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PubMed2004

Quantitative DNA methylation analysis based on four-dye trace data from direct sequencing of PCR amplificates.

MOTIVATION: Methylation of cytosines in DNA plays an important role in the regulation of gene expression, and the analysis of methylation patterns is fundamental for the understanding of cell differentiation, aging processes, diseases and cancer development. Such analysis has been limited, because technologies for detailed and efficient high-throughput studies have not been available. We have developed a novel quantitative methylation analysis algorithm and workflow based on direct DNA sequencing of PCR products from bisulfite-treated DNA with high-throughput sequencing machines. This technology is a prerequisite for success of the Human Epigenome Project, the first large genome-wide sequencing study for DNA methylation in many different tissues. Methylation in tissue samples which are compositions of different cells is a quantitative information represented by cytosine/thymine proportions after bisulfite conversion of unmethylated cytosines to uracil and PCR. Calculation of quantitative methylation information from base proportions represented by different dye signals in four-dye sequencing trace files needs a specific algorithm handling imbalanced and overscaled signals, incomplete conversion, quality problems and basecaller artifacts. RESULTS: The algorithm we developed has several key properties: it analyzes trace files from PCR products of bisulfite-treated DNA sequenced directly on ABI machines; it yields quantitative methylation measurements for individual cytosine positions after alignment with genomic reference sequences, signal normalization and estimation of effectiveness of bisulfite treatment; it works in a fully automated pipeline including data quality monitoring; it is efficient and avoids the usual cost of multiple sequencing runs on subclones to estimate DNA methylation. The power of our new algorithm is demonstrated with data from two test systems based on mixtures with known base compositions and defined methylation. In addition, the applicability is proven by identifying CpGs that are differentially methylated in real tissue samples.

Algorithms↗

PubMed2006

A summarization approach for Affymetrix GeneChip data using a reference training set from a large, biologically diverse database.

BACKGROUND: Many of the most popular pre-processing methods for Affymetrix expression arrays, such as RMA, gcRMA, and PLIER, simultaneously analyze data across a set of predetermined arrays to improve precision of the final measures of expression. One problem associated with these algorithms is that expression measurements for a particular sample are highly dependent on the set of samples used for normalization and results obtained by normalization with a different set may not be comparable. A related problem is that an organization producing and/or storing large amounts of data in a sequential fashion will need to either re-run the pre-processing algorithm every time an array is added or store them in batches that are pre-processed together. Furthermore, pre-processing of large numbers of arrays requires loading all the feature-level data into memory which is a difficult task even with modern computers. We utilize a scheme that produces all the information necessary for pre-processing using a very large training set that can be used for summarization of samples outside of the training set. All subsequent pre-processing tasks can be done on an individual array basis. We demonstrate the utility of this approach by defining a new version of the Robust Multi-chip Averaging (RMA) algorithm which we refer to as refRMA. RESULTS: We assess performance based on multiple sets of samples processed over HG U133A Affymetrix GeneChip arrays. We show that the refRMA workflow, when used in conjunction with a large, biologically diverse training set, results in the same general characteristics as that of RMA in its classic form when comparing overall data structure, sample-to-sample correlation, and variation. Further, we demonstrate that the refRMA workflow and reference set can be robustly applied to naïve organ types and to benchmark data where its performance indicates respectable results. CONCLUSION: Our results indicate that a biologically diverse reference database can be used to train a model for estimating probe set intensities of exclusive test sets, while retaining the overall characteristics of the base algorithm. Although the results we present are specific for RMA, similar versions of other multi-array normalization and summarization schemes can be developed.

Algorithms↗

PubMed2002

Process involved in reading imaging studies: workflow analysis and implications for workstation development.

