Automated extraction of DNA from blood and PCR setup using a Tecan Freedom EVO liquid handler for forensic genetic STR typing of reference samples

We have implemented and validated automated protocols for DNA extraction and PCR setup using a Tecan Freedom EVO liquid handler mounted with the Te-MagS magnetic separation device (Tecan, M?nnedorf, Switzerland). The protocols were validated for accredited forensic genetic work according to ISO 17025 using the Qiagen MagAttract DNA Mini M48 kit (Qiagen GmbH, Hilden, Germany) from fresh whole blood and blood from deceased individuals. The workflow was simplified by returning the DNA extracts to the original tubes minimizing the risk of misplacing samples. The tubes that originally contained the samples were washed with MilliQ water before the return of the DNA extracts. The PCR was setup in 96-well microtiter plates. The methods were validated for the kits: AmpF?STR Identifiler, SGM Plus and Yfiler (Applied Biosystems, Foster City, CA), GenePrint FFFL and PowerPlex Y (Promega, Madison, WI). The automated protocols allowed for extraction and addition of PCR master mix of 96 samples within 3.5h. In conclusion, we demonstrated that (1) DNA extraction with magnetic beads and (2) PCR setup for accredited, forensic genetic short tandem repeat typing can be implemented on a simple automated liquid handler leading to the reduction of manual work, and increased quality and throughput.     

Automation of three-dimensional cell culture in arrayed microfluidic devices

The increasing interest in studying the interactions between cells and the extracellular matrix (ECM) has created a need for high throughput low-cost three-dimensional (3D) culture systems. The recent development of tubeless microfluidics via passive pumping provides a high throughput microchannel culture platform compatible with existing high throughput infrastructures (e.g., automated liquid handlers). Here, we build on a previously reported high throughput two-dimensional system to create a robust automated system for 3D culture. Operational controls including temperature and sample handling have been characterized and automated. Human mammary fibroblasts (HMFs) suspended in type I collagen are loaded and cultured in microchannel arrays and used to optimize the system operational parameters. A Peltier cooler maintains the collagen as a liquid at 4 °C during cell seeding, followed by polymerization at 37 °C. Optimization of this platform is discussed (e.g., controlling collagen contraction, increasing cell viability, preventing the removal of microchannel contents), and 3D distribution of HMFs is examined by fluorescent microscopy. Finally, we validate the platform by automating a previously developed 3D breast carcinoma coculture assay. The platform allows more efficient 3D culture experiments and lays the foundation for high throughput studies of cell-ECM interactions.     

Incorporating Automation in Undergraduate Laboratories

Automated techniques and philosophies, which have become increasingly important in modern laboratories, are not traditionally covered in undergraduate chemistry curricula. To this end, we have been incorporating automated sample preparation methods using standard robotic workstations in our undergraduate analytical chemistry laboratory. Using a Benchmate™ II Workstation, an automated method has been developed and implemented for the solid phase extraction of capsaicins from commercial hot pepper sauces prior to liquid chromatographic analysis. This paper reports on pedagogical aspects of incorporating automation into the undergraduate curriculum as well as results obtained for manual and automated extractions conducted by students.     

Automatic liquid handling for life science: a critical review of the current state of the art

Liquid handling plays a pivotal role in life science laboratories. In experiments such as gene sequencing, protein crystallization, antibody testing, and drug screening, liquid biosamples frequently must be transferred between containers of varying sizes and/or dispensed onto substrates of varying types. The sample volumes are usually small, at the micro- or nanoliter level, and the number of transferred samples can be huge when investigating large-scope combinatorial conditions. Under these conditions, liquid handling by hand is tedious, time-consuming, and impractical. Consequently, there is a strong demand for automated liquid-handling methods such as sensor-integrated robotic systems. In this article, we survey the current state of the art in automatic liquid handling, including technologies developed by both industry and research institutions. We focus on methods for dealing with small volumes at high throughput and point out challenges for future advancements.     

