Semiautomated preparation of DNA templates for large-scale sequencing projects

The rate limiting step in a large-scale sequencing project is the generation of single-stranded DNA templates. We describe a fast, semiautomated procedure, using 96-well microtitre plates, in which 192 templates can be readily prepared in 1 day. The technique can be carried out manually or can be semiautomated using a robot pipetting device. We also provide evidence for the reliability and applicability of this method to a large-scale sequencing project.     

Quantification of cells cultured on 96-well plates

The method for cell number measurement in monolayer cultures by crystal violet staining published recently by Gillies et al. (R. G. Gillies, N. Didier, M. Denton (1986) Anal. Biochem. 159, 109-113) was modified and significantly improved. The procedure was adapted for use in 96-well plates since the method is inherently very sensitive. Modifications allowed fast and complete solubilization of dye adsorbed by cell nuclei during staining. Since light absorption of the unstained or destained cell layers is negligible, cell number measurements can be performed in the respective wells. Due to these features, multiple assays may be carried out rapidly using standard 96-well plate readers. In addition, it is shown that the sensitivity of the assay can be varied and easily controlled by choosing the appropriate pH during the staining procedure. This increases the flexibility of the method making it useful for determining cell density of a wide range of different cell types.     

Magnetic, microplate-format plasmid isolation protocol for high-yield, sequencing-grade DNA

We have developed a rapid, microplate-format plasmid isolation procedure to purify sequencing-grade DNA templates for high-throughput DNA sequencing operations. A modified lysozyme/boiling method is used to produce a plasmid-containing supernatant that is then purified by iron bead capture. After binding, the beads are pelleted in a magnetic field, washed and the DNA eluted in water. The method yields up to 10 micrograms plasmid DNA from a 1-mL overnight culture in a deep-well microplate. The procedure is suitable for large-scale experiments, amenable to automation and does not require expensive reagents or equipment. The entire protocol can be completed in as little as 2 h, and one technician with a 96-well pipetting station can process up to 48 plates per day. This protocol is ideal for any high-throughput operation in which template quantity, quality and reproducibility are of primary importance.     

Screening for soluble expression of recombinant proteins in a 96-well format

For future structural and functional genomics programs new tools will be required. The implementation of high-throughput (HTP) methods for protein production will be an essential element. Present HTP protein production developments in structural genomics are aimed at obtaining well-expressing and highly soluble proteins, which are preferred candidates for structure-function studies. Here, we describe a cheap and efficient procedure to identify well-expressing soluble proteins in Escherichia coli in a compact 96-well format. Reproducible lysis on filter plates, followed by a filtration step on 96-well filter plates, allows the efficient separation of inclusion bodies from the soluble fraction. In the following step a dot blot procedure using anti-RGS-His(4) antibody (Qiagen) to detect expression of recombinant His-tagged protein is applied allowing direct detection of the target protein in the soluble fraction. The method is well suited for automation and should be applicable to expression screening of most proteins and fusion domains to which specific antibodies are available.     

Measuring beta-galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays.

We describe a high-throughput procedure for measuring beta-galactosidase activity in bacteria. This procedure is unique because all manipulations, including bacterial growth and cell permeabilization, are performed in a 96-well format. Cells are permeabilized by chloroform/SDS treatment directly in the 96-well blocks and then transferred to 96-well microplates for standard colorimetric assay of beta-galactosidase activity as described by Miller [J. H. Miller (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]. Absorbance data are collected with a microplate reader and analyzed using a Microsoft Excel spreadsheet. The beta-galactosidase specific activity values obtained with the high-throughput procedure are identical to those obtained by the traditional single-tube method of Miller. Thus, values obtained with this procedure may be expressed as Miller units and compared directly to Miller units reported in the literature. The 96-well format for permeabilization and assay of enzyme specific activity together with the use of 12-channel and repeater pipettors enables efficient processing of hundreds of samples in an 8-h day.     

High throughput isolation of DNA and RNA in 96-well format using a paint shaker

Most steps in plant nucleic acid isolation are easily adapted to 96-well format; however, tissue disruption typically is performed on samples individually and often is the rate-limiting step in sample processing. We have found that DNA and RNA isolation from Arabidopsis tissue can be carried out in 96-well format using a paint shaker fitted with an adapter for tissue disruption.     

