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Demonstrate performance of a complete automation and reagent workflow for analysis of cfDNA from bodily fluids. The efficient extraction of cfDNA from bodily fluids is a unique challenge due to the very low concentrations of nucleic acid. The extraction process along with library preparation is a laborious workflow, where human variability can lead to increased variability in the downstream analysis. Integrated DNA Technology (IDT) and Beckman Coulter (BC) have teamed up to provide a complete automation and reagent workflow for analysis of low frequency variants in cfDNA. The Apostle MiniMax™ High Efficiency Isolation Kit from BC provides complex, utilized magnetic nanoparticles to effectively capture cfDNA. IDT's library prep kit utilizes novel chemistry to maximize conversion, suppress adapter-dimer formation, reduce chimera rates, and facilitate double strand consensus analysis to call ultra-low frequency variants. Finally, IDT's xGen™ hybrid capture products maintain high library diversity and on-target rates to enable low frequency variant calling regardless of panel size. The Biomek i5 and i7 Hybrid workstations bring out the best performance from these reagents. The Biomek NGS workstations protocol is written with a modular design with safe stop points, making it customizable for each lab. The automated protocol uses Beckman's Demonstrated Method Interface tools which include: Biomek Method Launcher to run the method without going into Biomek software, Method Options Selector to choose the run parameters with a user friendly interface, Guided labware Setup to set the deck with labware based on the run parameters, DeckOptix Final Check software to help reduce deck setup errors. We demonstrate the performance of this complete workflow with a range of plasma inputs (4-8 mL). Using control samples with known variant frequencies, the workflow yields high library complexity, 100% positive predictive value, and reliable detection of Citation Format: Nicole Roseman, Shilpa Parakh, Hsiao-Yun Huang, Kevin Lai, Timothy Barnes, Lyn Lewis, Ushati Das Chakravarty, Anastasia Potts, Alisa Jackson, Amy Yoder, Jessica Sheu, Tzu-Chun Chen. Improved conversion in extraction, library construction, and capture improve sensitivity for variants in liquid biopsy samples [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5863.

Aim The GenDx NGSgo workflow for HLA typing using Next-generation sequencing (NGS) consists of four practical steps: amplification, library preparation, sequencing and data analysis. Out of these four steps the library preparation is the most labor intensive and most interspersed by incubations. To decrease hands-on time and reduce the risk of human error, NGSgo library preparation can be automated from start to finish using a liquid handler workstation. Here we describe the results of the automation of the NGSgo library preparation for Illumina on a Beckman Coulter Biomek 4000 workstation. The workflow was fully automated from amplicon pool to finalized library, making it completely hands-off to accommodate overnight runs and allow the operator to step away from the machine. Methods The Biomek setup included a thermal cycler, 96-well plate magnet, two peltiers devices for cooling 96-well plates and 1.5 ml tubes, a gripper tool, reagent reservoirs and liquid waste disposal. Pipetting was performed by two pipet tools. This setup allowed for flexible processing of 4 to 24 samples per run, with any number of loci. Per run only three boxes of pipetting tips were used for 24 samples. Panels with variable numbers of samples and replicate panels were processed with the Biomek. Subsequently, the pooled libraries were sequenced on a MiSeq system and the resulting data was analyzed with NGSengine software (GenDx). Results MiSeq run parameters were indicative of a high-quality sequencing runs. Read depth, mappability and noise levels of the sequence data presented here showed that the automated workflow resulted in high quality reads and accurate typings. Results from the replicate panel showed a 100% typing match. Conclusions – NGSgo library preparation for 4 to 24 samples can be performed on the Biomek 4000 platform, without manual intervention. – The automated workflow results in high-quality reproducible libraries. – Performing automated NGSgo library preparation as described here reduces hands-on time from 1.5 h to 10 min as compared to manual execution. B. Luiken: 5. Employee; Company/Organization; GenDx. E. Tavkin: 5. Employee; Company/Organization; Beckman Coulter. S. Parakh: 5. Employee; Company/Organization; Beckman Coulter. J. Lu: 5. Employee; Company/Organization; Beckman Coulter. A. Jackson: 5. Employee; Company/Organization; Beckman Coulter. S. van Wageningen: 5. Employee; Company/Organization; GenDx. M. Penning: 5. Employee; Company/Organization; GenDx.

Human carbonic anhydrase II (CA II), a zinc metalloenzyme, was screened against 960 structurally diverse, biologically active small molecules. The assay monitored CA II esterase activity against the substrate 4-nitrophenyl acetate in a format allowing high-throughput screening. The assay proved to be robust and reproducible with a hit rate of approximately 2%. Potential hits were further characterized by determining their IC(50) and K(d) values and tested for nonspecific, promiscuous inhibition. Three known sulfonamide CA inhibitors were identified: acetazolamide, methazolamide, and celecoxib. Other hits were also found, including diuretics and antibiotics not previously identified as CA inhibitors, for example, furosemide and halazone. These results confirm that many sulfonamide drugs have CA inhibitory properties but also that not all sulfonamides are CA inhibitors. Thus many, but not all, sulfonamide drugs appear to interact with CA II and may target other CA isozymes. The screen also yielded several novel classes of nonsulfonamide inhibitors, including merbromin, thioxolone, and tannic acid. Although these compounds may function by some nonspecific mechanism (merbromin and tannic acid), at least 1 (thioxolone) appears to represent a genuine CA inhibitor. Thus, this study yielded a number of potentially new classes of CA inhibitors and preliminary experiments to characterize their mechanism of action.

