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PubMed1995

[A simple classification system for all skin flaps].

The arterial quality of a skin flap largely depends on the vascular connections to the surrounding tissue. We can differentiate three typical vascular structures underneath the body's surface: large supporting vessels running parallel to the body's surface, transporting vessels (perforators) with a segmental vascularization and leading to horizontal (reticular) nets of distribution. These structures can be found at the subcutaneous plane, all septofascial planes, and the vascular pattern of the muscles. All three structures can provide sufficient blood to partially separated and mobilized skin areas (flaps). Each skin flap can be classified by the type of enclosed tissue (subcutis, fascia, muscle) and the type of blood supply (axial, segmental, reticular). The quality of blood flow within a skin flap is not disturbed by separation and mobilization if the vascular continuity to the supporting horizontal vessels stays intact. This is the case in socalled axial pattern flaps (the supporting vessel is elevated with the flap) and in segmental pattern flaps (here continuity stays intact with the supporting vessel in deeper layers). In contrast, reticular pattern flaps are characterized by a decreased quality of blood flow (sensible to pressure and tension) because they lack a direct connection to the supporting vessels. Most skin flaps show a complex organization of their vascular structure: they either have a parallel support by several structures (improved quality of blood circulation) or they contain several structures in sequence within their longitudinal axis (decreased quality of blood circulation). This structural analysis does not take into account the hemodynamic conditions and their influence on the quality of blood circulation which are predisposed by the width and the amount of vessels.

Arteries↗
PubMed1995

Efficient, automatic detection of heterozygous bases during large-scale DNA sequence screening.

A crucial factor in the success of positional cloning efforts is the ability to screen rapidly many different candidate genes for mutations. By modifying standard software, we have improved the detection of heterozygous base positions in PCR products sequenced by cycle sequencing. A key element of the method is the incorporation of a modified heterozygote detection algorithm that permits the use of DNA sequence data derived from PCR and sequencing reactions that have not been fully optimized. This allows sequencing runs of average quality to be used. We demonstrate that the sensitivity and specificity of the method are well suited to mutation detection applications such as positional cloning.

Algorithms↗
PubMed1984

Use of unpurified synthetic deoxynucleotide primers for rapid dideoxynucleotide chain termination sequencing.

Synthetic oligodeoxyribonucleotides were used as primers for sequencing DNA by the dideoxy chain termination method. We report that the use of unpurified preparations of such oligonucleotides decreases the time involved in preparing the primers and yields a sequencing reaction of comparable quality to that obtained with pure preparations. It is possible to use unpurified primers if the yield per coupling step during manual or machine synthesis of oligonucleotides is greater than 98%. Also, a method for the rapid purification and desalting of synthetic DNAs is presented for cases in which the coupling is less efficient. Unpurified primers have been used to determine the sequence of a 1987-bp fragment with a single M13 recombinant clone. This DNA was first sequenced using the universal M13 primer. Subsequently, a primer homologous to the 3' end of the newly obtained sequence was synthesized and used to extend the sequence. New primers were synthesized until the sequence of the whole fragment was determined. Because only one clone is necessary, this method can be used to obtain the nucleotide sequence of large DNA fragments in a relatively short time and in an orderly fashion, simplifying analysis of the data.

Base Sequence↗
PubMed1994

Fully-automated, nonradioactive solid-phase sequencing of genomic DNA obtained from PCR.

Nonradioactive sequencing in combination with solid-phase template purification is a powerful method for sequence analysis, especially of PCR-generated fragments. To perform DNA sequencing under optimal conditions, it is necessary to obtain a well-purified and single-stranded (ss) DNA template. The disadvantage of ssDNA sequencing is that additional steps are required to generate ssDNA templates before any sequencing reactions can be carried out and is thus more time-consuming. Here we describe an automated solid-phase system for direct sequencing of ssPCR products that incorporates magnetic beads coated with streptavidin as solid support and a computer-controlled device (PolySeq) with heating, magnetic and mixing functions, all of which are integrated into a robotic workstation (Biomek 1000). This solid-phase method is extremely useful for rapid template purification and strand separation of DNA obtained from PCR as well as for high-quality dideoxyribonucleotide chain termination sequencing with fluorescently labeled primers. The system allows the complete automation of the sequencing procedure starting with PCR amplicons. DNA sequencing results obtained with this system were reproducible and gave an excellent length of readable sequences with low background and an analysis capacity of 30,000-40,000 bp per week.