Multiplexed array-based and in-solution genomic enrichment for flexible and cost-effective targeted next-generation sequencing

The unprecedented increase in the throughput of DNA sequencing driven by next-generation technologies now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. However, sample preparation and targeted enrichment of multiple samples has become a rate-limiting and costly step in high-throughput genetic analysis. Here we present an efficient protocol for parallel library preparation and targeted enrichment of pooled multiplexed bar-coded samples. The procedure is compatible with microarray-based and solution-based capture approaches. The high flexibility of this method allows multiplexing of 3-5 samples for whole-exome experiments, 20 samples for targeted footprints of 5 Mb and 96 samples for targeted footprints of 0.4 Mb. From library preparation to post-enrichment amplification, including hybridization time, the protocol takes 5-6 d for array-based enrichment and 3-4 d for solution-based enrichment. Our method provides a cost-effective approach for a broad range of applications, including targeted resequencing of large sample collections (e.g., follow-up genome-wide association studies), and whole-exome or custom mini-genome sequencing projects. This protocol gives details for a single-tube procedure, but scaling to a manual or automated 96-well plate format is possible and discussed.     

Comparison of plasma sample purification by manual liquid–liquid extraction, automated 96-well liquid–liquid extraction and automated 96-well solid-phase extraction for analysis by high-performance liquid chromatography with tandem mass spectrometry

Three extraction procedures were developed for the quantitative determination of a carboxylic acid containing analyte (I) in human plasma by high-performance liquid chromatography (HPLC) with negative ion electrospray tandem mass spectrometry (MS–MS). The first procedure was based on the manual liquid–liquid extraction (LLE) of the acidified plasma samples with methyl tert.-butyl ether. The second procedure was based on the automation of the manual LLE procedure using 96-well collection plates and a robotic liquid handling system. The third approach was based on automated solid-phase extraction (SPE) using 96-well SPE plates and a robotic liquid handling system. A lower limit of quantitation of 50 pg/ml was achieved using all three extraction procedures. The total time required to prepare calibration curve standards, aliquot the standards and plasma samples, and process a total of 96 standards and samples by manual LLE was three-times longer than the time required for 96-well SPE or 96-well LLE (4 h, 50 min vs. 1 h, 43 min). Even more importantly, the time the bioanalyst physically spent on the 96-well LLE or 96-well SPE procedure was only a small fraction of the time spent on the manual LLE procedure (<10 min vs. 4 h, 10 min). It should be noted that the 96-well SPE procedure incorporated the two steps of evaporation of the eluates to dryness and subsequent reconstitution of the dried extract. The total time required for the 96-well SPE could be reduced by 50% if the eluates were injected directly, eliminating the drying and reconstitution steps, which is achievable when sensitivity is less of an issue.     

Automated protein precipitation by filtration in the 96-well format

The use of automated protein precipitation by filtration in the 96-well format as a rapid sample preparation technique for high throughput bioanalysis using liquid chromatography tandem mass spectrometry is reported. A robotic sample processor is used to aspirate sequentially a plasma sample and acetonitrile separated by air gaps. These are then mixed by being dispensed into individual channels of a 96-well filter block. The resulting supernatant is separated from the precipitated plasma proteins by the application of gentle vacuum using a custom manifold. The filtered supernatants are collected into a deep well microtitre plate, evaporated to dryness using a heated 96-well dry down station and reconstituted in water prior to analysis. The efficiency of the extraction procedure is measured by the Lowry method for determining protein concentration. This method was used to optimise both the volume and the order of reagent addition, and to compare several prototype 96-well filter blocks. Using the optimised procedure a specific, precise and accurate method was developed for the β-agonist salbutamol in rabbit plasma with a calibration range of 1 to 100 ng/ml from 100 μl of sample.     