Automated purification of dye terminator sequencing reactions: an approach to high-throughput capillary electrophoresis sequencing of large templates

Demands for higher quantity and quality of sequence data during genome sequencing projects have led to a need for completely automated reagent systems designed to isolate, process, and analyze DNA samples. While much attention has been given to methodologies aimed at increasing the throughput of sample preparation and reaction setup, purification of the products of sequencing reactions has received less scrutiny despite the profound influence that purification has on sequence quality. Commonly used and commercially available sequencing reaction cleanup methods are not optimal for purifying sequencing reactions generated from larger templates, including bacterial artificial chromosomes (BACs) and those generated by rolling circle amplification. Theoretically, these methods would not remove the original template since they only exclude small molecules and retain large molecules in the sample. If the large template remains in the purified sample, it could understandably interfere with electrokinetic injection and capillary performance. We demonstrate that the use of MagneSil^® paramagnetic particles (PMPs) to purify ABI PRISM^® BigDye^® sequencing reactions increases the quality and read length of sequences from large templates. The high-quality sequence data obtained by our procedure is independent of the size of template DNA used and can be completely automated on a variety of automated platforms.     

Robotic liquid handling and automation in epigenetics

Automated liquid-handling robots and high-throughput screening (HTS) are widely used in the pharmaceutical industry for the screening of large compound libraries, small molecules for activity against disease-relevant target pathways, or proteins. HTS robots capable of low-volume dispensing reduce assay setup times and provide highly accurate and reproducible dispensing, minimizing variation between sample replicates and eliminating the potential for manual error. Low-volume automated nanoliter dispensers ensure accuracy of pipetting within volume ranges that are difficult to achieve manually. In addition, they have the ability to potentially expand the range of screening conditions from often limited amounts of valuable sample, as well as reduce the usage of expensive reagents. The ability to accurately dispense lower volumes provides the potential to achieve a greater amount of information than could be otherwise achieved using manual dispensing technology. With the emergence of the field of epigenetics, an increasing number of drug discovery companies are beginning to screen compound libraries against a range of epigenetic targets. This review discusses the potential for the use of low-volume liquid handling robots, for molecular biological applications such as quantitative PCR and epigenetics.     

Design of an automated enhanced-throughput platform for functional characterization of positive allosteric modulator-induced leftward shifts in apparent agonist potency in vitro

Prosecution of positive allosteric modulator (PAM) targets demands a specialized assay toolset. Many GPCR or ion channel targets are adaptable to functional assays whereby PAM efficacy can be inferred from left or rightward shifts in the concentration-response curves of orthosteric agonist. The inherent emphasis on throughput and occasional paucity of radioligands for a diverse array of allosteric modulator targets yields a need for an enhanced throughput agonist potency shift assay. Here, we describe a process by which such an assay was automated with robust, reproducible in vitro pharmacology. In direct comparison with a manual CRC shift assay, the enhanced throughput automated platform described here delivered near identical rank orders (r(2) = 0.75) at ~4-fold throughput/assay iteration. Correspondingly, average cycle time/plate decreased from 104 to 72 minutes. We also observed reductions in assay interference associated with compounds exhibiting ago-allosterism, which we attribute to preread compound incubation periods which are more precisely time-constrained under automation control. By leveraging automated laboratory technology, we have achieved meaningful throughput with no sacrifice of precision. Rather than to be target-class specific, the present process was specifically designed to serve as a platform template for a variety of cell-based functional allosteric modulation assays.     

A simple method for validation and verification of pipettes mounted on automated liquid handlers