Aim Holotype HLA is a powerful NGS-based method for HLA genotyping being adopted by HLA laboratories over legacy technologies such as SSO and Sanger SBT for its increased accuracy, resolution, repeatability and high-throughput nature. The elimination of human and random error is a desirable attribute of any diagnostic test and the implementation of a liquid handling system can significantly reduce both sources of error in an NGS workflow. Development of a protocol that streamlines multiple modules of the NGS process was the goal of this multi-center study. Methods The Beckman Coulter Biomek 4000 automated liquid handler is a small footprint solution for pre- and post-PCR processes in NGS-based HLA typing. During development, the protocol was optimized through comparison of multiple data points at measurable stopping points to provide a customized modular liquid handling solution for any HLA lab. Results Technologists and scientists from three sites performed multiple runs of 24 samples for 11 HLA loci using the Holotype HLA 24/11 configuration automated on the Biomek 4000 to generate data on the accuracy, specificity, repeatability and reproducibility of an automated Holotype HLA solution. Conclusion Data obtained from the three centers were analyzed using Omixon HLA Twin and assessed for accuracy, reproducibility, and allele assignment ambiguities to demonstrate that high-throughput NGS-based HLA typing is achievable in under 48 h from gDNA to genotype using the Beckman Coulter Biomek 4000 modular based NGS protocol for Holotype HLA 24/11.

The Human Leukocyte Antigen (HLA) system consists of 21 genes and their various alleles (over 12,500 alleles discovered as of January 2015) that allow the immune system to differentiate between native cells and non-native cells and pathogens. Accurate HLA typing is essential for tissue transplants, as a close match between donor and recipient reduces the risk of transplant rejection. Traditionally, HLA typing has been performed with a laborious series of Sanger sequencing based assays that limit sample throughput and sometimes provide ambiguous results due to high levels of homology within the HLA system. More recently, a number of vendors have developed HLA typing assays that utilize next generation sequencing (NGS) technologies. NGS-based HLA typing provides much higher resolution than Sanger sequencing and provides much higher sample throughput due to the massive number of sequencing reads generated in each run. However, preparing DNA samples for HLA typing utilizing NGS-based assays still requires a fairly complicated workflow that involves amplifying the HLA loci and then converting those amplicons to fragment libraries suitable for sequencing. This process necessarily involves a number of pipetting operations that leave open the possibility of pipetting errors, cross-contamination, and errors in maintaining data integrity. To reduce the complexity of the fragment-library preparation, provide greater laboratory efficiency, and prevent errors, we have developed automation protocols from a variety of reagent vendors for NGS-based HLA typing on the Biomek series of automated liquid handlers. These protocols are highly modular in design, allowing for customized installation across one or several automated liquid handlers depending on the laboratory requirements. These automation protocols also utilize Biomek Method Launcher, a new software package that streamlines method operation and instrument setup, as well as HTML-driven user interfaces that allow the user the opportunity to customize their workflows to provide greater efficiency in the laboratory. The Data Acquisition and Reporting Tool (DART) software can also be included to provide improved data handling and reporting to LIMS systems.

Early evaluation of new drug entities for their potential to cause mitochondrial dysfunction is becoming an important task for drug development. Multi-parametric high-content screening (mp-HCS) of mitochondrial toxicity holds promise as a lead in-vitro strategy for drug testing and safety evaluations. In this study, we have developed a mp-HCS and multi-parametric data analysis scheme for assessing cell responses to induced mitochondrial perturbation. The mp-HCS measurements are shown to be robust enough to allow for quantitative comparison of biological systems with different metabolic pathways simulated by alteration of growth media. Substitution of medium glucose for galactose sensitized cells to drug action and revealed novel response parameters. Each compound was quantitatively characterized according to induced phenotypic changes of cell morphology and functionality measured by fluorescent biomarkers for mitochondrial activity, plasma membrane permeability, and nuclear morphology. Descriptors of drug effects were established by generation of a SCRIT (Specialized-Cell-Response-to-Induced-Toxicity) vector, consisting of normalized statistical measures of each parameter at each dose and growth condition. The dimensionality of SCRIT vectors depends on the number of parameters chosen, which in turn depends on the hypothesis being tested. Specifically, incorporation of three parameters of response into SCRIT vectors enabled clustering of 84 training compounds with known pharmacological and toxicological activities according to the degree of toxicity and mitochondrial involvement. Inclusion of 6 parameters enabled the resolution of more subtle differences between compounds within a common therapeutic class; scoring enabled a ranking of statins in direct agreement with clinical outcomes. Comparison of drug-induced changes required variations in glucose for separation of mitochondrial dysfunction from other types of cytotoxicity. These results also demonstrate that the number of drugs in a training set, the choice of parameters used in analysis, and statistical measures are fundamental for specific hypothesis testing and assessment of quantitative phenotypic differences.