with chronic myelogenous leukemia (CML); identifying activating mutations in epidermal growth factor receptor (EGFR) for first-line therapy of new drugs in patients with lung cancer; and the monitoring of resistance mutations such as EFGR T790M in patients with non-small cell lung cancer (NSCLC).

  Clovis Oncology used a technology called BEAMing (Beads, Emulsions, Amplification, and Magnetics), a type of digital PCR for blood-based molecular testing, to perform EGFR testing on almost 250 patients in clinical trials. In BEAMing, individual EGFR gene copies from plasma are separated into individual water droplets in a water-in-oil emulsion. The gene copies are then amplified by PCR on magnetic beads.

  The beads are counted by flow cytometry using fluorescently labeled probes to distinguish mutant beads from wild-type. Because each bead can be traced to an individual EGFR molecule in the patient’s plasma, the method is highly quantitative.

  “BEAMing is particularly well-suited for the detection of known mutations in circulating tumor DNA. In this circumstance, the mutation of interest often occurs at low levels, perhaps only 1–2 copies per milliliter or even less, and in a high background of wild-type DNA that comes from normal tissue. BEAMing can detect one mutant molecule in a background of 5,000 wild-type molecules in clinical samples,” stated Andrew Allen, MRCP, Ph.D., chief medical officer, Clovis Oncology.

  In the studies, the EGFR-resistance mutation T790M could be identified in plasma 81% of the time that it was seen in the matched patient tumor biopsy. Additionally, about 10% of patients in the study had a T790M mutation in plasma that was not identified in tissue, presumably because of tumor heterogeneity. Another 5–10% of the patients did not provide an EGFR result, usually because the tissue biopsy had no tumor cells.

  In aggregate, these results suggest that plasma EGFR testing can be a valuable complement to tumor testing in the clinical management of NSCLC patients, and can provide an alternative when a biopsy is not available. Tumor biopsies may provide only limited tissue, if in fact any tissue is available, for molecular analysis. Also, mutations may be missed due to tumor heterogeneity. These mutations may be captured by sampling the blood, which acts as a reservoir for mutations from all parts of a patient’s tumor burden.

  In the last few years, a panoply of clinically actionable driver mutations have been identified for NSCLC, including mutations in EGFR, BRAF, and HER2, as well as ALK, ROS, and RET rearrangements. These driver mutations will migrate NSCLC molecular diagnostic testing in the next few years toward panel testing of relevant cancer genes using various digital technologies, including next-generation sequencing.

   

PCR Has a History of Amplifying Its Game

A GEN 35th Anniversary Retrospective

• Jeffrey S. Buguliskis, Ph.D.

PCR is a fast and inexpensive technique used to amplify segments of DNA that continues to adapt and evolve for the demanding needs of molecular biology researchers. This diagram shows the basic principles of PCR amplification. [NHGRI]

• The influence that the polymerase chain reaction (PCR) has had on modern molecular biology is nothing short of remarkable. This technique, which is akin to molecular photocopying, has been the centerpiece of everything from the OJ Simpson Trial to the completion of the Human Genome Project. Clinical laboratories use this DNA amplification method for infectious disease testing and tissue typing in organ transplantation. Most recently, with the explosion of the molecular diagnostics field and meteoric rise in the use of next-generation sequencing platforms, PCR has enhanced its standing as an essential pillar of genomic science.

  Let’s open the door to the past and take a look back around 35 years ago when GEN started reporting on the relatively new disciplines of genetic engineering and molecular biology. At that time, GEN was among the first to hear the buzz surrounding a new method to synthesize and amplify DNA in the laboratory. In reviewing the fascinating history of PCR, we will see how the molecular diagnostics field took shape and where it could be headed in the future.

• Some Like It Hot

  The biological sciences rarely advance within a vacuum—rather they rely on previous discoveries to promote directly or indirectly our understanding. The contributions made by scientists in the field of molecular biology that contributed to the functional pieces of PCR were numerous and spread out over more than two decades.

  It began with H. Gobind Khorana’s advances in understanding the genetic code, leading to the use of synthetic DNA oligonucleotides, continued through Kjell Kleepe’s 1971 vision of a two-primer system for replicating DNA segments, to Fredrick Sanger’s method of DNA sequencing—a process that would win him the Nobel prize in 1980—which utilized DNA oligo primers, nucleotide precursors, and a DNA synthesis enzyme.

