Loader Lite: a new software tool for the ABI PRISM 3700 DNA sequencer

Here we describe the development of a novel software tool entitled Loader Lite that generates plate records or sample sheetsfor the ABI PRISMs 3700 DNA sequencer. The major advantage of this program is that it enables the ongoing operation of sequencing instruments without reference to external network(s). The autonomous operation of sequencing instruments is critical if sample throughput is to be maintained during periods of network outage. Loader Lite employs a deliberate strategy of inputting anonymous tray barcodes at run time. After sequencing, the barcodes are reconciled with relevant project details by reference to a database. This software takes advantage of barcode scanning technology by creating plate records directly on the local computer, serving an individual sequencer, immediately before importing and linking. This real-time synthesis of the plate records at the point of loading all but eliminates loading errors. Loader Lite is user-friendly, fully configurable, and permits the running of partial or full 384-well sample trays, using any standard combinations of run modules, dye sets, mobility files, analysis modules, etc. The 96-well format is not supported; however, this capability will appear in subsequent versions that are currently under development. This application is designed as an added value, adjunct program to the regular ABI PRISM 3700 Data Collection software. We have successfully used Loader Lite over the past six months to load approximately 7 million sequencing reactions and believe its utility and functionality will prove to be attractive to the wider sequencing community.     

A computerized system for scoring primate behavior using standard keyboards

A scoring system has been developed for primate behavior which uses standard keyboards and minicomputers or microcomputers. The mnemonic, alphanumeric code used is easily learned, highly flexible, and can be recorded in longhand for later entry into a computer if a keyboard is not immediately available. The software consists of two programs, both of which can be written in BASIC. SCORE is used for data acquisition and appends the test time to each behavioral sequence. DATSUM decodes and summarizes the test data using table-driven logic. The minimum hardware required is a 16K microcomputer, an alphanumeric keyboard, a display, and cassette storage.     

Laboratory voice data entry system

We have assembled a system using a personal computer workstation equipped with standard office software, an audio system, speech recognition software and an inexpensive radio-based wireless microphone that permits laboratory workers to enter or modify data while performing other work. Speech recognition permits users to enter data while their hands are holding equipment or they are otherwise unable to operate a keyboard. The wireless microphone allows unencumbered movement around the laboratory without a "tether" that might interfere with equipment or experimental procedures. To evaluate the potential of voice data entry in a laboratory environment, we developed a prototype relational database that records the disposal of radionuclides and/or hazardous chemicals. Current regulations in our laboratory require that each such item being discarded must be inventoried and documents must be prepared that summarize the contents of each container used for disposal. Using voice commands, the user enters items into the database as each is discarded. Subsequently, the program prepares the required documentation.     

Time, a quality-control parameter in flow cytometry

Time has been used as a quality-control parameter in our flow cytometer. This parameter was automatically incorporated into the list-mode data base by hooking the computer clock directly into the acquisition logic. Quality control can then be checked by "replaying" fluorescence or light scatter data vs. time and any instrumental drift can be observed and degraded data can be excised. The technique is illustrated with a number of DNA data sets from a tissue culture cell line in which artificial perturbations were introduced during data acquisition to simulate potential causes of instrumental drift.     

JAX Colony Management System (JCMS): an extensible colony and phenotype data management system

The Jackson Laboratory Colony Management System (JCMS) is a software application for managing data and information related to research mouse colonies, associated biospecimens, and experimental protocols. JCMS runs directly on computers that run one of the PC Windows^® operating systems, but can be accessed via web browser interfaces from any computer running a Windows, Macintosh^®, or Linux^® operating system. JCMS can be configured for a single user or multiple users in small- to medium-size work groups. The target audience for JCMS includes laboratory technicians, animal colony managers, and principal investigators. The application provides operational support for colony management and experimental workflows, sample and data tracking through transaction-based data entry forms, and date-driven work reports. Flexible query forms allow researchers to retrieve database records based on user-defined criteria. Recent advances in handheld computers with integrated barcode readers, middleware technologies, web browsers, and wireless networks add to the utility of JCMS by allowing real-time access to the database from any networked computer.     