Software development for imaging workstations has lagged behind hardware availability. To guide development and to analyze work flow involved in interpretation of cross-sectional imaging studies, we assessed the cognitive and physical processes. We observed the performance and interpretation of body computed tomography (CT scans and recorded the events that occurred during this process. We studied work flow using a bottleneck analysis. Twenty-four ofa total of 54 cases (44%) involved comparing the images with those of prior scans. Forty-seven of 54 scans (87%) were viewed using windows other than soft tissue, or compared with precontrast scans. In 46 cases (85%), the interpretation stopped to return to a previous level for review. Measurement of lesions was performed in 24 of 54 (44%) cases, and in 15 (63%)of these cases, measurements were taken of lesions on old studies for comparison. Interpretation was interrupted in 14 of 54 cases (26%) by referring clinicians desiring consultation. The work flow analysis showed film folder retrieval by the film room to be the bottleneck for interpretation by film. For picture archiving and communication system (PACS) reading,the CT examination itself proved to be the bottleneck. We conclude that workstations for CT interpretation should facilitate movement within scans, comparison with prior examinations, and measuring lesions on these scans. Workstation design should consider means of optimizing time currently not used between interpretation sessions, minimizing interruptions and providing more automated functions currently requiring physician interaction.

Efficiency, Organizational↗

PubMed1997

Processes involved in reading imaging studies: workflow analysis and implications for workstation development.

Software development for imaging workstations has lagged behind hardware availability. To guide development and to analyze work flow involved in interpretation of cross-sectional imaging studies, we assessed the cognitive and physical processes. We observed the performance and interpretation of body computed tomography (CT) scans and recorded the events that occurred during this process. We studied work flow using a bottleneck analysis. Twenty-four of a total of 54 cases (44%) involved comparing the images with those of prior scans. Forty-seven of 54 scans (87%) were viewed using windows other than soft tissue, or compared with precontrast scans. In 46 cases (85%), the interpretation stopped to return to a previous level for review. Measurement of lesions was performed in 24 of 54 (44%) cases, and in 15 (63%) of these cases, measurements were taken of lesions on old studies for comparison. Interpretation was interrupted in 14 of 54 cases (26%) by referring clinicians desiring consultation. The work flow analysis showed film folder retrieval by the film room to be the bottleneck for interpretation by film. For picture archiving and communication system (PACS) reading, the CT examination itself proved to be the bottleneck. We conclude that workstations for CT interpretation should facilitate movement within scans, comparison with prior examinations, and measuring lesions on these scans. Workstation design should consider means of optimizing time currently not used between interpretation sessions, minimizing interruptions and providing more automated functions currently requiring physician interaction.

Cross-Sectional Studies↗

PubMed2026

Reconstruction of ancestral plant genomes for inter-crop translational research.

We present Ancestral Genome Reconstruction (AGR), an exploratory framework for the automated inference of "paleogenomes" from large-scale comparative datasets. By analyzing 84 extant angiosperm species, we reconstructed 10 key ancestral angiosperm genomes millions of years old. These reconstructed ancestors were instrumental in (1) estimating when angiosperms emerged, when major botanical families originated, and when shared ancestral whole-genome duplication events occurred; and (2) tracing the evolutionary trajectories of ancestral chromosomes and genes, especially those that may have driven the emergence of key life-history traits (e.g., woody vs. herbaceous, aquatic vs. terrestrial, C3 vs. C4, and symbiotic root-nodulating vs. non-nodulating species). We demonstrated that these paleogenomes serve as tractable backbones for inter-crop translational research. Through an open-access web tool, OrthoViewer, we identified orthologs that have retained the same ancestral genomic context, favoring the identification of genes associated with "phenologs"- orthologous genes across species driving analogous phenotypes, traits, or processes-exemplified by FUWA for yield components, FLC for flowering time, and DDM1 for DNA methylation. Taken together, this study provides a testable paleogenomic workflow, opening novel avenues for integrating evolutionary genomics data into modern climate-smart crop breeding and supporting the agroecological transition.

Genome, Plant↗

PubMed2025

Optimized GMP-grade production of non-viral Sleeping Beauty-generated CARCIK cells for enhanced fitness and clinical scalability.

