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PubMed1997

Solution structure and base pair opening kinetics of the i-motif dimer of d(5mCCTTTACC): a noncanonical structure with possible roles in chromosome stability.

BACKGROUND: Repetitive cytosine-rich DNA sequences have been identified in telomeres and centromeres of eukaryotic chromosomes. These sequences play a role in maintaining chromosome stability during replication and may be involved in chromosome pairing during meiosis. The C-rich repeats can fold into an 'i-motif' structure, in which two parallel-stranded duplexes with hemiprotonated C.C+ pairs are intercalated. Previous NMR studies of naturally occurring repeats have produced poor NMR spectra. This led us to investigate oligonucleotides, based on natural sequences, to produce higher quality spectra and thus provide further information as to the structure and possible biological function of the i-motif. RESULTS: NMR spectroscopy has shown that d(5mCCTTTACC) forms an i-motif dimer of symmetry-related and intercalated folded strands. The high-definition structure is computed on the basis of the build-up rates of 29 intraresidue and 35 interresidue nuclear Overhauser effect (NOE) connectivities. The i-motif core includes intercalated interstrand C.C+ pairs stacked in the order 2*.8/1.7*/1*.7/2.8* (where one strand is distinguished by an asterisk and the numbers relate to the base positions within the repeat). The TTTA sequences form two loops which span the two wide grooves on opposite sides of the i-motif core; the i-motif core is extended at both ends by the stacking of A6 onto C2.C8+. The lifetimes of pairs C2.C8+ and 5mC1.C7+ are 1 ms and 1 s, respectively, at 15 degrees C. Anomalous exchange properties of the T3 imino proton indicate hydrogen bonding to A6 N7 via a water bridge. The d(5mCCTTTTCC) deoxyoligonucleotide, in which position 6 is occupied by a thymidine instead of an adenine, also forms a symmetric i-motif dimer. However, in this structure the two TTTT loops are located on the same side of the i-motif core and the C.C+ pairs are formed by equivalent cytidines stacked in the order 8*.8/1.1*/7*.7/2.2*. CONCLUSIONS: Oligodeoxynucleotides containing two C-rich repeats can fold and dimerize into an i-motif. The change of folding topology resulting from the substitution of a single nucleoside emphasizes the influence of the loop residues on the i-motif structure formed by two folded strands.

Cytidine↗

PubMed2004

arrayMagic: two-colour cDNA microarray quality control and preprocessing.

UNLABELLED: arrayMagic is a software package for quality control and preprocessing of two-colour cDNA microarray data. The automated analysis pipeline comprises data import, normalization, replica merging, quality diagnostics and data export. The script-based processing combines reproducibility and flexibility at high-throughput and provides quality-assured and preprocessed microarray data to high-level follow-up analysis. AVAILABILITY: The R package arrayMagic is available with BSD license at http://www.bioconductor.org CONTACT: a.buness@dkfz.de SUPPLEMENTARY INFORMATION: The package contains documentation in the form of manual pages and a vignette with a guided tour of a typical workflow.

Algorithms↗

PubMed2001

Quality control in manufacturing oligo arrays: a combinatorial design approach.

The advent of the DNA microarray technology has brought with it the exciting possibility of simultaneously observing the expression levels of all genes in an organism. One such microarray technology, called "oligo arrays", manufactures short single strands of DNA (called probes) onto a glass surface using photolithography. An altered or missed step in such a manufacturing protocol can adversely affect all probes using this failed step, and is in general impossible to disentangle from experimental variation when using such a defective array. The idea of designing special quality control probes to detect a failed step was first formulated by Hubbell and Pevzner. We consider an alternative formulation of this problem and use a combinatorial design approach to solve it. Our results improve over prior work in guaranteeing coverage of all protocol steps and in being able to tolerate a greater number of unreliable probe intensities.

Combinatorial Chemistry Techniques↗

PubMed2002

Quality control in manufacturing oligo arrays: a combinatorial design approach.

