Automatic scanning of interphase FISH for prenatal diagnosis in uncultured amniocytes

Fluorescence in situ hybridization (FISH) of uncultured amniocytes using chromosome-specific DNA probes offers the opportunity for rapid aneuploidy screening. Between 80 and 95% of all chromosomal disorders expected in the second trimester of pregnancy can be discovered within 24 hr if DNA probes specific for chromosomes 21, 18, 13, X, and Y are used. Rapid results are crucial for clinical decision-making and are helpful in decreasing the anxiety level in most patients. One of the major factors that have been preventing the rapid FISH test from being broadly incorporated into the clinical setting is the limited staff in the cytogenetics laboratories. The present study demonstrates the use of an automated scanning system (Duet, BioView Ltd. Rehovot, Israel) for analyzing FISH in uncultured amniocytes. Fifty-six amniotic fluid samples were evaluated in parallel by karyotyping, manual FISH analysis, and automatic FISH scanning. Automatic scanning provided accurate results compared to both manual FISH scoring and karyotype analysis. The correlation between automatic and manual FISH scanning was found to be very high (r = 0.9, p < 0.0001). The availability of automation for aneuploidy screening in amniotic fluid samples will enable offering this test to a broader patient population while providing fast and reliable results.     

Automated scoring of multiprobe FISH in human spermatozoa.

In the case of chromosomal aneuploidy in sperm wherein the incident rate is low and a large number of cells require scoring, automated methods that rely on computer software to segment and to count fluorescence signals are particularly necessary due to countless hours spent in reading slides and to the potential for interoperator differences. The purpose of this pilot experiment was to determine whether there were significant differences in the estimates of disomy frequency produced by automated versus manual scoring of signals for chromosome X, Y, and 18 in human sperm. The frequency of X18, Y18, XX18, YY18, and XY18 were determined in four separate normozoospermic samples. Slides were hybridized using a standard sperm FISH protocol for centromere-specific probes. Between 500 and 564, DAPI positive nuclei were captured from each sample and scored using the automated system, and the same slides were scored by a trained cytogeneticist, who was blind to the purpose of the study and the automated system results. None of the estimated frequencies was significantly different between manual and automated methods, regardless of whether individual slides or pooled results across all samples were compared. To our knowledge, this is the first report examining the validity of automated cell scoring in human spermatozoa. The results from this pilot exploration of sperm FISH suggest the comparability between automated and manual methods for estimating sex chromosome disomy and provide evidence that automated laser scanning of multiprobe sperm FISH should be explored further.     

PCR candidate region mismatch scanning: adaptation to quantitative, high-throughput genotyping

Linkage and association analyses were performed to identify loci affecting disease susceptibility by scoring previously characterized sequence variations such as microsatellites and single nucleotide polymorphisms. Lack of markers in regions of interest, as well as difficulty in adapting various methods to high-throughput settings, often limits the effectiveness of the analyses. We have adapted the Escherichia coli mismatch detection system, employing the factors MutS, MutL and MutH, for use in PCR-based, automated, high-throughput genotyping and mutation detection of genomic DNA. Optimal sensitivity and signal-to-noise ratios were obtained in a straightforward fashion because the detection reaction proved to be principally dependent upon monovalent cation concentration and MutL concentration. Quantitative relationships of the optimal values of these parameters with length of the DNA test fragment were demonstrated, in support of the translocation model for the mechanism of action of these enzymes, rather than the molecular switch model. Thus, rapid, sequence-independent optimization was possible for each new genomic target region. Other factors potentially limiting the flexibility of mismatch scanning, such as positioning of dam recognition sites within the target fragment, have also been investigated. We developed several strategies, which can be easily adapted to automation, for limiting the analysis to intersample heteroduplexes. Thus, the principal barriers to the use of this methodology, which we have designated PCR candidate region mismatch scanning, in cost-effective, high-throughput settings have been removed.     