BACKGROUND: Strict adherence to GMP guidelines and regulatory compliance is crucial when transitioning from research to clinical-grade production of ATMPs like CAR T cells. The success of CAR T cell therapy in treating hematological malignancies highlights the need for closed or automated systems to ensure quality and efficacy. Recent evidence also suggests that ex vivo culture conditions can significantly impact CAR T cell functionality. METHODS: We present our optimized methodology for expanding Sleeping Beauty transposon-engineered Chimeric Antigen Receptor-Cytokine-Induced Killer (CARCIK) cells using G-Rex devices and evaluate its impact on CARCIK cell phenotype and T cell fitness. RESULTS: Building on our previously validated protocol, we introduced key simplifications to optimize the CARCIK differentiation process. Delaying the nucleofection step eliminated the need for feeder cells while maintaining efficient CAR expression and high cell viability. Transitioning from T-flasks to G-Rex bioreactors reduced operator hands-on time from 21 to 28 days to 14-17 days and resulted in a less differentiated CARCIK cell product. Metabolic and transcriptional analyses showed that the novel protocol improves CARCIK cell fitness and in vivo efficacy against B-cell lymphoma. The novel method was validated in Good Manufacturing Practices (GMP) conditions at our two Cell Factories and yielded enough numbers of CARCIK-CD19 cells for clinical use. CONCLUSIONS: Optimizing non-viral CARCIK cell production using G-Rex bioreactors and refined timing adjustments has streamlined the workflow, enhanced cell fitness, and resulted in a highly effective therapeutic product with demonstrated in vivo efficacy in mice. These improvements reduced manipulation and contamination risks, while optimizing logistics and space efficiency, facilitating allogeneic CARCIK generation for a current phase I/II clinical trial (NCT05869279) in patients with R/R CD19 + non-Hodgkin Lymphoma (B-cell NHL) and Chronic Lymphocytic Leukemia (CLL), confirming the approach's scalability and clinical potential.

Humans↗

PubMedDate not supplied

Radiology speech recognition: workflow, integration, and productivity issues.

Continuous voice recognition is now a reality, with at least 3 major vendors providing independent systems for radiology departments. However, to be truly useful, such systems must be integrated into the overall medical informatics architecture including order entry into the radiology information system, the ability to receive orders from the radiology information system, acceptance and transcription of dictation, and transmission of validated reports to the hospital information system (HIS)for billing capture. In addition, the system should permit sufficient flexibility to permit radiologists to operate in multiple modes: real-time recognition, batch processing, or spooling of audio files for later transcription by human agents, which is especially useful for very difficult accents. This article demonstrates the various modes of operation for 2 recognition systems that have been used in our center, describes the basics of their recognition models, examines the HL7 messages required for medical informatics integration, and finally illustrates the effect on report turnaround times.

Computer Communication Networks↗

PubMed2026

Combining Annotation Software to Identify Orthologous Genes (CASIO) Provides a New Dataset of Orthologous Genes for Swallowtail Butterflies.

With the massive increase in genomic resources, it is becoming increasingly popular to analyse thousands of loci across many species. However, many of the available genomes are not annotated, which hinders an efficient search for orthologous protein-coding genes. Here, we aim to develop a semi-automated pipeline and compare four genomic annotation methods (BRAKER2, BUSCO, Miniprot and Scipio). Our results highlight the importance of integrating multiple annotation tools to optimise ortholog detection and improve genomic studies. Each annotation method showed different strengths. BRAKER2 annotated a substantial number of genes. BUSCO, despite limitations inherent to its reference database, identified a higher number of orthologs. Miniprot exhibited notable flexibility in accommodating diverse protein datasets, whereas Scipio successfully recovered a considerable set of genes that were not detected by the other tools. The combination of these tools allowed for more comprehensive ortholog detection. Taking advantage of this pipeline, we developed a comprehensive dataset of orthologous genes for swallowtail butterflies (Lepidoptera: Papilionidae), called Papilionidae_odb, which will facilitate future studies, especially for a non-model group with abundant genomic data and few transcriptomic resources. We tested Papilionidae_odb by inferring a robust phylogenetic framework for Leptocircini using 142 complete genomes, which improved branch support for some phylogenetic relationships, although challenges remained in resolving relationships within certain species groups, likely due to rapid radiations. Our results highlight the complementary nature of the annotation methods and suggest that combining these tools can yield more accurate results in genomic research. This approach was implemented in a Snakemake workflow called CASIO (Combining Annotation Software to Identify Orthologous genes) and can easily be applied to other non-model groups to improve genomic datasets in diverse taxa where transcriptomic resources are still limited.