  All of the previous discoveries were essential to PCR’s birth, yet it would be an egregious mistake to begin a retrospective on PCR and not discuss the enzyme upon which the entire reaction hinges upon—DNA polymerase. In 1956, Nobel laureate Arthur Kornberg and his colleagues discovered DNA polymerase (Pol I), in Escherichia coli. Moreover, the researchers described the fundamental process by which the polymerase enzyme copies the base sequence of a DNA template strand. However, it would take biologists another 20 years to discover a version of DNA polymerase that was stable enough for use for any meaningful laboratory purposes.

  That discovery came in 1976 when a team of researchers from the University of Cincinnati described the activity of a DNA polymerase (Taq) they isolated from the extreme thermophile bacteria, Thermus aquaticus, which lives in hot springs and hydrothermal vents. The fact that this enzyme could withstand typical protein-denaturing temperatures and function optimally around 75–80°C fortuitously set the stage for the development of PCR.

  By 1983, all of the ingredients to bake the molecular cake were sitting in the biological cupboard waiting to be assembled in the proper order. At that time, Nobel laureate Kary Mullis was working as a scientist for the Cetus Corporation trying to perfect oligonucleotide synthesis. Mullis stumbled upon the idea of amplifying segments of DNA using multiple rounds of replication and the two primer system—essentially modifying and expanding upon Sanger’s sequencing reaction. Mullis discovered that the temperatures for each step (melting, annealing, and extension) in the reaction would need to be painstakingly controlled by hand. In addition, he realized that since the reactions were using a non-thermostable DNA polymerase, fresh enzyme would need to be “spiked in” after each successive cycle.

  Mullis’ hard work and persistence paid off as the reaction was successful at amplifying a particular segment of DNA that was flanked by two opposing nucleotide primer molecules. Two years later, the Cetus team presented their work at the annual meeting of the American Society for Human Genetics, and the first mention of the method was published in Science that same year; however, that article did not go into detail about the specifics of the newly developed PCR method—a paper that would be rejected by roughly 15 journals and would not be published until 1987.

  Although scientists were a bit slow on the uptake for the new method, the researchers at Cetus were developing ways to improve upon the original assay. In 1986, the scientists substituted the original heat-liable DNA polymerase for the temperature-resistant Taq polymerase, removing the need to spike in enzyme and dramatically reducing errors while increasing sensitivity. A year later, PerkinElmer launched their creation of a thermal cycler, allowing scientists to regulate the heating and cooling parts of the PCR reaction with greater efficiency.

  Extremely soon after the introduction of Taq and the launch of the thermal cycler, the PCR reaction exploded exponentially among research laboratories and not only vaulted molecular biology to the pinnacle of researcher interests, it also launched a molecular diagnostics revolution that continues today and shows no signs of slowing down.

• Molecular Workhorse

  In the years since PCR first burst onto the scene, there have been a number of significant advancements to the technique that have widely improved the overall method. For example, in 1991, a new DNA polymerase from the hyperthermophilic bacteria Pyrococcus furiosus, or Pfu, was introduced as a high-fidelity alternative enzyme to Taq. Unlike Taq polymerase, Pfu has built in 3′ to 5′ exonuclease proofreading activity, which allows the enzyme to correct nucleotide incorporation errors on the fly—dramatically increasing base specificity, albeit at a reduced rate of amplification versusTaq.

  In 1995, two advancements were introduced to PCR users. The first, called antibody “hot-start” PCR, utilized an immunoglobulin molecule that is directed to the DNA polymerase and inhibits its activity until the first 95°C melt stage, denaturing the antibody and allowing the polymerase to become active. Although this process was effective in increasing the specificity of the PCR reaction, many researchers found that the technique was time consuming and often caused cross-contamination of samples.

  The second innovation introduced that year began another revolution for molecular biology and the PCR method. Real-time PCR, or quantitative PCR (qPCR), allowed researchers to quantitatively create DNA templates for PCR amplification from RNA transcripts through the use of the reverse-transcriptase enzyme and specifically incorporated fluorescent reporter dyes. The technique is still widely used by researchers to monitor gene expression extremely accurately. Over the past 20 years, many companies have spent many R&D dollars to create more accurate, higher throughput, and simple qPCR machines to meet researcher demands.

  With the advent of next-generation sequencing techniques—and the rise of techniques that started commanding the attention of more and more researchers—PCR machines and methods needed to evolve and modernize to keep pace. PCR remained the lynchpin in almost all the next-generation sequencing reactions that came along, but the traditional technique wasn’t nearly as precise as required.