The advent of the DNA microarray technology has brought with it the exciting possibility of simultaneously observing the expression levels of all genes in an organism. One such microarray technology, called "oligo arrays," manufactures short single strands of DNA (called probes) onto a glass surface using photolithography. An altered or missed step in such a manufacturing protocol can adversely affect all probes using this failed step and is in general impossible to disentangle from experimental variation when using such a defective array. The idea of designing special quality control probes to detect a failed step was first formulated by Hubbell and Pevzner (1999). We consider an alternative formulation of this problem and use a combinatorial design approach to solve it. Our results improve over prior work in guaranteeing coverage of all protocol steps and in being able to tolerate a greater number of unreliable probe intensities.

Combinatorial Chemistry Techniques↗

PubMed1994

[MRT of the female pelvis: turbo-spin-echo (TSE) sequences compared to conventional spin-echo sequences at 0.5 tesla].

Markedly T2-weighted MR images of the pelvis can be obtained with TSE sequences using short acquisition times. In order to determine whether TSE sequences at 0.5 Tesla produce diagnostically valuable images, 9 normals and 53 patients with suspected gynaecological tumours were examined with TSE and SE sequences. Visual comparison showed a considerable superiority of the TSE sequences in most respects. The quality of the TSE images was regarded as very good in 82% and good in 18% (SE sequences: 27% very good, 55% good, 18% moderate), delineation of anatomical structures 81% very good and 19% good (SE sequence: 31% very good, 53% good, 16% moderate). TSE sequences were also superior in the detection of pathological changes (87% very good, 13% good) compared with SE sequences (56% very good, 37% good, 7% moderate). Venous plexuses in the parametrium were better demonstrated by SE sequences. There were significantly higher signal to noise ratios (SNR) with TSE sequences for fat, bone marrow (each p < 0.001), and urine (p < 0.05). Using muscle as reference tissue, there were significantly higher signal-difference to noise ratios (SDR) for fat (p < 0.001), and bone marrow (p < 0.0001), the other pelvic organs and tumours showed no significant differences between the SNR and SDR. TSE sequences are well suited to replace SE sequences in the MR diagnosis of gynaecological tumours.

Adolescent↗

PubMed2000

The genome sequence of Drosophila melanogaster.

The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.

Animals↗

PubMed2006

Assessment of the relationship between pre-chip and post-chip quality measures for Affymetrix GeneChip expression data.

BACKGROUND: Gene expression microarray experiments are expensive to conduct and guidelines for acceptable quality control at intermediate steps before and after the samples are hybridised to chips are vague. We conducted an experiment hybridising RNA from human brain to 117 U133A Affymetrix GeneChips and used these data to explore the relationship between 4 pre-chip variables and 22 post-chip outcomes and quality control measures. RESULTS: We found that the pre-chip variables were significantly correlated with each other but that this correlation was strongest between measures of RNA quality and cRNA yield. Post-mortem interval was negatively correlated with these variables. Four principal components, reflecting array outliers, array adjustment, hybridisation noise and RNA integrity, explain about 75% of the total post-chip measure variability. Two significant canonical correlations existed between the pre-chip and post-chip variables, derived from MAS 5.0, dChip and the Bioconductor packages affy and affyPLM. The strongest (CANCOR 0.838, p < 0.0001) correlated RNA integrity and yield with post chip quality control (QC) measures indexing 3'/5' RNA ratios, bias or scaling of the chip and scaling of the variability of the signal across the chip. Post-mortem interval was relatively unimportant. We also found that the RNA integrity number (RIN) could be moderately well predicted by post-chip measures B_ACTIN35, GAPDH35 and SF. CONCLUSION: We have found that the post-chip variables having the strongest association with quantities measurable before hybridisation are those reflecting RNA integrity. Other aspects of quality, such as noise measures (reflecting the execution of the assay) or measures reflecting data quality (outlier status and array adjustment variables) are not well predicted by the variables we were able to determine ahead of time. There could be other variables measurable pre-hybridisation which may be better associated with expression data quality measures. Uncovering such connections could create savings on costly microarray experiments by eliminating poor samples before hybridisation.

Artifacts↗

PubMed2003

Identification of mitochondrial DNA substitutions related to meat quality in Japanese Black cattle.

