Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Sequencing quality”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 631 records · Page 35Linked to original sources
PubMed2006

A Sanger/pyrosequencing hybrid approach for the generation of high-quality draft assemblies of marine microbial genomes.

Since its introduction a decade ago, whole-genome shotgun sequencing (WGS) has been the main approach for producing cost-effective and high-quality genome sequence data. Until now, the Sanger sequencing technology that has served as a platform for WGS has not been truly challenged by emerging technologies. The recent introduction of the pyrosequencing-based 454 sequencing platform (454 Life Sciences, Branford, CT) offers a very promising sequencing technology alternative for incorporation in WGS. In this study, we evaluated the utility and cost-effectiveness of a hybrid sequencing approach using 3730xl Sanger data and 454 data to generate higher-quality lower-cost assemblies of microbial genomes compared to current Sanger sequencing strategies alone.

Biotechnology↗
PubMed2002

Strategies for multiple sequence alignment.

We present an overview of multiple sequence alignments to outline the practical consequences for the choices among different techniques and parameters. We begin with a discussion of the scoring methods for quantifying the quality of a multiple sequence alignment, followed by a discussion of the algorithms implemented within a variety of multiple sequence alignment programs. We also discuss additional alignment details such as gap penalty and distance metrics. The paper concludes with a discussion on how to improve alignment quality and the limitations of the techniques described in this paper

Algorithms↗
PubMed2004

Plant genomics: the third wave.

Completing the primary genomic sequence of Arabidopsis thaliana was a major milestone, being the first plant genome and only the third high-quality finished eukaryotic genome sequence. Understanding how the genome sequence comprehensively encodes developmental programs and environmental responses is the next major challenge for all plant genome projects. This requires fully characterizing the genes, the regulatory sequences, and their functions. We discuss several functional genomics approaches to decode the linear sequence of the reference plant Arabidopsis thaliana, including full-length cDNA collections, microarrays, natural variation, knockout collections, and comparative sequence analysis. Genomics provides the essential tools to speed the work of the traditional molecular geneticist and is now a scientific discipline in its own right.

Arabidopsis↗
PubMed2005

Draft versus finished sequence data for DNA and protein diagnostic signature development.

Sequencing pathogen genomes is costly, demanding careful allocation of limited sequencing resources. We built a computational Sequencing Analysis Pipeline (SAP) to guide decisions regarding the amount of genomic sequencing necessary to develop high-quality diagnostic DNA and protein signatures. SAP uses simulations to estimate the number of target genomes and close phylogenetic relatives (near neighbors or NNs) to sequence. We use SAP to assess whether draft data are sufficient or finished sequencing is required using Marburg and variola virus sequences. Simulations indicate that intermediate to high-quality draft with error rates of 10(-3)-10(-5) (approximately 8x coverage) of target organisms is suitable for DNA signature prediction. Low-quality draft with error rates of approximately 1% (3x to 6x coverage) of target isolates is inadequate for DNA signature prediction, although low-quality draft of NNs is sufficient, as long as the target genomes are of high quality. For protein signature prediction, sequencing errors in target genomes substantially reduce the detection of amino acid sequence conservation, even if the draft is of high quality. In summary, high-quality draft of target and low-quality draft of NNs appears to be a cost-effective investment for DNA signature prediction, but may lead to underestimation of predicted protein signatures.

Computational Biology↗
PubMed2002

Identification of mixups among DNA sequencing plates.

MOTIVATION: During the process of high-throughput genome sequencing there are opportunities for mixups of reagents and data associated with particular projects. The sequencing templates or sequence data generated for an assembly may become contaminated with reagents or sequences from another project, resulting in poorer quality and inaccurate assemblies. RESULTS: We have developed a system to assess sequence assemblies and monitor for laboratory mixups. We describe several methods for testing the consistency of assemblies and resolving mixed ones. We use statistical tests to evaluate the distribution of sequencing reads from different plates into contigs, and a graph-based approach to resolve situations where data has been inappropriately combined. While these methods have been designed for use in a high-throughput DNA sequencing environment processing thousands of clones, they can be applied in any situation where distinct sequencing projects are performed at redundant coverage.

Algorithms↗
PubMed2005

Simpleaffy: a BioConductor package for Affymetrix Quality Control and data analysis.

UNLABELLED: Quality Control is a fundamental aspect of successful microarray data analysis. Simpleaffy is a BioConductor package that provides access to a variety of QC metrics for assessing the quality of RNA samples and of the intermediate stages of sample preparation and hybridization. Simpleaffy also offers fast implementations of popular algorithms for generating expression summaries and detection calls. AVAILABILITY: Simpleaffy can be downloaded from http://www.bioconductor.org. SUPPLEMENTARY INFORMATION: Additional information can be found on the supplementary website located at http://bioinformatics.picr.man.ac.uk.

Computational Biology↗
PubMed2003

Contrast normalization of oligonucleotide arrays.

Affymetrix high-density oligonucleotide array is a tool that has the capacity to simultaneously measure the abundance of thousands of mRNA sequences in biological samples. In order to allow direct array-to-array comparisons, normalization is a necessity. When deciding on an appropriate normalization procedure there are a couple questions that need to be addressed, e.g., on which level should the normalization be performed: On the level of feature intensities or on the level of expression indexes? Should all features/expression indexes be used or can we choose a subset of features likely to be unregulated? Another question is how to actually perform the normalization: normalize using the overall mean intensity or use a smooth normalization curve? Most of the currently used normalization methods are linear; e.g., the normalization method implemented in the Affymetrix software GeneChip is based on the overall mean intensity. However, along with alternative methods of summarizing feature intensities into an expression index, nonlinear methods have recently started to appear. For many of these alternative methods, the natural choice is to normalize on the level of feature intensities, either using all feature intensities or only perfect match intensities. In this report, a nonlinear normalization procedure aimed for normalizing feature intensities is proposed.

