Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR.

Despite the interest in characterizing genomic variation, the presence of large repeats at the breakpoints hinders the analysis of many structural variants. This is especially problematic for inversions, since there is typically no gain or loss of DNA. Here, we tested novel linkage-based droplet digital PCR (ddPCR) assays to study 20 inversions ranging from 3.1 to 742 kb flanked by inverted repeats (IRs) up to 134 kb long. Of those, we validated 13 inversions predicted by different genome-wide techniques. In addition, we obtained new experimental human population information across 95 African, European, and East Asian individuals for 16 inversions, including four already validated variants without high-throughput genotyping methods. Through comparison with previous data, independent replicates and both inversion breakpoints, we demonstrate that the technique is highly accurate and reproducible. Most studied inversions are widespread across continents, and their frequency is negatively correlated with genetic length. Moreover, all except two show clear signs of being recurrent, and we could better define the factors affecting recurrence levels and estimate the inversion rate across the genome. Finally, the generated genotypes have allowed us to check inversion functional effects, validating gene expression differences reported before for two inversions and finding new candidate associations. Therefore, the developed methodology makes it possible to screen these and other complex genomic variants quickly in a large number of samples for the first time, highlighting the importance of direct genotyping to assess their potential consequences and clinical implications.

International Interlaboratory Digital PCR Study Demonstrating High Reproducibility for the Measurement of a Rare Sequence Variant.

This study tested the claim that digital PCR (dPCR) can offer highly reproducible quantitative measurements in disparate laboratories. Twenty-one laboratories measured four blinded samples containing different quantities of a KRAS fragment encoding G12D, an important genetic marker for guiding therapy of certain cancers. This marker is challenging to quantify reproducibly using quantitative PCR (qPCR) or next generation sequencing (NGS) due to the presence of competing wild type sequences and the need for calibration. Using dPCR, 18 laboratories were able to quantify the G12D marker within 12% of each other in all samples. Three laboratories appeared to measure consistently outlying results; however, proper application of a follow-up analysis recommendation rectified their data. Our findings show that dPCR has demonstrable reproducibility across a large number of laboratories without calibration. This could enable the reproducible application of molecular stratification to guide therapy and, potentially, for molecular diagnostics.

Screening newborn blood spots for 22q11.2 deletion syndrome using multiplex droplet digital PCR.

BACKGROUND: The diagnosis of 22q11 deletion syndrome (22q11DS) is often delayed or missed due to the wide spectrum of clinical involvement ranging from mild to severe, often life-threatening conditions. A delayed diagnosis can lead to life-long health issues that could be ameliorated with early intervention and treatment. Owing to the high impact of 22q11DS on public health, propositions have been made to include 22q11DS in newborn screening panels; however, the method of choice for detecting 22q11DS, fluorescent in situ hybridization, requires specialized equipment and is cumbersome for most laboratories to implement as part of their routine screening. We sought to develop a new genetic screen for 22q11DS that is rapid, cost-effective, and easily used by laboratories currently performing newborn screening. METHODS: We evaluated the accuracy of multiplex droplet digital PCR (ddPCR) in the detection of copy number of 22q11DS by screening samples from 26 patients with 22q11DS blindly intermixed with 1096 blood spot cards from the general population (total n = 1122). RESULTS: Multiplex ddPCR correctly identified all 22q11DS samples and distinguished between 1.5- and 3-Mb deletions, suggesting the approach is sensitive and specific for the detection of 22q11DS. CONCLUSIONS: These data demonstrate the utility of multiplex ddPCR for large-scale population-based studies that screen for 22q11DS. The use of samples from blood spot cards suggests that this approach has promise for newborn screening of 22q11DS, and potentially for other microdeletion syndromes, for which early detection can positively impact clinical outcome for those affected.

Droplet Digital™ PCR quantitation of HER2 expression in FFPE breast cancer samples.

UNLABELLED: The human epidermal growth factor receptor 2 (HER2, also known as erbB2) gene is involved in signal transduction for cell growth and differentiation. It is a cell surface receptor tyrosine kinase and a proto-oncogene. Overexpression of HER2 is of clinical relevance in breast cancer due to its prognostic value correlating elevated expression with worsening clinical outcome. At the same time, HER2 assessment is also of importance because successful anti-tumor treatment with Herceptin® is strongly correlated with HER2 overexpression in the tumor (approximately 30% of all breast tumors overexpress HER2). In a comprehensive national study, Wolff et al. [1] state that "Approximately 20% of current HER2 testing may be inaccurate" which underscores the importance of developing more accurate methods to determine HER2 status. Droplet Digital™ PCR (ddPCR™) has the potential to improve upon HER2 measurements due to its ability to quantitate DNA and RNA targets with high precision and accuracy. Here we present a study which investigates whether ddPCR can be used to assess HER2 transcript levels in formalin-fixed paraffin embedded (FFPE) human breast tumors and whether these ddPCR measurements agree with prior assessments of these same samples by pathologists using immunohistochemistry (IHC) and in some cases fluorescence in situ hybridization (FISH). We also determined the copy number of HER2 in these samples as compared to the CEP17 reference gene. RESULTS: Clinical FFPE samples were successfully studied using ddPCR and compared to results from standard FISH and IHC methodology. The results demonstrate that ddPCR can rank order the samples in complete agreement with the current standard methods and that ddPCR has the added benefit of providing quantitative results, rather than relying on the expert skill of a seasoned pathologist for determination.

Full-scale walk-in containerized lithium-ion battery energy storage system fire test data.

Three installation-level lithium-ion battery (LIB) energy storage system (ESS) tests were conducted to the specifications of the UL 9540A standard test method [1]. Each test included a mocked-up initiating ESS unit rack and two target ESS unit racks installed within a standard size 6.06 m (20 ft) International Organization for Standardization (ISO) container. All tests were conducted with an identical LIB configuration. The initiating unit rack included nine modules (2,430 individual 18650 form factor cells) with a total capacity of 28.9 kWh. The target unit racks were loaded to one-third capacity of the initiating unit with nine partial modules and a total capacity of 9.6 kWh. All cells in the container were charged to 100% state-of-charge and none were electrically connected. Within the initiating mock-up unit, a flexible film heater was wrapped around an individual 18650 form factor cell. This instrumented 18650 cell was heated at a rate of 6°C/min to initiate thermal runaway. Test 1 was a baseline performance test and did not utilize any active fire suppression systems. Test 2 included a Novec 1230 system designed for an 8.3 vol% concentration discharged upon activation of two smoke detectors installed inside the container. Test 3 incorporated a dry pipe water suppression system to provide a uniform 20.8 mm/min (0.5 gpm/ft2) spray density delivered at the top of the ESS unit enclosures. Thermocouples were used to measure the cell temperatures in the initiating unit rack and module surface temperatures for the initiating unit and target unit racks. Thermocouples were located throughout the ISO container to measure gas temperatures and wall temperatures. Schmidt-Boelter heat flux gauges were installed to measure incident heat flux to each of the target unit racks as well as the walls adjacent to the initiating rack. Smoke detectors and smoke obscuration meters were used to identify the presence of smoke and characterize opacity of the smoke in the container. Various laboratory- and industrial-grade sensors were used to characterize the gas composition throughout container.