Complete sequences of mitochondrial (mt) genomes of eight Japanese Black cattle were determined to investigate the relationships between mt deoxyribonucleic acid (DNA) displacement loop (D-loop) types and other mtDNA regions and to identify the variation in the coding region that may influence the economic traits. The survey of mitochondrial sequences in the encoding region revealed 14 substitutions including six antonymous substitutions and one in 16S ribosomal ribonucleic acid (rRNA). Three methods of polymorphic DNA analyses (polymerase chain reaction [PCR]-restriction fragment length polymorphism [RFLP], mismatch PCR-RFLP, PCR-single-strand conformation polymorphism [SSCP]) were performed on these seven candidate substitutions (base pair [bp] 2,232, 12,158, 12,908, 13,310, 14,122, 14,140, and 14,565) for 202 Japanese Black cattle. The substitution of bp 13,310 was observed in all samples, but not in the reference sequence, indicating that this is a minor substitution or a sequencing mistake in the reference sequence. The substitutions at bp 14,122, 14,140, and 14,565 were observed in only a few samples, suggesting that these were also minor substitutions. The substitutions at bp 2,232 (16S rRNA), 12,158, and 12,908 (reduced nicotinamide adenine dinucleotide-ubiquinone oxidoreductase chain-5) were closely related to mitochondrial D-loop types that have previously been related to differences in the carcass traits of Japanese Black cattle. Evaluation of the effects on six carcass traits with mixed model procedures suggests that the bp 2,232 substitution affects longissimus muscle area and beef marbling score. The substitution at bp 2,232 is a strong candidate for the mitochondrial effect on meat quality.

Animals↗

PubMed2003

Whole genome shotgun sequencing guided by bioinformatics pipelines--an optimized approach for an established technique.

While the sequencing of bacterial genomes has become a routine procedure at major sequencing centers, there are still a number of genome projects at small- or medium-size facilities. For these facilities a maximum of control over sequencing, assembling and finishing is essential. At the same time, facilities have to be able to co-operate at minimum costs for the overall project. We have established a pipeline for the distributed sequencing of Alcanivorax borkumensis SK2, Azoarcus sp. BH72, Clavibacter michiganensis subsp. michiganensis NCPPB382, Sorangium cellulosum So ce56 and Xanthomonas campestris pv. vesicatoria 85-10. Our pipeline relies on standard tools (e.g. PHRED/PHRAP, CAP3 and Consed/Autofinish) wherever possible, supplementing them with new tools (BioMake and BACCardI) to achieve the aims described above.

Algorithms↗

PubMed2002

Optimal gene expression analysis by microarrays.

DNA microarrays make possible the rapid and comprehensive assessment of the transcriptional activity of a cell, and as such have proven valuable in assessing the molecular contributors to biological processes and in the classification of human cancers. The major challenge in using this technology is the analysis of its massive data output, which requires computational means for interpretation and a heightened need for quality data. The optimal analysis requires an accounting and control of the many sources of variance within the system, an understanding of the limitations of the statistical approaches, and the ability to make sense of the results through intelligent database interrogation.

Algorithms↗

PubMed2003

ESTprep: preprocessing cDNA sequence reads.

MOTIVATION: High accuracy of data always governs the large-scale gene discovery projects. The data should not only be trustworthy but should be correctly annotated for various features it contains. Sequence errors are inherent in single-pass sequences such as ESTs obtained from automated sequencing. These errors further complicate the automated identification of EST-related sequencing. A tool is required to prepare the data prior to advanced annotation processing and submission to public databases. RESULTS: This paper describes ESTprep, a program designed to preprocess expressed sequence tag (EST) sequences. It identifies the location of features present in ESTs and allows the sequence to pass only if it meets various quality criteria. Use of ESTprep has resulted in substantial improvement in accurate EST feature identification and fidelity of results submitted to GenBank. AVAILABILITY: The program is freely available for download from http://genome.uiowa.edu/pubsoft/software.html

Algorithms↗

PubMed2002

Sequence diversity of Jeryl Lynn strain of mumps virus: quantitative mutant analysis for vaccine quality control.