Animals↗

PubMed2026

Challenges for reproducibility in species delimitation.

Species richness is a foundational metric for comparing biodiversity among clades and regions in ecology, evolution, and conservation. As the biodiversity crisis accelerates, taxonomists face increasing pressure to delimit and name species rapidly, often relying on automated or semi-automated methods that prioritize speed over thoroughness. Yet the reproducibility of species delimitation (the degree to which independent experts reach consistent conclusions given the same evidence) remains largely unquantified, and its consequences for estimates of species richness have never been assessed at the scale of an entire fauna. This gap is consequential: if species delimitation is highly variable among practitioners, then published species counts may reflect the idiosyncrasies of individual taxonomists as much as the true biological structure of diversity within and among clades. Here we evaluate the precision, or reproducibility, of species delimitation in Neotropical freshwater fishes, the most species-rich continental vertebrate assemblage. We provided identical morphological and molecular datasets for species representing four genera to 40 taxonomic experts (10 per genus), who were asked to delimit species using (1) their preferred analytical approaches and (2) standardized analytical outputs. Total variance in species delimitation was partitioned into variance attributable to data analysis and interpretation of results. Total discordance was high (35.0%), and although standardization of analytical methods reduced discordance, substantial variance remained (23.5%) due to interpretative differences. Deviations from modal species estimates were not explained by any of seven expert attributes assessed, including taxonomic experience, publication record, geographic location, taxonomic concepts, or analytical methods. These results demonstrate that species delimitation can be subject to considerable subjectivity, even among experienced taxonomists working with identical data. Improving the precision of species delimitation will require coordinated advances across the full taxonomic workflow, including greater standardization of data acquisition and analysis, and clearer interpretative frameworks that explicitly define the evidentiary thresholds required to recognize species boundaries. Community-wide adoption of transparent reporting standards, analogous to those developed in genomics and clinical research, would help expose the sources of interpretative disagreement and facilitate more consistent application of species concepts across taxa and research groups. Developing benchmark datasets and shared reference taxonomies, against which new delimitation hypotheses can be evaluated, represents a tractable near-term goal for the systematic community. Ultimately, however, reproducible taxonomy cannot be achieved through procedural standardization alone. High-quality revisionary taxonomy must be grounded in experienced character evaluation and homology assessment, concept delimitation, and contingent analytical judgment, skills that are developed over years of immersive engagement with natural history collections, primary literature, and fieldwork. The decline of training opportunities in classical systematics therefore poses a direct threat not only to taxonomic productivity but to taxonomic consistency. Continued investment in a well-trained community of systematists, supported by institutions, collections, and funding agencies, remains the most reliable foundation for consistent and accurate species delimitation. Our results underscore that biodiversity metrics widely used in ecology, conservation planning, and macroevolutionary research are sensitive to practitioner variation in ways that have not previously been quantified, and that addressing this variation requires both methodological reform and sustained commitment to systematic expertise.

biodiversity↗

PubMed2004

[Automatic milking systems--quality assurance of milk for drinking].