A semi-automated protein assay for cell cultures

A semi-automated modification of the protein determination procedure of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem. 193, 265-275) is described. The assay is well suited to the analysis of the protein of adherent cultured cells. The procedure is carried out in 96-well microtest plates on protein solutions of 50 microliter or less, and can detect less than 0.5 micrograms of protein (equivalent to about 10(3) cultured cells). Optical densities are read and printed by an automatic microplate reader capable of processing 96 samples in less than 2 min.     

A recombineering based approach for high-throughput conditional knockout targeting vector construction

Functional analysis of mammalian genes in vivo is primarily achieved through analysing knockout mice. Now that the sequencing of several mammalian genomes has been completed, understanding functions of all the genes represents the next major challenge in the post-genome era. Generation of knockout mutant mice has currently been achieved by many research groups but only by making individual knockouts, one by one. New technological advances and the refinements of existing technologies are critical for genome-wide targeted mutagenesis in the mouse. We describe here new recombineering reagents and protocols that enable recombineering to be carried out in a 96-well format. Consequently, we are able to construct 96 conditional knockout targeting vectors simultaneously. Our new recombineering system makes it a reality to generate large numbers of precisely engineered DNA constructs for functional genomics studies.     

DNA sequencing using 96-capillary array electrophoresis

Practical DNA sequencing in a rugged capillary array electrophoresis system coupled directly to 96-well microtiter plates is demonstrated. A CCD detector was used to monitor all capillaries simultaneously with laser-induced fluorescence at 1.75 frames per second. The reconstructed electropherograms show good signal-to-noise ratios and resolution for the entire capillary array. The system used standard dye labeling and image splitting to obtain fluorescence intensities in two wavelength regions to allow calling up to 410 bases for the DNA sequence. The use of a replaceable poly(ethylene oxide) matrix and a protective poly(vinylpyrrolidone) coating allows high separation speed and short turnaround time for high throughput DNA sequencing. Critical evaluation of the system performance over repeated runs with base calling is presented.     

LoPPS: a long PCR product sequencing method for rapid characterisation of long amplicons

Here, we propose an optimised protocol (LoPPS, long PCR product sequencing) which allows the fast, cost-attractive, and high-throughput sequencing of long PCR products. LoPPS constitutes an alternative to the primer-walking technology which is expensive and time consuming but remains the current standard procedure. It is based on the ultrasonic shearing, polishing, and cloning of PCR or RT-PCR products and is compatible with 96- or 384-well microplate systems in which bacterial growth, preparation of plasmid DNA, and sequencing can be automated. We present results obtained from 24 different RT-PCR products (2.5-4.8 kbp long) obtained from various RNA viruses and fully sequenced using LoPPS. The method proved to be robust and fast. It was successfully used on a low amount of DNA and allowed each target nucleotide position to be controlled twice or more, with a final cost which is one-third of that of primer-walking.     

An enzymatic fluorescent assay for the quantification of phosphite in a microtiter plate format

A sensitive fluorometric assay for the quantification of phosphite has been developed. The assay uses the enzymatic oxidation of phosphite to phosphate by a recombinant phosphite dehydrogenase with NAD⁺ as cosubstrate to produce the highly fluorescent reaction product resorufin. The optimized assay can be carried out in a 96-well microtiter plate format for high-throughput screening purposes and has a detection limit of 0.25 nmol phosphite. We used the method to quantify phosphite levels in plant tissue extracts and to determine phosphite dehydrogenase activity in transgenic plants. The assay is suitable for other biological or environmental samples. Because phosphite is a widely used fungicide to protect plants from pathogenic oomycetes, the assay provides a cost-effective and easy-to-use method to monitor the fate of phosphite following application.     

Fast response temperature measurement and highly reproducible heating methods for 96-well plates

Hyperthermia, the procedure of exposing cells to a temperature between 42 degrees and 49 degrees C, has been shown to be a promising approach for cancer treatment. To understand the underlying mechanisms of hyperthermic killing of cancer cells, it is critical to have an accurate temperature measurement technique and a heating method with high reproducibility. To this end, we have developed a method using fine thermocouples with fast response time to measure the temperatures in multiple wells of a 96-well plate. The accuracy of temperature measurement was +/- 0.2 degree C. Such a capability allows a complete record of the time and temperature of the treatment procedure and helps define an accurate thermal dose. We have also compared several methods for heating 96-well plates and found that use of copper blocks in contact with the lower surface of the 96-well plate in an incubator provides a highly reproducible heating method. The common method of using water bath to heat cells in vitro resulted in a decrease of cell viability even at the control temperature of 37 degrees C and a decrease in the reproducibility of certain biological assays. In summary, using these improved techniques, proposed thermal dose can be defined more precisely, and highly reproducible heating in vitro can be achieved.     