The Jeryl Lynn strain of mumps vaccine live (MVL) was developed in 1966 by Merck Co. and has been widely used in the U.S. and other countries since the early 1970s. Partial sequencing has recently shown that the vaccine contains a mixture of two substrains with substantially different nucleotide sequences. We have determined the complete genomic sequences of both substrains and identified 414 nucleotide differences (2.69%), leading to 87 amino acid substitutions (1.67%). We used this information to develop methods for quantification of the substrain components in vaccine samples based on PCR and restriction enzyme cleavage and oligonucleotide microarray hybridization and monitored their dynamics in viral populations propagated in different conditions. Passaging Jeryl Lynn strain in Vero or CEF cell cultures resulted in rapid selection of the major component JL1, while growth in embryonated chicken eggs (ECE) favored accumulation of the minor component JL2. Based on the findings presented here, it is proposed that the substrain composition of Jeryl Lynn vaccine can be monitored as a part of its quality control to ensure consistency of the vaccine.

Amino Acid Sequence↗

PubMed2006

MASQOT-GUI: spot quality assessment for the two-channel microarray platform.

UNLABELLED: MASQOT-GUI provides an open-source, platform-independent software pipeline for two-channel microarray spot quality control. This includes gridding, segmentation, quantification, quality assessment and data visualization. It hosts a set of independent applications, with interactions between the tools as well as import and export support for external software. The implementation of automated multivariate quality control assessment, which is a unique feature of MASQOT-GUI, is based on the previously documented and evaluated MASQOT methodology. Further abilities of the application are outlined and illustrated. AVAILABILITY: MASQOT-GUI is Java-based and licensed under the GNU LGPL. Source code and installation files are available for download at http://masqot-gui.sourceforge.net/

Computer Graphics↗

PubMed2026

ImpuT2T: Pangenome-Based Patching for Human Genome Assemblies.

With improvements in sequencing and assembly have come many high-quality telomere-to-telomere assemblies and reference pangenomes. However, the long-read sequencing recipes needed for high quality assemblies are expensive, and out of reach for many research groups. Here we propose ImpuT2T, a method that takes an assembly produced via inexpensive HiFi sequencing reads, and uses a panel of T2T (or near-T2T) assemblies to scaffold and fill ("patch") the gaps between the HiFi contigs. Benchmarking against reference assemblies demonstrates that ImpuT2T is highly effective at patching human HiFi assemblies, consistently outperforming existing patching approaches. Moreover, we show that including more haplotypes in the pangenome improves the quality of the patched assemblies, with the greatest gains achieved using the full HPRC Release 2 pangenome.

Journal Article↗

PubMed2005

[Comparison of a T1-weighted inversion-recovery-, gradient-echo- and spin-echo sequence for imaging of the brain at 3.0 Tesla].

PURPOSE: The increased T1 relaxation times at 3.0 Tesla lead to a reduced T1 contrast, requiring adaptation of imaging protocols for high magnetic fields. This prospective study assesses the performance of three techniques for T1-weighted imaging (T1w) at 3.0 T with regard to gray-white differentiation and contrast-to-noise-ratio (CNR). MATERIALS AND METHODS: Thirty-one patients were examined at a 3.0 T system with axial T1 w inversion recovery (IR), spin-echo (SE) and gradient echo (GE) sequences and after contrast enhancement (CE) with CE-SE and CE-GE sequences. For qualitative analysis, the images were ranked with regard to artifacts, gray-white differentiation, image noise and overall diagnostic quality. For quantitative analysis, the CNR was calculated, and cortex and basal ganglia were compared with the white matter. RESULTS: In the qualitative analysis, IR was judged superior to SE and GE for gray-white differentiation, image noise and overall diagnostic quality, but inferior to the GE sequence with regard to artifacts. CE-GE proved superior to CE-SE in all categories. In the quantitative analysis, CNR of the basal ganglia was highest for IR, followed by GE and SE. For the CNR of the cortex, no significant difference was found between IR (16.9) and GE (15.4) but both were superior to the SE (9.4). The CNR of the cortex was significantly higher for CE-GE compared to CE-SE (12.7 vs. 7.6, p < 0.001), but the CNR of the basal ganglia was not significantly different. CONCLUSION: For unenhanced T1 w imaging at 3.0 T, the IR technique is, despite increased artifacts, the method of choice due to its superior gray-white differentiation and best overall image quality. For CE-studies, GE sequences are recommended. For cerebral imaging, SE sequences give unsatisfactory results at 3.0 T.