  Digital PCR (dPCR) was introduced as a refinement of the conventional method, with the first real commercial system emerging around 2006. dPCR can be used to quantify directly and clonally amplify DNA or RNA.

  The apparatus carries out a single reaction within a sample. The sample, however, is separated into a large number of partitions. Moreover, the reaction is performed in each partition individually—allowing a more reliable measurement of nucleic acid content. Researchers often use this method for studying gene-sequence variations, such as copy number variants (CNV), point mutations, rare-sequence detection, and microRNA analysis, as well as for routine amplification of next-generation sequencing samples.

• Future of PCR: Better, Faster, Stronger!

  It is almost impossible to envision a future laboratory setting that wouldn’t utilize PCR in some fashion, especially due to the heavy reliance of next-generation sequencing techniques for accurate PCR samples and at the very least using the method as a simple amplification tool for creating DNA fragments of interest.

  Yet there is at least one new next-generation sequencing technique that can identify native DNA sequences without an amplification step—nanopore sequencing. Although this technique has performed well in many preliminary trials, it is in its relative infancy. It will probably undergo additional development lasting several years before it approaches large-scale adoption by researchers. Even then, PCR has become so engrained into daily laboratory life that to try to phase out the technique would be like asking molecular biologists to give up their pipettes or restriction enzymes.

  Most PCR equipment manufacturers continue to seek ways to improve the speed and sensitivity of their thermal cyclers, while biologists continue to look toward ways to genetically engineer better DNA polymerase molecules with even greater fidelity than their naturally occurring cousins. Whatever the new advancements are, and wherever they lead the life sciences field, you can count on us at GEN to continue to provide our readers with detailed information for another 35 years … at least!

   

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Twitter is Becoming a Powerful Tool in Science and Medicine

Posted in Academic Publishing, Clinical Diagnostics, Computational Biology/Systems and Bioinformatics, Conference Coverage with Social Media, Curation, Curation methodology, Digital HealthCare – biotech & internet joint ventures, Drug Toxicity, Evolutionary cognition, FDA, FDA Regulatory Affairs, Global Partnering & Biotech Investment, Health Economics and Outcomes Research, HealthCare IT, Interviews with Scientific Leaders, Investment in Technological Breakthrough, Personal Health Applications: Tech Innovations serves HealhCare, Pharmaceutical Analytics, Pharmaceutical Discovery, Pharmacologic toxicities, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Scientific & Biotech Conferences: Press Coverage, tagged #mobilehealth, #science2_0, @science 2_0, Adverse event, Aviva Lev-Ari, Cancer - General, cancer patient management, Clinical trial, Conditions and Diseases, Conference Coverage with Social Media, Content Curation, Curation methodology, Food and Drug Administration, health, https://twitter.com/#!/pharma_BI, medical devices and digital health, medicine, mobile applications, patient outcomes, patient-interaction, Pharmacovigilance, research, science, Social media, Social network, Twitter on November 6, 2014| 1 Comment »

Twitter is Becoming a Powerful Tool in Science and Medicine

 Curator: Stephen J. Williams, Ph.D.

Updated 4/2016

Twitter.com

A recent Science article (Who are the science stars of Twitter?; Sept. 19, 2014) reported the top 50 scientists followed on Twitter. However, the article tended to focus on the use of Twitter as a means to develop popularity, a sort of “Science Kardashian” as they coined it. So the writers at Science developed a “Kardashian Index (K-Index) to determine scientists following and popularity on Twitter.

Now as much buzz Kim Kardashian or a Perez Hilton get on social media, their purpose is solely for entertainment and publicity purposes, the Science sort of fell flat in that it focused mainly on the use of Twitter as a metric for either promotional or public outreach purposes. A notable scientist was mentioned in the article, using Twitter feed to gauge the receptiveness of his presentation. In addition, relying on Twitter for effective public discourse of science is problematic as:

• Twitter feeds are rapidly updated and older feeds quickly get buried within the “Twittersphere” = LIMITED EXPOSURE TIMEFRAME
• Short feeds may not provide the access to appropriate and understandable scientific information (The Science Communication Trap) which is explained in The Art of Communicating Science: traps, tips and tasks for the modern-day scientist. “The challenge of clearly communicating the intended scientific message to the public is not insurmountable but requires an understanding of what works and what does not work.” – from Heidi Roop, G.-Martinez-Mendez and K. Mills