Evaluation of an automated in vitro micronucleus assay in CHO-K1 cells

In this paper, we describe the evaluation of an automated in vitro micronucleus assay using CHO-K1 cells in 96-well plates. CHO-K1 cells were pre-loaded with a cell dye that stains the cytoplasm, after which the cells were treated with the test compounds for either 3h (for the +S9 condition) or 24h (for the -S9 condition). A total of 10 concentrations were tested, of which the top five concentrations were scored (limited by either cytotoxicity or solubility). At the end of the incubation period the cells were fixed and their DNA was stained with Hoechst. The visualization and scoring of the cells was done using an automated fluorescent microscope coupled with proprietary automated image analysis software provided by Cellomics (Pittsburg, PA). A total of 46 compounds were used in this evaluation, including 8 aneugens and 25 clastogens with varied mechanisms of action. Thirteen non-genotoxic compounds were also included. The automated scoring had a sensitivity of 88% and a specificity of 100%, with a predictive value positive of 100% and a predictive value negative of 76%, compared to data from the literature that was obtained with manual scoring. We also describe the incorporation of a metabolic activation system using rat liver S9 homogenates, and the use of cell number counts as a cytotoxicity index which is complementary to the CBPI- (cytokinesis-block proliferation index) based index. Finally, we also discuss the potential for artefactual findings due to fluorescent precipitate, which should be carefully monitored to prevent false positive results. In conclusion, the automated in vitro micronucleus scoring is a valid alternative to the manual scoring of slides, and it has the advantage of generating data in a rapid and consistent manner, and with low compound requirements, which makes it well suited as a screening assay in the early stages of compound development.     

Evaluating CASP4 predictions with physical energy functions

Physical energy scoring functions based on implicit solvation models are tested by evaluating predictions from the most recent CASP4 competition. The best performing scoring functions are identified along with the best protocol for preparing structures before energies are evaluated. Ranking of structures with the best scoring functions is compared across CASP4 targets to establish when physical scoring functions can be expected to reliably distinguish structures that are most similar to the native fold in a set of misfolded or unfolded protein conformations. The results are used to interpret previous studies where scoring functions were tested on the standard decoy sets by Park, Levitt, and Baker. We show that the best physical scoring functions can be applied successfully in automated consensus scoring applications where a single best conformation has to be selected from a set of structures from different sources. Finally, the potential for better protein structure scoring functions is discussed with a suggestion for an empirically parameterized linear combination of energy components.     

Assembling genes from predicted exons in linear time with dynamic programming.

In a number of programs for gene structure prediction in higher eukaryotic genomic sequences, exon prediction is decoupled from gene assembly: a large pool of candidate exons is predicted and scored from features located in the query DNA sequence, and candidate genes are assembled from such a pool as sequences of nonoverlapping frame-compatible exons. Genes are scored as a function of the scores of the assembled exons, and the highest scoring candidate gene is assumed to be the most likely gene encoded by the query DNA sequence. Considering additive gene scoring functions, currently available algorithms to determine such a highest scoring candidate gene run in time proportional to the square of the number of predicted exons. Here, we present an algorithm whose running time grows only linearly with the size of the set of predicted exons. Polynomial algorithms rely on the fact that, while scanning the set of predicted exons, the highest scoring gene ending in a given exon can be obtained by appending the exon to the highest scoring among the highest scoring genes ending at each compatible preceding exon. The algorithm here relies on the simple fact that such highest scoring gene can be stored and updated. This requires scanning the set of predicted exons simultaneously by increasing acceptor and donor position. On the other hand, the algorithm described here does not assume an underlying gene structure model. Indeed, the definition of valid gene structures is externally defined in the so-called Gene Model. The Gene Model specifies simply which gene features are allowed immediately upstream which other gene features in valid gene structures. This allows for great flexibility in formulating the gene identification problem. In particular it allows for multiple-gene two-strand predictions and for considering gene features other than coding exons (such as promoter elements) in valid gene structures.     

Recognizing complex, asymmetric functional sites in protein structures using a Bayesian scoring function

The increase in known three-dimensional protein structures enables us to build statistical profiles of important functional sites in protein molecules. These profiles can then be used to recognize sites in large-scale automated annotations of new protein structures. We report an improved FEATURE system which recognizes functional sites in protein structures. FEATURE defines multi-level physico-chemical properties and recognizes sites based on the spatial distribution of these properties in the sites' microenvironments. It uses a Bayesian scoring function to compare a query region with the statistical profile built from known examples of sites and control nonsites. We have previously shown that FEATURE can accurately recognize calcium-binding sites and have reported interesting results scanning for calcium-binding sites in the entire Protein Data Bank. Here we report the ability of the improved FEATURE to characterize and recognize geometrically complex and asymmetric sites such as ATP-binding sites and disulfide bond-forming sites. FEATURE does not rely on conserved residues or conserved residue geometry of the sites. We also demonstrate that, in the absence of a statistical profile of the sites, FEATURE can use an artificially constructed profile based on a priori knowledge to recognize the sites in new structures, using redoxin active sites as an example.     