We have implemented a simple, inexpensive, and fast procedure for validation and verification of the performance of pipettes mounted on automated liquid handlers (ALHs) as necessary for laboratories accredited under ISO 17025. A six- or seven-step serial dilution of OrangeG was prepared in quadruplicates in a flat-bottom 96-well microtiter plate, manually using calibrated pipettes. Each pipette of the liquid handler (1-8) dispensed a selected volume (1-200 μL) of OrangeG eight times into the wells of the microtiter plate. All wells contained a total of 200 μL liquid. The absorbance was read, and the dispensed volume of each pipette was calculated based on a plot of volume and absorbance of a known set of OrangeG dilutions. Finally, the percent inaccuracy (%d) and the imprecision (%CV) of each pipette were calculated. Using predefined acceptance criteria, each pipette was then either approved or failed. Failed pipettes were either repaired or the volume deviation was compensated for by applying a calibration curve in the liquid-handler software. We have implemented the procedure on a Sias Xantus, an MWGt TheONYX, four Tecan Freedom EVO, a Biomek NX Span-8, and four Biomek 3000 robots, and the methods are freely available. In conclusion, we have set up a simple, inexpensive, and fast solution for the continuous validation of ALHs used for accredited work according to the ISO 17025 standard. The method is easy to use for aqueous solutions but requires a spectrophotometer that can read microtiter plates.     

Web浏览器历史数据自动分类取证系统

为提高取证的自动化程度,提出了一种基于页面自动分类技术的浏览器历史数据取证算法,并设计实现了一个原型系统。该系统在获取浏览器历史数据的基础上,自动对其进行特征提取、页面分类。实验结果表明该系统有效提高了取证人员的效率和准确度。     

Dispensing Precision for the SciClone™ Automated Liquid Handling Workstation for 96-Channel Pipetting

One of the first steps in drug discovery involves identification of novel compounds that interfere with therapeutically relevant biological processes.

Identification of ‘lead’ compounds in all therapeutic areas included in a drug discovery program requires labor-intensive evaluation of numerous samples in a battery of therapy targeted biological assays. To accelerate the identification of ‘lead’ compounds, Janssen Research Foundation (JRF) has developed in the past an automated high throughput screening (HTS) based on the unattended operation of a custom Zymark tracked robot system. Automation of enzymatic and cellular assays was realized with this system adapted to the handling of microtiter plates. The microtiter plate technology is the basis of our screening. All compounds within our chemical library are stored and distributed in micronic tube racks or microtiter plates for screening. An efficient in-house developed mainframe based laboratory information management system supported all screening activities. Our experience at JRF has shown that the preparation of test compounds and serial dilutions has been a rate-limiting step in the overall screening process. In order to increase compound throughput, it was necessary both to optimize the robotized assays and to automate the compound supply processes. In HTS applications, one of the primary requirements is highly accurate and precise pipetting of microliter volumes of samples into microplates. The SciClone™ is an automated liquid handling workstation capable of both 96- and 384-channel high precision pipetting. For high throughput applications, the SciClone™ instrumentation is able to pipette a variety of liquid solutions with a high degree of accuracy and precision between microplates (inter-plate variability) and tip-to-tip (intra-plate variability) within a single plate. The focus of this presentation is to review the liquid handling performance of the SciCloneTM system as a multipurpose instrument for pipetting aqueous or organic solutions, and virus suspensions into 96- and 384-well microplates. The capabilities of the system and the resulting benefits for our screening activities will be described.     

Optimizing memory allocation for multistage scheduling including setup times

Mapping linear workflow applications onto a set of homogeneous processors can be optimally solved in polynomial time for the throughput objective with fewer processors than stages. This result holds true even when setup times occur in the execution and homogeneous buffers are available for the storage of intermediate results. In this kind of application, several computation stages are interconnected as a linear application graph, and each stage holds a buffer of limited size where intermediate results are stored and a processor setup time occurs when passing from one stage to another. In this paper, we tackle the problem in which the buffer sizes are not given beforehand and must be fixed before the execution to maximize the throughput within each processor. The goal of this work is to minimize the cost induced by the setup times by allocating buffers that are proportinal in size to each other. We present a closed formula to compute the optimal buffer allocation in the case of nondecreasing setup costs in the linear application. For the case of unsorted setup times, we provide competitive heuristics that are validated via extensive simulation. Three nonscalable brute force algorithms are also provided to compare heuristic approaches to optimal ones for small applications and to evaluate the relevance of our approach.     