Base Sequence↗
PubMed2003

Noise sampling method: an ANOVA approach allowing robust selection of differentially regulated genes measured by DNA microarrays.

MOTIVATION: A crucial step in microarray data analysis is the selection of subsets of interesting genes from the initial set of genes. In many cases, especially when comparing a specific condition to a reference, the genes of interest are those which are differentially expressed. Two common methods for gene selection are: (a) selection by fold difference (at least n fold variation) and (b) selection by altered ratio (at least n standard deviations away from the mean ratio). RESULTS: The novel method proposed here is based on ANOVA and uses replicate spots to estimate an empirical distribution of the noise. The measured intensity range is divided in a number of intervals. A noise distribution is constructed for each such interval. Bootstrapping is used to map the desired confidence levels from the noise distribution corresponding to a given interval to the measured log ratios in that interval. If the method is applied on individual arrays having replicate spots, the method can calculate an overall width of the noise distribution which can be used as an indicator of the array quality. We compared this method with the fold change and unusual ratio method. We also discuss the relationship with an ANOVA model proposed by Churchill et al. In silico experiments were performed while controlling the degree of regulation as well as the amount of noise. Such experiments show the performance of the classical methods can be very unsatisfactory. We also compared the results of the 2-fold method with the results of the noise sampling method using pre and post immortalization cell lines derived from the MDAH041 fibroblasts hybridized on Affymetrix GeneChip arrays. The 2-fold method reported 198 genes as upregulated and 493 genes as downregulated. The noise sampling method reported 98 gene upregulated and 240 genes downregulated at the 99.99% confidence level. The methods agreed on 221 genes downregulated and 66 genes upregulated. Fourteen genes from the subset of genes reported by both methods were all confirmed by Q-RT-PCR. Alternative assays on various subsets of genes on which the two methods disagreed suggested that the noise sampling method is likely to provide fewer false positives.

Algorithms↗
PubMed2003

Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management.

The spa gene of Staphylococcus aureus encodes protein A and is used for typing of methicillin-resistant Staphylococcus aureus (MRSA). We used sequence typing of the spa gene repeat region to study the epidemiology of MRSA at a German university hospital. One hundred seven and 84 strains were studied during two periods of 10 and 4 months, respectively. Repeats and spa types were determined by Ridom StaphType, a novel software tool allowing rapid repeat determination, data management and retrieval, and Internet-based assignment of new spa types following automatic quality control of DNA sequence chromatograms. Isolates representative of the most abundant spa types were subjected to multilocus sequence typing and pulsed-field gel electrophoresis. One of two predominant spa types was replaced by a clonally related variant in the second study period. Ten unique spa types, which were equally distributed in both study periods, were recovered. The data show a rapid dynamics of clone circulation in a university hospital setting. spa typing was valuable for tracking of epidemic isolates. The data show that disproval of epidemiologically suggested transmissions of MRSA is one of the main objectives of spa typing in departments with a high incidence of MRSA.

Germany↗
PubMed2001

New advances in brain tumor imaging.