StreamingTrim 1.0: a Java software for dynamic trimming of 16S rRNA sequence data from metagenetic studies

Next‐generation sequencing technologies are extensively used in the field of molecular microbial ecology to describe taxonomic composition and to infer functionality of microbial communities. In particular, the so‐called barcode or metagenetic applications that are based on PCR amplicon library sequencing are very popular at present. One of the problems, related to the utilization of the data of these libraries, is the analysis of reads quality and removal (trimming) of low‐quality segments, while retaining sufficient information for subsequent analyses (e.g. taxonomic assignment). Here, we present StreamingTrim, a DNA reads trimming software, written in Java, with which researchers are able to analyse the quality of DNA sequences in fastq files and to search for low‐quality zones in a very conservative way. This software has been developed with the aim to provide a tool capable of trimming amplicon library data, retaining as much as taxonomic information as possible. This software is equipped with a graphical user interface for a user‐friendly usage. Moreover, from a computational point of view, StreamingTrim reads and analyses sequences one by one from an input fastq file, without keeping anything in memory, permitting to run the computation on a normal desktop PC or even a laptop. Trimmed sequences are saved in an output file, and a statistics summary is displayed that contains the mean and standard deviation of the length and quality of the whole sequence file. Compiled software, a manual and example data sets are available under the BSD‐2‐Clause License at the GitHub repository at https://github.com/GiBacci/StreamingTrim/ .     

Severe interference between retinal angiography and automated four-color flow cytometry analysis of blood mononuclear cells.

BACKGROUND: Retinal angiography has become a widely used diagnostic tool. It requires the intravenous administration of the fluorescent dyes fluorescein and indocyanin green. We recently received blood taken 8 h after retinal angiography, without our knowing it. We describe the failure of an automated flow cytometry system in the enumeration of lymphocyte subpopulations in this sample. METHODS: Cell enumeration was achieved by the use of the lyse-no wash MultiTEST procedure (Becton-Dickinson) together with the FACSCalibur cytometer. Absolute cell counts were obtained using TruCount beads. Data were analyzed automatically by the MultiSET and manually with the CellQuest softwares. RESULTS: The dot plots obtained with this sample looked quite abnormal. All monuclear cells stained brightly in the FITC channel irrespective of anti-CD3-FITC conjugate binding. This resulted in a major undercompensation for the increased spillover of the fluorescein emission into the PE-channel. PE-labeled cell and TruCount bead events coalesced. The MultiSET software failed to draw proper gatings and proved useless. Alternative manual gatings could partially rescue the analysis. CONCLUSIONS: Clinicians and cytometrists should be aware that, because of dye entry or binding, blood mononuclear cells collected shortly after retinal angiography are not suitable even for common cytometry applications.     

Plato: a genetic algorithm approach to run-time reconfiguration in?autonomic computing systems

Increasingly, applications need to be able to self-reconfigure in response to changing requirements and environmental conditions. Autonomic computing has been proposed as a means for automating software maintenance tasks. As the complexity of adaptive and autonomic systems grows, designing and managing the set of reconfiguration rules becomes increasingly challenging and may produce inconsistencies. This paper proposes an approach to leverage genetic algorithms in the decision-making process of an autonomic system. This approach enables a system to dynamically evolve target reconfigurations at run time that balance tradeoffs between functional and non-functional requirements in response to changing requirements and environmental conditions. A key feature of this approach is incorporating system and environmental monitoring information into the genetic algorithm such that specific changes in the environment automatically drive the evolutionary process towards new viable solutions. We have applied this genetic-algorithm based approach to the dynamic reconfiguration of a collection of remote data mirrors, demonstrating an effective decision-making method for diffusing data and minimizing operational costs while maximizing data reliability and network performance, even in the presence of link failures.     