Implementation of Fault-Tolerant GridRPC Applications

A task parallel application is implemented with Ninf-G, a GridRPC system. A series of experiments are conducted on the Grid testbed in Asia Pacific for three months. Through tens of long executions, typical fault patterns were collected, and instability of the network throughput was determined to be a major reason of the faults. Several important points are stressed to avoid task throughput decline due to the fault-recovery operations: Timeout minimization for fault detection, background recovery, redundant task assignments, and so on. This study also issues a steer for design of the automated fault-tolerant mechanism in an upper layer of the GridRPC framework.     

Microfiltration isolation of human urinary exosomes for characterization by MS

Purpose: The purpose of this study was to address the hypothesis that small vesicular urinary particles known as exosomes could be selectively microfiltered using low protein‐binding size exclusion filters, thereby simplifying their use in clinical biomarker discovery studies. Experimental design: We characterized a microfiltration approach using a low protein binding, hydrophilized polyvinylidene difluoride membrane to easily and efficiently isolate urinary exosomes from fresh, room temperature or 4°C urine, with a simultaneous depletion of abundant urinary proteins. Using LC‐MS, immunoblot analysis, and electron microscopy methods, we demonstrate this method to isolate intact exosomes and thereby enrich for a low abundant urinary proteome. Results: In comparison to other standard methods of exosome isolation including ultracentrifugation and nanofiltration, we demonstrate equivalent enrichment of the exosome proteome with reduced co‐purification of abundant urinary proteins. Conclusion and clinical relevance: In conclusion, we demonstrate a microfiltration isolation method that preserves the exosome structure, reduces contamination from higher abundant urinary proteins, and can be easily implemented into mass spectrometry analysis for biomarker discovery efforts or incorporation into routine clinical laboratory applications to yield higher sample throughput.     

Simulation as a Tool for Optimizing Automated Laboratory Equipment and Processes?

In the design of complex automated manufacturing equipment simulation technology has increasingly developed into an essential planning tool which is highly used in the field of semiconductor industry. In the field of laboratory technology a similar trend towards automated systems or automated sequencing of systems can be found. This results in the question if the use of simulation can help detecting optimization potentials in the design of laboratory equipment as well as in laboratory processes. By means of a case study out of the field of clean production the elaborated results help to show the benefits of simulation as a planning tool.With the help of simulation a material flow- and throughput optimized equipment design and therefore required equipment control strategies were ascertained in several iterative runs. The system installation, being in its development stage, was represented realistically in a detailed simulation model. The performance of the system in operation was visualized and then analyzed. Thus, bottlenecks could be localized easily and the equipment design and dimensioning could be reviewed.With the selective simulative examination of different optimization approaches the equipment manufacturer was provided with important data for economical- and investment decisions. The use of simulation technology provides a high planning reliability and helps to avoid planning mistakes already in the stage of system development.This advantages could also be used in future developments in the field of laboratory automation.     

Liquid-handling robotic workstations for functional genomics

More and more functional genomics laboratories are willing to invest in robotic workstations due to the higher throughput liquid-handling intensive nature of the work. In this report, the features of robotic workstations important for functional genomics are discussed. Workstations for functional genomics are useful for replication of clone sets, PCR and sequencing set-up and clean-up, hit picking, gel loading, and nucleic acid purification procedures. Workstations not only increase throughput, but also ensure that assay steps are performed consistently, human error- and learning curve-free from run to run. Workstations fit on a laboratory bench and may have several robotic arms in different configurations. Available configurations include grippers and single-, oligo-, or multi-probe heads, with uniform or independent spanning, and liquid-level detection or tracking options. Most hardware and software packages can be designed for integration with other automated devices, building towards a fully automated system.     

Automation of Serological Diagnosis for Herpes B Virus Infections Using Robot-Assisted Integrated Workstations

A robot-assisted automated facility was established within a biosafety level 3 laboratory located on a university campus. A unique integration of a Genesis (TECAN) liquid handler into the SAGIAN Core System (Beckman-Coulter) enabled tube liquid handling and transfer of reagents from tubes to microplates. An automated enzyme immunoassay (ELISA) for detecting herpes B virus antibodies was developed. Repetition experiments of the automated ELISA and comparison to the manual ELISA demonstrated the efficacy and high reproducibility of the automated method. It is anticipated that research and education activities will benefit from the establishment of the automated diagnostic laboratory in an academic environment.     