Rapid automated simultaneous screening of (G1691A) Factor V, (G20210A) prothrombin,and (C677T) methylenetetrahydrofolate reductase variants by multiplex PCR using fluorescence scanning technology

The Factor V Leiden mutation (G1691A), and mutations in the prothrombin (G20210A) and 5,10-methylenetetrahydrofolate reductase (C677T) genes are common hereditary risk factors associated with venous thrombosis. The aim of this study was to develop an automated, PCR-based genotyping assay for rapid simultaneous screening of these three mutations. We adapted multiplex PCR, using primer modifications to introduce cleavage sites for restriction endonucleases into the fragments bearing each of the mutations. The three mutations were analyzed in a single tube by fluorescence scanning. An internal digestion control was introduced to prevent false-negative results due to incomplete digestion or a total lack of digestion. DNA fragment analysis was carried out using an automated capillary electrophoresis instrument (ABI310). This reliable, efficient, easy-to-use assay can be applied to specimens from large clinical trials and epidemiological surveys.     

Automated evaluation of frequencies of aneuploid sperm by laser-scanning cytometry (LSC)

BACKGROUND: Laser-scanning cytometry (LSC) allows fast automated scoring of fluorescence signals directly on microscopic slides. Frequencies of spontaneous aneuploidies in murine and human sperm were evaluated by using this new LSC technique. Rapid detection may be of great interest in reproductive toxicology, as certain chemicals act as aneugens during meiosis, increasing the production of aneuploid germ cells. Materials and Methods Selected chromosomes were detected by using fluorescence in situ hybridization (FISH) and fluorochrome-labeled DNA-probes. Sperm chromatin was counterstained with propidium iodide. By scanning across the slide, fluorescence signals within sperm nuclei were detected and counted. RESULTS: In murine sperm, the frequencies of disomies for chromosomes 8 and X were 0.019% and 0.021%, respectively. The automated assessment in human sperm resulted in disomy frequencies of 0.061% and 0.090% for chromosomes 13 and X, respectively. These results were comparable to data obtained from the same samples by manual microscopic scoring and to literature data. CONCLUSIONS: Frequencies of genotypically abnormal sperm were not significantly different between automated and manual scoring. In conclusion, sperm aneuploidy was reliably determined and disomic sperm were successfully relocated by LSC. By virtue of rapid and reliable analyses, LSC has the powerful potential to replace manual microscopic FISH analysis in molecular cytogenetics.     

Comparison of manual and automated methods for identifying target sounds in audio recordings of Pileated, Pale-billed, and putative Ivory-billed woodpeckers

ABSTRACT.   Although offering many benefits over manual recording and survey techniques for avian field studies, automated sound recording systems produce large datasets that must be carefully examined to locate sounds of interest. We compared two methods for locating target sounds in continuous sound recordings: (1) a manual method using computer software to provide a visual representation of the recording as a sound spectrogram and (2) an automated method using sound analysis software preprogrammed to identify specific target sounds. For both methods, we examined the time required to process a 24-h recording, scanning accuracy, and scanning comprehensiveness using four different target sounds of Pileated Woodpeckers ( Dryocopus pileatus ), Pale-billed Woodpeckers ( Campephilus guatemalensis ), and putative Ivory-billed Woodpeckers ( Campehilus principalis ). We collected recordings from the bottomland forests of Florida and the Neotropical dry forests of Costa Rica, and compared manual versus automated cross-correlation scanning techniques. The automated scanning method required less time to process sound recordings, but made more false positive identifications and was less comprehensive than the manual method, identifying significantly fewer target sounds. Although the automated scanning method offers a fast and economic alternative to traditional manual efforts, our results indicate that manual scanning is best for studies requiring an accurate account of temporal patterns in call frequency and for those involving birds with low vocalization rates.     

Multicolor fluorescent differential display

Differential display and DNA microarray have emerged as the two most popular methods for gene expression profiling. Here, we developed a multicolor fluorescent differential display (FDD) method that combines the virtues of both differential display in signal amplification and DNA microarray in signal analysis. As in DNA microarray, RNA samples being compared can be labeled with either a red or green fluorescent dye and displayed in a single lane, allowing convenient scoring and quantification of the differentially expressed messages. In addition, the multicolor FDD has a built-in signal proofreading capability that is achieved by labeling each RNA sample from a comparative study with both red and green fluorescent dyes followed by their reciprocal mixings in color. Thus, the multicolor FDD provides a platform upon which a sensitive and accurate gene expression profiling by differential display can be automated and digitally analyzed. It is envisioned that cDNAs generated by the multicolor FDD may also be used directly as probes for DNA microarray, allowing an integration of the two most widely used technologies for comprehensive analysis of gene expression.     