A rapid,flexibel method for biochemical assays using a microtiter plate reader and a microcomputer. Application for assays of protein,Na,K-Atpase and K-p-Nitrophenylphosphatase

A computerized system which greatly accelerates and eases the collection, storage, and analysis of data has been applied to several standard biochemical assays. The system uses a commercially available microtiter plate reader connected to an apple IIe microcomputer via a standard serial port. Data are transmitted automatically from the reader to the microcomputer, where they can be viewed, printed, further analyzed immediately, or stored on a diskette for later retrieval and processing. Some or all of the data may be entered manually. The program calculates a linear least squares best fit to a standard curve after correcting all data for blanks, then determines the quantities of substrate or product contained in each well of a microtiter plate. Data from two plates may be combined, enabling calculation of enzyme specific activities. This system can be adapted to any assay whose final step can be performed by a microtiter plate reader. Its use is described for determination of protein concentration, Na,K-ATPase activity, and K-stimulated p-nitrophenylphosphatase activity.     

An innovation in flow cytometry data collection and analysis producing a correlated multiple sample analysis in a single file

The problems associated with rapid analysis and interpretation of data from multicolor immunofluorescence panels have been a formidable barrier to their routine use. Using present flow cytometry concepts, a panel of 11 tubes each containing multiple phenotypic markers or controls requires postdata acquisition manipulation of many multiparameter histogram and listmode files. We have developed a method that compresses all of the information from such a panel into a single listmode data file during run time. A single data file is used to record the entire phenotypic analysis for a particular patient or series within an experiment. This is accomplished by the incorporation of a tube identifier parameter (TIP) as well as the fluorescence and light scatter parameters normally collected. The TIP can then be used for gating discrimination of any tube or set of tubes within a panel. When the TIP is correlated with the PRISM parameter the entire patient phenotypic image can be represented within a single two-parameter histogram we have called a phenogram. This phenogram can be generated in real time, providing on-line preprocessing of a complex multicolor experiment. By examining the image created by the phenogram it is possible to rapidly flag abnormalities such as incorrect gating. This procedure was carried out on an EPICS Elite flow cytometer in its standard configuration with the addition of hardware to provide an input for the TIP.     

智能化鼠多功能行为训练系统的研究

目的：研制一种微机控制的大鼠多功能行为训练检测系统－梯度电压自动驱动大鼠及微机适时显示,记录和分析实验结果。方法：采用仿windows界面,以TurboC2．0编制,利用TC直接对硬件端口操作,完成信号的采集,处理和对实验仪器的控制。结果：本系统实现了：①梯度电压自动驱动;②声,光,电不同条件刺激;③多路径自动设置;④所没数据建立数据库以SAS软件处理其结果。结论：本系统自动化程度高,操作简便,数据处理科学、准确,尤其是还可用于听觉、视觉等特殊鼠记忆模型的建立。     

Automated sample mounting and alignment system for biological crystallography at a synchrotron source

High-throughput data collection for macromolecular crystallography requires an automated sample mounting and alignment system for cryo-protected crystals that functions reliably when integrated into protein-crystallography beamlines at synchrotrons. Rapid mounting and dismounting of the samples increases the efficiency of the crystal screening and data collection processes, where many crystals can be tested for the quality of diffraction. The sample-mounting subsystem has random access to 112 samples, stored under liquid nitrogen. Results of extensive tests regarding the performance and reliability of the system are presented. To further increase throughput, we have also developed a sample transport/storage system based on "puck-shaped" cassettes, which can hold sixteen samples each. Seven cassettes fit into a standard dry shipping Dewar. The capabilities of a robotic crystal mounting and alignment system with instrumentation control software and a relational database allows for automated screening and data collection to be developed.     

Time interval gating for analysis of cell function using flow cytometry.