However, as highlighted below, Twitter, and other social media platforms are being used in creative ways to enhance the research, medical, and bio investment collaborative, beyond a simple news-feed.  And the power of Twitter can be attributed to two simple features

1. Ability to organize – through use of the hashtag (#) and handle (@), Twitter assists in the very important task of organizing, indexing, and ANNOTATING content and conversations. A very great article on Why the Hashtag in Probably the Most Powerful Tool on Twitter by Vanessa Doctor explains how hashtags and # search may be as popular as standard web-based browser search. Thorough annotation is crucial for any curation process, which are usually in the form of database tags or keywords. The use of # and @ allows curators to quickly find, index and relate disparate databases to link annotated information together. The discipline of scientific curation requires annotation to assist in the digital preservation, organization, indexing, and access of data and scientific & medical literature. For a description of scientific curation methodologies please see the following links:

Please read the following articles on CURATION

The Methodology of Curation for Scientific Research Findings

Power of Analogy: Curation in Music, Music Critique as a Curation and Curation of Medical Research Findings – A Comparison

Science and Curation: The New Practice of Web 2.0

2. Information Analytics –

Multiple analytic software packages have been made available to analyze information surrounding Twitter feeds, including Twitter feeds from #chat channels one can set up to cover a meeting, product launch etc.. Some of these tools include:

Twitter Analytics – measures metrics surrounding Tweets including retweets, impressions, engagement, follow rate, …

Twitter Analytics – Hashtags.org – determine most impactful # for your Tweets For example, meeting coverage of bioinvestment conferences or startup presentations using #startup generates automatic retweeting by Startup tweetbot @StartupTweetSF.

 

3. Tweet Sentiment Analytics

Examples of Twitter Use

A. Scientific Meeting Coverage

• The 2013 Society of Teachers of Family Medicine Annual Spring Conference. integrated social media with meeting proceedings to enhance conference engagement

In a paper entitled Twitter Use at a Family Medicine Conference: Analyzing #STFM13 authors Ranit Mishori, MD, Frendan Levy, MD, and Benjamin Donvan analyzed the public tweets from the 2013 Society of Teachers of Family Medicine (STFM) conference bearing the meeting-specific hashtag #STFM13. Thirteen percent of conference attendees (181 users) used the #STFM13 to share their thoughts on the meeting (1,818 total tweets) showing a desire for social media interaction at conferences but suggesting growth potential in this area. As we have also seen, the heaviest volume of conference-tweets originated from a small number of Twitter users however most tweets were related to session content.

However, as the authors note, although it is easy to measure common metrics such as number of tweets and retweets, determining quality of engagement from tweets would be important for gauging the value of Twitter-based social-media coverage of medical conferences.

Thea authors compared their results with similar analytics generated by the HealthCare Hashtag Project, a project and database of medically-related hashtag use, coordinated and maintained by the company Symplur.  Symplur’s database includes medical and scientific conference Twitter coverage but also Twitter usuage related to patient care. In this case the database was used to compare meeting tweets and hashtag use with the 2012 STFM conference.

These are some of the published journal articles that have employed Symplur (www.symplur.com) data in their research of Twitter usage in medical conferences.

• The impact of social media on a major international emergency medicine conference.
  Neill, A., Cronin, J. J., Brannigan, D., O’Sullivan, R., & Cadogan, M. (2013). The impact of social media on a major international emergency medicine conference. Emergency Medicine Journal, emermed-2012. Chicago
• Social media in radiology: early trends in Twitter microblogging at radiology’s largest international meeting.
  Hawkins, C. M., Duszak, R., & Rawson, J. V. (2014). Social Media in Radiology: Early Trends in Twitter Microblogging at Radiology’s Largest International Meeting. Journal of the American College of Radiology, 11(4), 387-390.
• International Urology Journal Club via Twitter: 12-Month Experience.
  Thangasamy, I. A., Leveridge, M., Davies, B. J., Finelli, A., Stork, B., & Woo, H. H. (2014). International Urology Journal Club via Twitter: 12-Month Experience. European urology.
• Use of social media in urology: data from the American Urological Association (AUA).
  Loeb, S., Bayne, C. E., Frey, C., Davies, B. J., Averch, T. D., Woo, H. H., … & Eggener, S. E. (2014). Use of social media in urology: data from the American Urological Association (AUA). BJU international, 113(6), 993-998. Chicago.
• Social media: A tool to spread information: A case study analysis of Twitter conversation at the Cardiac Society of Australia & New Zealand 61st Annual Scientific Meeting 2013.
  Ferguson, C., Inglis, S. C., Newton, P. J., Cripps, P. J., Macdonald, P. S., & Davidson, P. M. (2014). Social media: A tool to spread information: A case study analysis of Twitter conversation at the Cardiac Society of Australia & New Zealand 61st Annual Scientific Meeting 2013. Collegian.