During the period of this review, there has been continued use and development of neuroimaging techniques in brain tumor diagnosis and management. Although no monumental developments or improvements in neuroimaging techniques or technology have occurred, important studies continue to be published showing the clinical impact and utility of various neuroimaging techniques to improve the care of patients with brain tumors. Several studies have shown the power of functional neuroimaging techniques with both magnetic resonance imaging (MRI) and positron-emission tomography (PET) to map eloquent cortex and assist in the planning of surgical and radiation therapy. New nuclear imaging radiopharmaceuticals, including various PET ligands and single photon emission computed tomography (SPECT) agents, have also been developed and show their potential power in the evaluation of brain tumor patients. New MRI pulse sequences to improve image quality and shorten imaging time have also been developed. Several excellent reviews of the use of fluorodeoxyglucose (FDG)-PET and MRI techniques were also published. This article reviews the relevant and important neuroimaging literature related to brain tumor that was published during the defined time period of November 1, 1999 to October 31, 2000. Discussion is organized by modality, including nuclear imaging techniques (SPECT and PET) and MRI (pulse sequence development, contrast agent development, functional MRI developments, and general MRI-related information).

Brain Neoplasms↗
PubMed1995

A computer-controlled conformal radiotherapy system. II: Sequence processor.

PURPOSE: A sequence processor (SP) is described as part of a larger computer-controlled conformal radiotherapy system (CCRS). The SP provides the means to accept and then translate highly sophisticated radiation therapy treatment plans into vendor specific instructions to control treatment delivery on a computer-controlled treatment machine. METHODS AND MATERIALS: The sequence processor (SP) is a small workstation computer that interfaces to the control computer of computer-controlled treatment machines, and to other parts of the larger CCRS system. The system reported here has been interfaced to a computer-controlled racetrack microtron with two treatment gantries, and also to other linear accelerator treatment machines equipped with multileaf collimators. An extensive design process has been used in defining the role of the SP within the context of the larger CCRS project. Flexibility and integration with various components of the project, including databases, treatment planning system, graphical simulator, were key factors in the development. In conjunction with the planned set of treatment fields, a procedural scripting language is used to define the sequence of treatment events that are performed, including operator interactions, communications to other systems such as dosimetry and portal imaging devices, and database management. RESULTS: A flexible system has been developed to allow investigation into procedural steps required for simulating and delivering complex radiation treatments. The system has been used to automate portions of the acceptance testing for the control system of the microtron, and is used for routine daily quality assurance testing. The sequence processor system described here has been used to deliver all clinical treatments performed on the microtron system in 2 years of clinical treatment (more than 200 patients treated to a variety of treatment sites). CONCLUSIONS: The sequence processor system has enabled the delivery of complex treatment using computer-controlled treatment machines. The flexibility of the system allows integration with secondary devices and modification of procedural steps, making it possible to develop effective techniques for insuring safe and efficient computer-controlled conformal radiation therapy treatments.

Computer Simulation↗
PubMed2000

Processing and quality control of DNA array hybridization data.

MOTIVATION: The technology of hybridization to DNA arrays is used to obtain the expression levels of many different genes simultaneously. It enables searching for genes that are expressed specifically under certain conditions. However, the technology produces large amounts of data demanding computational methods for their analysis. It is necessary to find ways to compare data from different experiments and to consider the quality and reproducibility of the data. RESULTS: Data analyzed in this paper have been generated by hybridization of radioactively labeled targets to DNA arrays spotted on nylon membranes. We introduce methods to compare the intensity values of several hybridization experiments. This is essential to find differentially expressed genes or to do pattern analysis. We also discuss possibilities for quality control of the acquired data. AVAILABILITY: http://www.dkfz.de/tbi CONTACT: M.Vingron@dkfz-heidelberg.de

Animals↗
PubMed2004

me-PCR: a refined ultrafast algorithm for identifying sequence-defined genomic elements.

We have adapted the originally described electronic PCR (e-PCR) algorithm to perform string searches more accurately and much more rapidly than previously possible. Our implementation [multithreaded e-PCR (me-PCR)] runs sufficiently fast to allow even desktop machines to query quickly large genomes with very large genomic element sets. In addition, me-PCR is multithreaded, interprets all IUPAC nucleotide symbols, allows searches with elements specified by long sequences (such as SNPs), accepts ranges in the expected PCR size input field, requires substantially less memory for analysis of large sequences and corrects a number of minor flaws causing misreporting of hits in exceptional cases. Thus, me-PCR provides increased annotation capabilities for complex genomes to non-expert laboratories.