We propose a method which significantly shortens the time required for both the collection and analysis of data derived from multiple sample, flow cytometric kinetic assays. We have defined the term Time Interval Gating (TIG) to describe this method. TIG effectively allows one flow cytometer to concurrently monitor several samples over the course of a kinetic assay. Data for all samples are stored in a single FCS 2.0 compatible listmode data file which we refer to as the TIG data file. TIG is adaptable to most commerical flow cytometers. Standard listmode analysis software can be used to analyze the TIG data files and correlate any combination of tubes and/or time intervals from the assay. Results for the entire assay can be displayed on a single two parameter plot. This paper describes how TIG is applied to neutrophil oxidative burst measurement using a standard EPICS Elite flow cytometer. In this assay, 11 samples were each monitored for 30 min to identify the extent to which volatile organic chemicals (VOCs) inhibited the oxidation of DCFH in stimulated neutrophils. TIG makes the oxidative burst assay practical for high volume screening by reducing the overall flow cytometer and analysis time required by a factor of ten. In addition, TIG provides an organized approach to managing data acquisition on instruments equipped with automated sampling systems.     

Cheburator Software for Automatically Calculating Drug Inhibitory Concentrations from In Vitro Screening Assays

In the process of new cancer drug development, as the first step of their assessment, their activities are usually studied in vitro against a panel of cancer cell lines. The results of these in vitro drug screening assays are commonly expressed as inhibitory concentration 50% (IC₅₀): the concentration of the tested agent that inhibits the proliferation of the cancer cell population to 50% of the theoretically possible effect (absolute IC₅₀) or maximum effect practically achieved by the drug (relative IC₅₀). The currently available software for calculating IC₅₀ values requires manual data entry, is time consuming, and is prone to calculation errors. Thus, we have developed open source, free, easy-to-use software for performing standardized data evaluations and automatically calculating the IC₅₀. This software eliminates the laborious and error-prone manual entry of data, substantially reduces the amount of time spent for data analysis. It has been extensively used in our department as the main tool for in vitro data processing during the past several years and can be useful for other research groups working in the area of anticancer drug discovery, either alone or combined with other software packages. The current version of our program, Cheburator, together with sample data, source code, and documentation, is freely available at the following URL: http://www.cheburator.nevozhay.com (it is free for academic use, but a license is required for commercial use).     

Design and first results of CytoBuoy: a wireless flow cytometer for in situ analysis of marine and fresh waters.

BACKGROUND:The high costs of microscopical determination and counting of phytoplankton often limit sampling frequencies below an acceptable level for the monitoring of dynamic ecosystems. Although having a limited discrimination power, flow cytometry allows the analysis of large numbers of samples to a level that is sufficient for many basic monitoring jobs. For this purpose, flow cytometers should not be restricted to research laboratories. We report here on the development of an in situ flow cytometer for autonomous operation inside a small moored buoy or on other platforms. METHODS AND RESULTS: Operational specifications served a wide range of applications in the aquatic field. Specific conditions had to be met with respect to the operation platform and autonomy. A small, battery-operated flow cytometer resulted, requiring no external sheath fluid supply. Because it was designed to operate in a buoy, we call it CytoBuoy. Sampling, analysis, and radio transmission of the data proceed automatically at user-defined intervals. A powerful feature is the acquisition and radio transmission of full detector pulse shapes of each particle. This provides valuable morphological information for particles larger than the 5-microm laser focus. CONCLUSIONS:CytoBuoy allows on-line in situ particle analysis, estimation of phytoplankton biomass, and discrimination between different phytoplankton groups. This will increase the applicability of flow cytometry in the field of environmental monitoring.     