• Here, at Leaders in Pharmaceutical Business Intelligence (LBPI) we have integrated our web site, Twitter handle (@pharma_BI), and meeting specific hashtags, with a unique methodology, to monitor and measure meeting participant engagement for various international meetings (please see our Press Coverage section of our site for more information). These meetings included the 2nd Annual Sachs Associates Cancer BioInvestment & Partnering Forum and the 14^th Annual Sachs Associates Global Forum.

B. Twitter Usage for Patient Care and Engagement

Although the desire of patients to use and interact with their physicians over social media is increasing, along with increasing health-related social media platforms and applications, there are certain obstacles to patient-health provider social media interaction, including lack of regulatory framework as well as database and security issues. Some of the successes and issues of social media and healthcare are discussed in the post Can Mobile Health Apps Improve Oral-Chemotherapy Adherence? The Benefit of Gamification.

However there is also a concern if social media truly engages the patient and improves patient education. In a study of Twitter communications by breast cancer patients Tweeting about breast cancer, authors noticed Tweeting was a singular event. The majority of tweets did not promote any specific preventive behavior. The authors concluded “Twitter is being used mostly as a one-way communication tool.” (Using Twitter for breast cancer prevention: an analysis of breast cancer awareness month. Thackeray R¹, Burton SH, Giraud-Carrier C, Rollins S, Draper CR. BMC Cancer. 2013;13:508).

In addition a new poll by Harris Interactive and HealthDay shows one third of patients want some mobile interaction with their physicians.

Some papers cited in Symplur’s HealthCare Hashtag Project database on patient use of Twitter include:

• e-Patients in Twitter Hashtag Communities.
  Harmel M, Young K. e-Patients in twitter hashtag communities . J Participat Med. 2013 Jan 16; 5:e22
• Nurses and Twitter: The good, the bad, and the reluctant.
  Wilson, R., Ranse, J., Cashin, A., & McNamara, P. (2013). Nurses and Twitter: The good, the bad, and the reluctant. Collegian. Chicago.
• Using social media for continuous professional development.
  Moorley, C., & Chinn, T. (2014). Using social media for continuous professional development. Journal of advanced nursing.
• Networking opportunities for learning disability nurses.
  Abdulla, S., Marsden, D., Wilson, S., & Parker, M. (2013). Networking opportunities for learning disability nurses: Samuel Abdulla and colleagues explain why social media offer professionals new opportunities for information sharing, discussion and peer support. Learning Disability Practice, 16(5), 30-32.
• Using Twitter for professional knowledge.
  Kraft, M. A. (2013). Using Twitter for professional knowledge. Journal of the European Association for Health Information and Libraries, 9(4), 10.
• A Study on the Influence of Semantics on the Analysis of Micro-blog Tags in the Medical Domain.
  Vicient, C., & Moreno, A. (2013). A Study on the Influence of Semantics on the Analysis of Micro-blog Tags in the Medical Domain. In Availability, Reliability, and Security in Information Systems and HCI (pp. 446-459). Springer Berlin Heidelberg.
• Twitter for Public Health: An Open-source Data Solution.
  Nghiema, S., Mehtab, P., & Taoc, L. Twitter for Public Health: An Open-source Data Solution. Chicago.

C. Twitter Use in Pharmacovigilance to Monitor Adverse Events

Pharmacovigilance is the systematic detection, reporting, collecting, and monitoring of adverse events pre- and post-market of a therapeutic intervention (drug, device, modality e.g.). In a Cutting Edge Information Study, 56% of pharma companies databases are an adverse event channel and more companies are turning to social media to track adverse events (in Pharmacovigilance Teams Turn to Technology for Adverse Event Reporting Needs). In addition there have been many reports (see Digital Drug Safety Surveillance: Monitoring Pharmaceutical Products in Twitter) that show patients are frequently tweeting about their adverse events.