Algorithms↗
PubMed2006

A proposed metric for assessing the measurement quality of individual microarrays.

BACKGROUND: High-density microarray technology is increasingly applied to study gene expression levels on a large scale. Microarray experiments rely on several critical steps that may introduce error and uncertainty in analyses. These steps include mRNA sample extraction, amplification and labeling, hybridization, and scanning. In some cases this may be manifested as systematic spatial variation on the surface of microarray in which expression measurements within an individual array may vary as a function of geographic position on the array surface. RESULTS: We hypothesized that an index of the degree of spatiality of gene expression measurements associated with their physical geographic locations on an array could indicate the summary of the physical reliability of the microarray. We introduced a novel way to formulate this index using a statistical analysis tool. Our approach regressed gene expression intensity measurements on a polynomial response surface of the microarray's Cartesian coordinates. We demonstrated this method using a fixed model and presented results from real and simulated datasets. CONCLUSION: We demonstrated the potential of such a quantitative metric for assessing the reliability of individual arrays. Moreover, we showed that this procedure can be incorporated into laboratory practice as a means to set quality control specifications and as a tool to determine whether an array has sufficient quality to be retained in terms of spatial correlation of gene expression measurements.

Algorithms↗
PubMed2006

arrayQCplot: software for checking the quality of microarray data.

UNLABELLED: arrayQCplot is a software for the exploratory analysis of microarray data. This software focuses on quality control and generates newly developed plots for quality and reproducibility checks. It is developed using R and provides a user-friendly graphical interface for graphics and statistical analysis. Therefore, novice users will find arrayQCplot as an easy-to-use software for checking the quality of their data by a simple mouse click. AVAILABILITY: arrayQCplot software is available from Bioconductor at http://www.bioconductor.org. A more detailed manual is available at http://bibs.snu.ac.kr/software/arrayQCplot CONTACT: tspark@stats.snu.ac.kr.

Algorithms↗
PubMed2003

AltAVisT: comparing alternative multiple sequence alignments.

UNLABELLED: We introduce a WWW-based tool that is able to compare two alternative multiple alignments of a given sequence set. Regions where both alignments coincide are color-coded to visualize the local agreement between the two alignments and to identify those regions that can be considered to be reliably aligned. AVAILABILITY: http://bibiserv.techfak.uni-bielefeld.de/altavist/.

Color↗
PubMed2007

Pyrosequencing: a simple method for accurate genotyping.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained in this chapter.

DNA Primers↗
PubMed2003

Use of a three-color cDNA microarray platform to measure and control support-bound probe for improved data quality and reproducibility.

Construction methodologies for cDNA microarrays lack the ability to determine array integrity prior to hybridization, leaving the array itself a source of uncontrolled experimental variation. We solved this problem through development of a three-color cDNA array platform whereby printed probes are tagged with fluorescein and are compatible with Cy3 and Cy5 target labeling dyes when using confocal laser scanners possessing narrow bandwidths. Here we use this approach to: (i) develop a tracking system to monitor the printing of probe plates at predicted coordinates; (ii) define the quantity of immobilized probe necessary for quality hybridized array data to establish pre-hybridization array selection criteria; (iii) investigate factors that influence probe availability for hybridization; and (iv) explore the feasibility of hybridized data filtering using element fluorescein intensity. A direct and significant relationship (R2 = 0.73, P < 0.001) between pre-hybridization average fluorescein intensity and subsequent hybridized replicate consistency was observed, illustrating that data quality can be improved by selecting arrays that meet defined pre-hybridization criteria. Furthermore, we demonstrate that our three-color approach provides a means to filter spots possessing insufficient bound probe from hybridized data sets to further improve data quality. Collectively, this strategy will improve microarray data and increase its utility as a sensitive screening tool.