A computerized respiratory system including test functions of lung and circulation

The design of a microcomputer-controlled ventilator for automatic performance of lung function and circulatory tests has been described. It incorporates the characteristics of normal mechanical ventilation and also allows one to perform a multitude of test procedures for lung function and circulatory studies in paralyzed animals. The major components of the setup are a pump assembly with solenoid valves to direct gas flow, an electromechanical servo system, and a MS-DOS microcomputer system. The pump assembly has been constructed as a relatively simple device. Great versatility is created by the use of a microcomputer for the control of the ventilator. The software can be easily adapted to several other types of experimental studies. Besides the keyboard input the ventilator can be controlled by a remote computer system. This allows one to run an experimental protocol automatically and to use it in closed-loop servo ventilation. The flexibility in the choice of the respiratory parameters makes the ventilator suitable for lung function and circulatory studies during artificial ventilation. The ventilator has been successfully used in different animal studies during the last 6 yr.     

CytoAccess, a relational laboratory information management system for a clinical cytogenetics laboratory

We developed a CytoAccess laboratory management system based on the widely used Microsoft Access software to facilitate data processing, result reporting, and quality management for a full-service cytogenetics laboratory. The CytoAccess system consists of four functional modules. The data entry module is for logging in patient information. The result entry module is used to generate chromosome, fluorescent in situ hybridization (FISH), and array comparative genomic hybridization (aCGH) reports. The administrative module enables periodic monitoring of quality control and quality improvement (QA/QI) parameters and produces billing forms. The maintenance module allows users to update clinical demographics, report templates, code tables, and to refresh data links. We have integrated into this system over 15,000 chromosome and FISH results from prenatal, postnatal, and cancer cases for the past six years. This system is cost-effective, user-friendly, flexible in updating, and potentially adaptable for data mining.     

Easily assembled digital data acquisition system for the analytical ultracentrifuge

A system for the acquisition of digital data from the analytical ultracentrifuge which uses a commercially available data acquisition board, a standard IBM compatible personal computer (PC), and an interface circuit has been developed. The system uses the signal from the standard Beckman scanner. Preliminary analysis and data reduction are performed at the PC within minutes of data acquisition using simple commercially available software, and final data fitting is performed with a mainframe computer. Procedures are described which allow approach to equilibrium to be followed and attainment of equilibrium to be demonstrated. Data density of 200 points per millimeter column height (500 points per 100 μ1 of sample) allows the use of short columns and hence short run times. Only 2 min are required to collect a complete scan, which is recorded in a format suitable for direct analysis by standard spreadsheet software. This allows multiple sequential scans to be quickly recorded at equilibrium and averaged to reduce noise prior to analysis. The combination of characteristics allows molecular weight determinations to be performed relatively quickly with only a few micrograms of protein. The system is inexpensive and easy to assemble given the centrifuge and a PC.     

ABI sequencing analysis

The ABI Sequencing Analysis application is designed specifically for the analysis of data produced by the ABI DNA Sequencer. The ABI sequencer is a laser-based instrument that utilizes fluorescent labels to analyze the products of a sequencing reaction as they migrate through a gel. After the data are collected from a sequencing run, the Analysis program identifies and tracks the sample lanes of the gel and subsequently normalizes and integrates the raw data into a chromatogram of the final sequence. For the use, there are basically two types of files that can be manipulated to potentially improve the analysis results. The Gel File consists of a computer generated image of the sequencing gel with the fluorescent DNA banding patterns. This image allows the user to view and edit the tracking lines generated and used by Analysis to collect data points for each sample. Individual Sample Files are stored for each of the samples analyzed and include the chromatogram, raw data, and annotations and information regarding the sample and sequence run. Generally, the products of a sequencing reaction are easily resolved and the Analysis software interprets the correct nucleotide sequence. Ambiguous base calls tend to occur near the end of the sequence and may be either edited or deleted by the user before exporting the data for further comparisons or alignments. Occasionally the tracking lines within the gel image may need to be adjusted or moved. The sample data are then reextracted from the Gel File and analyzed again. This review explains the general operation of Analysis in terms of viewing and editing a chromatogram, retracking the lanes of a Gel File, and analyzing the final sample data. The three versions 1.2.1, 2.1.2, and 3.3 are discussed.