Web interface-driven cooperative exception handling in ADOME workflow management system

Exception handling in workflow management systems (WFMSs) is a very important problem since it is not possible to specify all possible outcomes and alternatives. Effective reuse of existing exception handlers can greatly help in dealing with workflow exceptions. On the other hand, cooperative support for user-driven computer supported resolution of unexpected exceptions and workflow evolution at run-time is vital for an adaptive WFMS. We have been developing ADOME-WFMS as a comprehensive framework in which the problem of workflow exception handling can be adequately addressed. In this article, we present an adaptive exception manager and its web-based interface for ADOME-WFMS with procedures for supporting the following: reuse of exception handlers, thorough and automated resolution of expected exceptions, effective management of Problem Solving Agents, cooperative exception handling, user-driven computer supported resolution of unexpected exceptions, and workflow evolution.     

Automated in situ hybridisation using Tecan robotics: a kick-start for Functional Genomics

High throughput parallel organic synthesis is now a common practice in discovery chemistry research. Argonaut's modular reaction cassette technology is aimed at parallel synthesis of focused small molecule libraries. The Trident™ automated platform has been designed to perform high throughput organic synthesis using this modular reaction cassette under truly inert conditions and in a robust, reproducible manner. This enables one to generate high quality libraries of small molecules and to access chemistry not normally amenable to automation.Recently we have introduced a Sample Processing Station (SPS), which enhances the upstream and downstream sample handling capabilities of the Trident™ platform. Enhanced software capabilities also provide the Trident™ synthesizer with powerful reaction development and optimization capabilities. Application of this technology in the synthesis of a small library of 1,2-diarylbenzimidazoles using solution-phase chemistry is presented. A number of product purification and isolation protocols using polymer-bound scavengers are discussed.     

Semi-automated sample preparation for plasma proteomics

The discovery of new biomarkers will be an essential step to enhance our ability to better diagnose and treat human disease. The proteomics research community has recently increased its use of human blood (plasma/serum) as a sample source for these discoveries. However, while blood is fairly non-invasive and readily available as a specimen, it is not easily analyzed by liquid chromatography (LC)/mass spectrometry (MS), because of its complexity. Therefore, sample preparation is a crucial step prior to the analysis of blood. This sample preparation must also be standardized in order to gain the most information from these valuable samples and to ensure reproducibility. We have designed a semi-automated and highly parallel procedure for the preparation of human plasma samples. Our process takes the samples through eight successive steps before analysis by LC/MS: (1) receipt, (2) reformatting, (3) filtration, (4) depletion, (5) concentration determination and normalization, (6) digestion, (7) extraction, and (8) randomization, triplication, and lyophilization. These steps utilize a number of different liquid handlers and liquid chromatography (LC) systems. This process enhances our ability to discover new biomarkers from human plasma.     

Engineering An Experimental Platform For High Throughput Reactivity Screening

The genomics revolution coupled to advances in computational power, informatics and robotics is driving drug discovery programs to produce drug candidates faster. This need has resulted in advances in high throughput methods for performing organic chemistry such as combinatorial and parallel synthesis. Yet there has not been a corollary advance in the ability to collect quantitative information on reactions that can be used to produce these drug candidates. This lack of an efficient and robust analytical method has resulted in a significant chemistry bottleneck. This work outlines a set of methods that helps address this chemistry bottleneck by using analytical constructs to detect and quantify reaction outcomes. To accomplish this, an integrated experimentalcheminformatics platform has been developed which couples an experimental design system, automated high throughput parallel and combinatorial synthesis methodology, sample processing, quantitative mass spectroscopy and automated data analysis. This platform is being used to optimize single reactions and the syntheses of whole libraries of compounds, and to generate large databases on specific reaction classes.