Color↗
PubMed2006

Novel design and controls for focused DNA microarrays: applications in quality assurance/control and normalization for the Health Canada ToxArray.

BACKGROUND: Microarray normalizations typically apply methods that assume absence of global transcript shifts, or absence of changes in internal control features such as housekeeping genes. These normalization approaches are not appropriate for focused arrays with small sets of genes where a large portion may be expected to change. Furthermore, many microarrays lack control features that can be used for quality assurance (QA). Here, we describe a novel external control series integrated with a design feature that addresses the above issues. RESULTS: An EC dilution series that involves spike-in of a single concentration of the A. thaliana chlorophyll synthase gene to hybridize against spotted dilutions (0.000015 to 100 microM) of a single complimentary oligonucleotide representing the gene was developed. The EC series is printed in duplicate within each subgrid of the microarray and covers the full range of signal intensities from background to saturation. The design and placement of the series allows for QA examination of frequently encountered problems in hybridization (e.g., uneven hybridizations) and printing (e.g., cross-spot contamination). Additionally, we demonstrate that the series can be integrated with a LOWESS normalization to improve the detection of differential gene expression (improved sensitivity and predictivity) over LOWESS normalization on its own. CONCLUSION: The quality of microarray experiments and the normalization methods used affect the ability to measure accurate changes in gene expression. Novel methods are required for normalization of small focused microarrays, and for incorporating measures of performance and quality. We demonstrate that dilution of oligonucleotides on the microarray itself provides an innovative approach allowing the full dynamic range of the scanner to be covered with a single gene spike-in. The dilution series can be used in a composite normalization to improve detection of differential gene expression and to provide quality control measures.

Animals↗
PubMed1994

Multiple protein structure alignment.

A method was developed to compare protein structures and to combine them into a multiple structure consensus. Previous methods of multiple structure comparison have only concatenated pairwise alignments or produced a consensus structure by averaging coordinate sets. The current method is a fusion of the fast structure comparison program SSAP and the multiple sequence alignment program MULTAL. As in MULTAL, structures are progressively combined, producing intermediate consensus structures that are compared directly to each other and all remaining single structures. This leads to a hierarchic "condensation," continually evaluated in the light of the emerging conserved core regions. Following the SSAP approach, all interatomic vectors were retained with well-conserved regions distinguished by coherent vector bundles (the structural equivalent of a conserved sequence position). Each bundle of vectors is summarized by a resultant, whereas vector coherence is captured in an error term, which is the only distinction between conserved and variable positions. Resultant vectors are used directly in the comparison, which is weighted by their error values, giving greater importance to the matching of conserved positions. The resultant vectors and their errors can also be used directly in molecular modeling. Applications of the method were assessed by the quality of the resulting sequence alignments, phylogenetic tree construction, and databank scanning with the consensus. Visual assessment of the structural superpositions and consensus structure for various well-characterized families confirmed that the consensus had identified a reasonable core.

Amino Acid Sequence↗
PubMed2006

Evaluation of DNA microarray results with quantitative gene expression platforms.

We have evaluated the performance characteristics of three quantitative gene expression technologies and correlated their expression measurements to those of five commercial microarray platforms, based on the MicroArray Quality Control (MAQC) data set. The limit of detection, assay range, precision, accuracy and fold-change correlations were assessed for 997 TaqMan Gene Expression Assays, 205 Standardized RT (Sta)RT-PCR assays and 244 QuantiGene assays. TaqMan is a registered trademark of Roche Molecular Systems, Inc. We observed high correlation between quantitative gene expression values and microarray platform results and found few discordant measurements among all platforms. The main cause of variability was differences in probe sequence and thus target location. A second source of variability was the limited and variable sensitivity of the different microarray platforms for detecting weakly expressed genes, which affected interplatform and intersite reproducibility of differentially expressed genes. From this analysis, we conclude that the MAQC microarray data set has been validated by alternative quantitative gene expression platforms thus supporting the use of microarray platforms for the quantitative characterization of gene expression.

Equipment Design↗