German consumers trust the safety and the quality of milk and milk products. Compared with other animal products, e. g. meat and meat products, their confidence is justified in so far as milk and milk products cause only few foodborne diseases in Germany, although 80 percent of all German cows develop at least one case of mastitis per lactation. Due to financial reasons, more and more German dairy farmers are forced to initiate a time-saving rationalization of their workflow. Therefore, automatic milking systems (AMS) are used increasingly, even though the costs of purchase result in a slow pick-up in sales. Moreover, AMS do not comply with legal requirements. Thus, an additional regular, the so called "catalogue of measures", had to be enacted to ensure the hygienic harmlessness of milk produced by AMS. This is the first time, that udder health at individual cow level was related to milk quality beyond merely clinical signs. Together with technical innovations for an improved health monitoring at cow and herd level as well as the implementation of quality assurance programs based on prevention, this improvement offers good prospects to produce not only a hygienically harmless, but also a physiologically composed milk and therefore a qualitatively high-grade milk from healthy cows. Being the vehicle of the most recent improvements in technology, AMS do have the potential to make a crucial contribution to this development.

Animal Husbandry↗

PubMedDate not supplied

Strengthening your ties to referring physicians through RIS/PACS integration.

Many imaging centers are turning to technology solutions to increase refering physician satisfaction, implementing such enhancements as automated report distribution, picture archiving and communications system (PACS), radiology information systems (RIS), and web-based results access. However, without seamless integration, these technology investments don't address the challenge at its core: convenient and reliable, two-way communication and interaction with referring physicians. In an integrated RIS/PACS solution, patient tracking in the RIS and PACS study status are logged and available to users. The time of the patient's registration at the imaging center, the exam start and completion time, the patient's departure time from the imaging center, and results status are all tracked and logged. An integrated RIS/PACS solution provides additional support to the radiologist, a critical factor that can improve the turnaround time of results to referring physicians. The RIS/PACS enhances the interpretation by providing the patient's history, which gives the radiologist additional insight and decreases the likelihood of missing a diagnostic element. In a tightly integrated RIS/PACS solution, results information is more complete. Physicians can view reports with associated images selected by the radiologist. They will also have full order information and complete imaging history including prior reports and images. Referring physicians can access and view images and exam notes at the same time that the radiologist is interpreting the exam. Without the benefit of an integrated RIS/PACS system, the referring physician would have to wait for the signed transcription to be released. In a seamlessly integrated solution, film-tracking modules within the RIS are fused with digital imaging workflow in the PACS. Users can see at a glance if a historical exam is available on film and benefit when a complete study history--both film-based and digital--is presented with the current case. It is up to the imaging center to market the benefits of reduced errors, reduced turnaround times, and a higher level of service to referring physician community, and encourage them to take advantage of the convenience it provides. The savvy imaging center will also regard the integrated RIS/PACS as a valuable marketing tool for use in attracting radiologists.

Appointments and Schedules↗

PubMed2003

Validation of a novel, fully integrated and flexible microarray benchtop facility for gene expression profiling.

Here we describe a novel microarray platform that integrates all functions needed to perform any array-based experiment in a compact instrument on the researcher's laboratory benchtop. Oligonucle otide probes are synthesized in situ via a light- activated process within the channels of a three-dimensional microfluidic reaction carrier. Arrays can be designed and produced within hours according to the user's requirements. They are processed in a fully automatic workflow. We have characterized this new platform with regard to dynamic range, discrimination power, reproducibility and accuracy of biological results. The instrument detects sample RNAs present at a frequency of 1:100 000. Detection is quantitative over more than two orders of magnitude. Experiments on four identical arrays with 6398 features each revealed a mean coefficient of variation (CV) value of 0.09 for the 6398 unprocessed raw intensities indicating high reproducibility. In a more elaborate experiment targeting 1125 yeast genes from an unbiased selection, a mean CV of 0.11 on the fold change level was found. Analyzing the transcriptional response of yeast to osmotic shock, we found that biological data acquired on our platform are in good agreement with data from Affymetrix GeneChips, quantitative real-time PCR and--albeit somewhat less clearly--to data from spotted cDNA arrays obtained from the literature.

Automation↗