There have been concerns with using Twitter and social media to monitor for adverse events. For example FDA funded a study where a team of researchers from Harvard Medical School and other academic centers examined more than 60,000 tweets, of which 4,401 were manually categorized as resembling adverse events and compared with the FDA pharmacovigilance databases. Problems associated with such social media strategy were inability to obtain extra, needed information from patients and difficulty in separating the relevant Tweets from irrelevant chatter.  The UK has launched a similar program called WEB-RADR to determine if monitoring #drug_reaction could be useful for monitoring adverse events. Many researchers have found the adverse-event related tweets “noisy” due to varied language but had noticed many people do understand some principles of causation including when adverse event subsides after discontinuing the drug.

However Dr. Clark Freifeld, Ph.D., from Boston University and founder of the startup Epidemico, feels his company has the algorithms that can separate out the true adverse events from the junk. According to their web site, their algorithm has high accuracy when compared to the FDA database. Dr. Freifeld admits that Twitter use for pharmacovigilance purposes is probably a starting point for further follow-up, as each patient needs to fill out the four-page forms required for data entry into the FDA database.

D. Use of Twitter in Big Data Analytics

Published on Aug 28, 2012

http://blogs.ischool.berkeley.edu/i29…

Course: Information 290. Analyzing Big Data with Twitter
School of Information
UC Berkeley

Lecture 1: August 23, 2012

Course description:
How to store, process, analyze and make sense of Big Data is of increasing interest and importance to technology companies, a wide range of industries, and academic institutions. In this course, UC Berkeley professors and Twitter engineers will lecture on the most cutting-edge algorithms and software tools for data analytics as applied to Twitter microblog data. Topics will include applied natural language processing algorithms such as sentiment analysis, large scale anomaly detection, real-time search, information diffusion and outbreak detection, trend detection in social streams, recommendation algorithms, and advanced frameworks for distributed computing. Social science perspectives on analyzing social media will also be covered.

This is a hands-on project course in which students are expected to form teams to complete intensive programming and analytics projects using the real-world example of Twitter data and code bases. Engineers from Twitter will help advise student projects, and students will have the option of presenting their final project presentations to an audience of engineers at the headquarters of Twitter in San Francisco (in addition to on campus). Project topics include building on existing infrastructure tools, building Twitter apps, and analyzing Twitter data. Access to data will be provided.

Other posts on this site on USE OF SOCIAL MEDIA AND TWITTER IN HEALTHCARE and Conference Coverage include:

Methodology for Conference Coverage using Social Media: 2014 MassBio Annual Meeting 4/3 – 4/4 2014, Royal Sonesta Hotel, Cambridge, MA

Strategy for Event Joint Promotion: 14th ANNUAL BIOTECH IN EUROPE FORUM For Global Partnering & Investment 9/30 – 10/1/2014 • Congress Center Basel – SACHS Associates, London

REAL TIME Cancer Conference Coverage: A Novel Methodology for Authentic Reporting on Presentations and Discussions launched via Twitter.com @ The 2nd ANNUAL Sachs Cancer Bio Partnering & Investment Forum in Drug Development, 19th March 2014 • New York Academy of Sciences • USA

PCCI’s 7th Annual Roundtable “Crowdfunding for Life Sciences: A Bridge Over Troubled Waters?” May 12 2014 Embassy Suites Hotel, Chesterbrook PA 6:00-9:30 PM

CRISPR-Cas9 Discovery and Development of Programmable Genome Engineering – Gabbay Award Lectures in Biotechnology and Medicine – Hosted by Rosenstiel Basic Medical Sciences Research Center, 10/27/14 3:30PM Brandeis University, Gerstenzang 121

Tweeting on 14th ANNUAL BIOTECH IN EUROPE FORUM For Global Partnering & Investment 9/30 – 10/1/2014 • Congress Center Basel – SACHS Associates, London

http://pharmaceuticalintelligence.com/press-coverage/

Statistical Analysis of Tweet Feeds from the 14th ANNUAL BIOTECH IN EUROPE FORUM For Global Partnering & Investment 9/30 – 10/1/2014 • Congress Center Basel – SACHS Associates, London

1st Pitch Life Science- Philadelphia- What VCs Really Think of your Pitch

What VCs Think about Your Pitch? Panel Summary of 1st Pitch Life Science Philly

How Social Media, Mobile Are Playing a Bigger Part in Healthcare

Can Mobile Health Apps Improve Oral-Chemotherapy Adherence? The Benefit of Gamification.

Medical Applications and FDA regulation of Sensor-enabled Mobile Devices: Apple and the Digital Health Devices Market

E-Medical Records Get A Mobile, Open-Sourced Overhaul By White House Health Design Challenge Winners

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