Simultaneous assay of sildenafil and desmethylsildenafil in human plasma using liquid chromatography-tandem mass spectrometry on silica column with aqueous-organic mobile phase

A liquid chromatography-tandem mass spectrometry method was developed for the analysis of sildenafil (SIL) and its metabolite desmethylsildenafil (DMS) in human plasma. Samples were accurately transferred to 96-well plates using a liquid handler (Multiprobe II). Solid-phase extraction was carried out on a 96-channel programmable liquid handling workstation (Quadra 96) using a C8 and cation-exchange mixed-mode sorbent. The extract was injected onto a silica column with an aqueous-organic mobile phase, a combination that was novel for improving the method sensitivity. The low limit of quantitation was 1.0 ng/ml for both SIL and DMS. The method was validated to meet the criteria of current industrial guidance for quantitative bioanalytical methods.     

Development and evaluation of an automated workstation for single nucleotide polymorphism discrimination using bacterial magnetic particles

We designed an automated workstation for magnetic particle-based single nucleotide polymorphism (SNP) discrimination of ALDH genotypes. Bacterial magnetic particles (BMPs) extracted from Magnetospirillum magneticum AMB-1 were used as DNA carriers. The principle for SNP discrimination in this study was based on fluorescence resonance energy transfer (FRET) between FITC (donor) and POPO-3 (acceptor) bound to double-stranded DNA. The workstation is equipped with a 96-way automated pipetter which collects and dispenses fluids as it moves in x- and z-directions. The platform contains a disposable tip rack station, a reagent vessel serving as a stock for POPO-3 and FITC-labeled probes and a reaction station for a 96-well microtiter plate. BMPs were collected by attaching a neodymium iron boron sintered (Nd-Fe-B) magnet on the bottom of the microtiter plate. This system permits the simultaneous heating and magnetic separation of 96 samples per assay. The genotypes ALDH2*1 and ALDH2*2 were discriminated by calculating the relative fluorescence intensities on BMPs.     

Protocols and Programs for High-Throughput Growth and Aging Phenotyping in Yeast

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting “Colony Forming Units”. To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.     

High throughput flow cytometry based yeast two-hybrid array approach for large-scale analysis of protein-protein interactions

The analysis of protein-protein interactions is a key focus of proteomics efforts. The yeast two-hybrid system (Y2H) has been the most commonly used method in genome-wide searches for protein interaction partners. However, the throughput of the current yeast two-hybrid array approach is hampered by the involvement of the time-consuming LacZ assay and/or the incompatibility of liquid handling automation due to the requirement for selection of colonies/diploids on agar plates. To facilitate large-scale Y2H assays, we report a novel array approach by coupling a GFP reporter based Y2H system with high throughput flow cytometry that enables the processing of a 96-well plate in as little as 3 min. In this approach, the yEGFP reporter has been established in both AH109 (MATa) and Y187 (MATα) reporter cells. It not only allows the generation of two copies of GFP reporter genes in diploid cells, but also allows the convenient determination of self-activators generated from both bait and prey constructs by flow cytometry. We demonstrate a Y2H array assay procedure that is carried out completely in liquid media in 96-well plates by mating bait and prey cells in liquid YPD media, selecting the diploids containing positive interaction pairs in selective media and analyzing the GFP reporter directly by flow cytometry. We have evaluated this flow cytometry based array procedure by showing that the interaction of the positive control pair P53/T is able to be reproducibly detected at 72 hr postmating compared with the negative control pairs. We conclude that our flow cytometry based yeast two-hybrid approach is robust, convenient, quantitative, and is amenable to large-scale analysis using liquid-handling automation.