Optimizing automated AFLP scoring parameters to improve phylogenetic resolution

The amplified fragment length polymorphism (AFLP) technique is an increasingly popular component of the phylogenetic toolbox, particularly for plant species. Technological advances in capillary electrophoresis now allow very precise estimates of DNA fragment mobility and amplitude, and current AFLP software allows greater control of data scoring and the production of the binary character matrix. However, for AFLP to become a useful modern tool for large data sets, improvements to automated scoring are required. We design a procedure that can be used to optimize AFLP scoring parameters to improve phylogenetic resolution and demonstrate it for two AFLP scoring programs (GeneMapper and GeneMarker). In general, we found that there was a trade-off between getting more characters of lower quality and fewer characters of high quality. Conservative settings that gave the least error did not give the best phylogenetic resolution, as too many useful characters were discarded. For example, in GeneMapper, we found that bin width was a crucial parameter, and that although reducing bin width from 1.0 to 0.5 base pairs increased the error rate, it nevertheless improved resolution due to the increased number of informative characters. For our 30-taxon data sets, moving from default to optimized parameter settings gave between 3 and 11 extra internal edges with >50% bootstrap support, in the best case increasing the number of resolved edges from 14 to 25 out of a possible 27. Nevertheless, improvements to current AFLP software packages are needed to (1) make use of replicate profiles to calibrate the data and perform error calculations and (2) perform tests to optimize scoring parameters in a rigorous and automated way. This is true not only when AFLP data are used for phylogenetics, but also for other applications, including linkage mapping and population genetics.     

High-throughput AFLP analysis using infrared dye-labeled primers and an automated DNA sequencer

Amplified fragment length polymorphism (AFLP) analysis is currently the most powerful and efficient technique for the generation of large numbers of anonymous DNA markers in plant and animal genomes. We have developed a protocol for high-throughput AFLP analysis that allows up to 70,000 polymorphic marker genotype determinations per week on a single automated DNA sequencer. This throughput is based on multiplexed PCR amplification of AFLP fragments using two different infrared dyelabeled primer combinations. The multiplexed AFLPs are resolved on a two-dye, model 4200 LI-COR automated DNA sequencer, and the digital images are scored using semi-automated scoring software specifically designed for complex AFLP banding patterns (AFLP-Quantar). Throughput is enhanced by using high-quality genomic DNA templates obtained by a 96-well DNA isolation procedure.     

A rapid, semi-automated method for scoring micronuclei in mononucleated mouse lymphoma cells

A semi-automated scoring system has been developed to provide rapid, accurate assessment of micronuclei in preparations of mononuclear mouse lymphoma L5178Y cells. Following exposure to a range of test agents, flat, single-cell preparations were produced from exponentially growing cultures by cytocentrifugation. Following staining with 4'-6-diamidino-2-phenylindole (DAPI), cells were scanned by use of the MicroNuc module of Metafer 4 v 3.4.102, after modifying the classifier developed for selecting micronuclei in binucleate cells to increase its sensitivity. The image gallery of all cells was then sorted to bring aberrant cells to the top of the gallery to assess visually the numbers of cells with micronuclei, as distinct from other debris. Slide quality was shown to be paramount in obtaining accurate results from an automated scan and the data obtained compared very well with the incidence of micronuclei scored conventionally by microscopy. Compared with manual scoring the time saving is considerable, as more than 2000 images are captured in approximately 2min, with subsequent visual assessment of aberrant cells in the image gallery taking about 1-2min/slide. By scanning all aberrant cells, the system also captures additional information on necrotic, apoptotic and fragmented cells. Although optimised for mouse lymphoma cells, it should be simple to adapt the method for any cell type growing in suspension.     