Brain↗

PubMed1995

Protein structure prediction by threading methods: evaluation of current techniques.

This paper evaluates the results of a protein structure prediction contest. The predictions were made using threading procedures, which employ techniques for aligning sequences with 3D structures to select the correct fold of a given sequence from a set of alternatives. Nine different teams submitted 86 predictions, on a total of 21 target proteins with little or no sequence homology to proteins of known structure. The 3D structures of these proteins were newly determined by experimental methods, but not yet published or otherwise available to the predictors. The predictions, made from the amino acid sequence alone, thus represent a genuine test of the current performance of threading methods. Only a subset of all the predictions is evaluated here. It corresponds to the 44 predictions submitted for the 11 target proteins seen to adopt known folds. The predictions for the remaining 10 proteins were not analyzed, although weak similarities with known folds may also exist in these proteins. We find that threading methods are capable of identifying the correct fold in many cases, but not reliably enough as yet. Every team predicts correctly a different set of targets, with virtually all targets predicted correctly by at least one team. Also, common folds such as TIM barrels are recognized more readily than folds with only a few known examples. However, quite surprisingly, the quality of the sequence-structure alignments, corresponding to correctly recognized folds, is generally very poor, as judged by comparison with the corresponding 3D structure alignments. Thus, threading can presently not be relied upon to derive a detailed 3D model from the amino acid sequence. This raises a very intriguing question: how is fold recognition achieved? Our analysis suggests that it may be achieved because threading procedures maximize hydrophobic interactions in the protein core, and are reasonably good at recognizing local secondary structure.

Amino Acid Sequence↗

PubMed2002

Diversity and complexity of HIV-1 drug resistance: a bioinformatics approach to predicting phenotype from genotype.

Drug resistance testing has been shown to be beneficial for clinical management of HIV type 1 infected patients. Whereas phenotypic assays directly measure drug resistance, the commonly used genotypic assays provide only indirect evidence of drug resistance, the major challenge being the interpretation of the sequence information. We analyzed the significance of sequence variations in the protease and reverse transcriptase genes for drug resistance and derived models that predict phenotypic resistance from genotypes. For 14 antiretroviral drugs, both genotypic and phenotypic resistance data from 471 clinical isolates were analyzed with a machine learning approach. Information profiles were obtained that quantify the statistical significance of each sequence position for drug resistance. For the different drugs, patterns of varying complexity were observed, including between one and nine sequence positions with substantial information content. Based on these information profiles, decision tree classifiers were generated to identify genotypic patterns characteristic of resistance or susceptibility to the different drugs. We obtained concise and easily interpretable models to predict drug resistance from sequence information. The prediction quality of the models was assessed in leave-one-out experiments in terms of the prediction error. We found prediction errors of 9.6-15.5% for all drugs except for zalcitabine, didanosine, and stavudine, with prediction errors between 25.4% and 32.0%. A prediction service is freely available at http://cartan.gmd.de/geno2pheno.html.

Computational Biology↗

PubMed2005

A human ImmunoChip cDNA microarray provides a comprehensive tool to study immune responses.

DNA microarray technology has developed rapidly in recent years and has become an essential tool, providing novel approaches to biomedical research. In this paper, we describe a self-designed ImmunoChip cDNA array for immunological research. With a comprehensive selection of genes of interest, we can focus on key signalling pathways and molecular mechanisms at relatively low cost compared to commercial platforms which are usually targeted at global screening of gene expression. To validate the efficiency of the ImmunoChip, we studied T helper cell polarization to functionally distinct subsets (Th1 and Th2). We also developed a tool for quality control of cDNA microarrays that assesses the technical quality of an ImmunoChip. The information produced with the quality control tool is shown to be valuable for extracting correct information from cDNA microarrays. Gene expression measurements with ImmunoChip are in agreement with the results obtained using oligonucleotide microarrays and with published quantitative RT-PCR data. The ImmunoChip provides reliable measurements and gives new insights into various aspects of human immune responses.

DNA, Complementary↗