Gene structure prediction by spliced alignment of genomic DNA with protein sequences: increased accuracy by differential splice site scoring

Gene identification in genomic DNA from eukaryotes is complicated by the vast combinatorial possibilities of potential exon assemblies. If the gene encodes a protein that is closely related to known proteins, gene identification is aided by matching similarity of potential translation products to those target proteins. The genomic DNA and protein sequences can be aligned directly by scoring the implied residues of in-frame nucleotide triplets against the protein residues in conventional ways, while allowing for long gaps in the alignment corresponding to introns in the genomic DNA. We describe a novel method for such spliced alignment. The method derives an optimal alignment based on scoring for both sequence similarity of the predicted gene product to the protein sequence and intrinsic splice site strength of the predicted introns. Application of the method to a representative set of 50 known genes from Arabidopsis thaliana showed significant improvement in prediction accuracy compared to previous spliced alignment methods. The method is also more accurate than ab initio gene prediction methods, provided sufficiently close target proteins are available. In view of the fast growth of public sequence repositories, we argue that close targets will be available for the majority of novel genes, making spliced alignment an excellent practical tool for high-throughput automated genome annotation.     

Assessment of DNA damage in Down Syndrome patients by means of a new, optimised single cell gel electrophoresis technique

Single cell gel electrophoresis (SCGE), also known as comet assay is a widely used method to detect DNA damage. Its use is nonetheless subjected to some pitfalls, due to differences in experimental set-up, to operator-dependent variability and to quantification of the comets, which is usually accomplished by visual scoring or by image-analysis software. Biological variability in the extent of DNA damage must be taken into account particularly regarding in vivo studies. In the present paper we propose an improved methodology where major features are: a) cryopreservation of lymphocytes collected at different time points and simultaneous analysis in a single run; b) use of an internal control on each slide; c) development of a custom-made software with semi - automated image analysis in order to overcome operator dependent variability. Cryopreservation was accomplished by storing lymphocytes in liquid nitrogen in a solution commonly used for preserving vital cells to be reinfused. We found that this procedure did not alter DNA after 2 and 4 months of storage. The use of quality control from a batch of aliquoted lymphocytes from a healthy donor on each slide, enabled to highlight possible experimental anomalies as well as verify inter-experimental variability. Moreover, by using a newly developed software able to automatically recognise comets we minimised operator-dependent variability in the scoring process. This improved methodology is proposed for longitudinal in vivo studies and in the present work its application made it possible to assess a significant increase of DNA in pediatric Down Syndrome patients compared to healthy controls of the same age.     

AFLP technology for DNA fingerprinting

The AFLP technique is a powerful DNA fingerprinting technology applicable to any organism without the need for prior sequence knowledge. The protocol involves the selective PCR amplification of restriction fragments of a total digest of genomic DNA, typically obtained with a mix of two restriction enzymes. Two limited sets of AFLP primers are sufficient to generate a large number of different primer combinations (PCs), each of which will yield unique fingerprints. Visualization of AFLP fingerprints after gel electrophoresis of AFLP products is described using either a conventional autoradiography platform or an automated LI-COR system. The AFLP technology has been used predominantly for assessing the degree of variability among plant cultivars, establishing linkage groups in crosses and saturating genomic regions with markers for gene landing efforts. AFLP fragments may also be used as physical markers to determine the overlap and positions of genomic clones and to integrate genetic and physical maps. Crucial characteristics of the AFLP technology are its robustness, reliability and quantitative nature. This latter feature has been exploited for co-dominant scoring of AFLP markers in sample collections such as F2 or back-cross populations using appropriate AFLP scoring software. This protocol can be completed in 2-3 d.     

Apples to apples: improving the performance of motif finders and their significance analysis in the Twilight Zone

MOTIVATION: Effective algorithms for finding relatively weak motifs are an important practical necessity while scanning long DNA sequences for regulatory elements. The success of such an algorithm hinges on the ability of its scoring function combined with a significance analysis test to discern real motifs from random noise. RESULTS: In the first half of the paper we show that the paradigm of relying on entropy scores and their E-values can lead to undesirable results when searching for weak motifs and we offer alternate approaches to analyzing the significance of motifs. In the second half of the paper we reintroduce a scoring function and present a motif-finder that optimizes it that are more effective in finding relatively weak motifs than other tools. AVAILABILITY: The GibbsILR motif finder is available at http://www.cs.cornell.edu/~keich.     

BEAR, a novel virtual screening methodology for drug discovery

BEAR (binding estimation after refinement) is a new virtual screening technology based on the conformational refinement of docking poses through molecular dynamics and prediction of binding free energies using accurate scoring functions. Here, the authors report the results of an extensive benchmark of the BEAR performance in identifying a smaller subset of known inhibitors seeded in a large (1.5 million) database of compounds. BEAR performance proved strikingly better if compared with standard docking screening methods. The validations performed so far showed that BEAR is a reliable tool for drug discovery. It is fast, modular, and automated, and it can be applied to virtual screenings against any biological target with known structure and any database of compounds.