Algorithms↗
PubMed2003

Statistical design of reverse dye microarrays.

MOTIVATION: In cDNA microarray experiments all samples are labelled with either Cy3 dye or Cy5 dye. Certain genes exhibit dye bias-a tendency to bind more efficiently to one of the dyes. The common reference design avoids the problem of dye bias by running all arrays 'forward', so that the samples being compared are always labelled with the same dye. But comparison of samples labelled with different dyes is sometimes of interest. In these situations, it is necessary to run some arrays 'reverse'-with the dye labelling reversed-in order to correct for the dye bias. The design of these experiments will impact one's ability to identify genes that are differentially expressed in different tissues or conditions. We address the design issue of how many specimens are needed, how many forward and reverse labelled arrays to perform, and how to optimally assign Cy3 and Cy5 labels to the specimens. RESULTS: We consider three types of experiments for which some reverse labelling is needed: paired samples, samples from two predefined groups, and reference design data when comparison with the reference is of interest. We present simple probability models for the data, derive optimal estimators for relative gene expression, and compare the efficiency of the estimators for a range of designs. In each case, we present the optimal design and sample size formulas. We show that reverse labelling of individual arrays is generally not required.

Algorithms↗
PubMed2003

New normalization methods for cDNA microarray data.

MOTIVATION: The focus of this paper is on two new normalization methods for cDNA microarrays. After the image analysis has been performed on a microarray and before differentially expressed genes can be detected, some form of normalization must be applied to the microarrays. Normalization removes biases towards one or other of the fluorescent dyes used to label each mRNA sample allowing for proper evaluation of differential gene expression. RESULTS: The two normalization methods that we present here build on previously described non-linear normalization techniques. We extend these techniques by firstly introducing a normalization method that deals with smooth spatial trends in intensity across microarrays, an important issue that must be dealt with. Secondly we deal with normalization of a new type of cDNA microarray experiment that is coming into prevalence, the small scale specialty or 'boutique' array, where large proportions of the genes on the microarrays are expected to be highly differentially expressed. AVAILABILITY: The normalization methods described in this paper are available via http://www.pi.csiro.au/gena/ in a software suite called tRMA: tools for R Microarray Analysis upon request of the authors. Images and data used in this paper are also available via the same link.

Algorithms↗
PubMed2002

Comparative genome sequencing for discovery of novel polymorphisms in Bacillus anthracis.

Comparison of the whole-genome sequence of Bacillus anthracis isolated from a victim of a recent bioterrorist anthrax attack with a reference reveals 60 new markers that include single nucleotide polymorphisms (SNPs), inserted or deleted sequences, and tandem repeats. Genome comparison detected four high-quality SNPs between the two sequenced B. anthracis chromosomes and seven differences among different preparations of the reference genome. These markers have been tested on a collection of anthrax isolates and were found to divide these samples into distinct families. These results demonstrate that genome-based analysis of microbial pathogens will provide a powerful new tool for investigation of infectious disease outbreaks.

Animals↗
PubMed2006

Modeling, docking, and simulation of the major facilitator superfamily.

X-ray structures are known for three members of the Major Facilitator Superfamily (MFS) of membrane transporter proteins, thus enabling the use of homology modeling to extrapolate to other MFS members. However, before employing such models for, e.g., mutational or docking studies, it is essential to develop a measure of their quality. To aid development of such metrics, two disparate MFS members (NupG and GLUT1) have been modeled. In addition, control models were created with shuffled sequences, to mimic poor quality homology models. These models and the template crystal structures have been examined in terms of both static and dynamic indicators of structural quality. Comparison of the behavior of modeled structures with the crystal structures in molecular dynamics simulations provided a metric for model quality. Docking of the inhibitor forskolin to GLUT1 and to a control model revealed significant differences, indicating that we may identify accurate models despite low sequence identity between target sequences and templates.

Binding Sites↗
PubMed1993

[The chemical shift imaging of liver].

Tissue characterization of different pathologies of the liver can be achieved by differences of relaxation time on changing of pulse sequences in magnetic resonance imaging (MRI). The usefulness of MRI for detection of liver disease is limited when the pathological change is subtle. Chemical shift is a fundamental characteristics in nuclear magnetic resonance (NMR). Chemical shift imaging (CSI) in MRI is based on the different Larmor frequency of proton in water and fat; and therefore it was able to enhance the effectiveness of pathology. For this study, Dixon's method was used to detect liver pathologies and compare its detectability with conventional pulse sequences. Forty cases were enrolled for study; they included 5 health volunteers, 15 hepatomas, 1 cholangiocarcinoma, 5 metastatic hepatic tumors and 14 fatty livers. In hepatic tumors, the lesion number, tumor margination and lesion-to-liver contrast in images were read and analyzed. Signal intensities, signal-to-noise ratio and contrast-to-noise ratio were compared, after measurement, from stored data. In fatty livers, the relative change of signal intensities in different areas of the liver in in-phase and out-phase images were compared with the back muscle and spleen to find where the fatty metamorphosis happened. CSI in spin echo or gradient echo pulse sequences was found to be adequate and valuable for detecting fatty liver, when compared with conventional MRI. CSI not only identified the extension of disease itself but also characterized the fatty change in liver parenchyma. Though CSI affords no further advantages than conventional pulse sequences for detection of hepatic tumors, occasionally, when the image quality of the conventional pulse sequences is not satisfactory or equivocal in lesion detection, the use of CSI might be attempted.

Fatty Liver↗
PubMed2002

Theoretical and experimental comparisons of gene expression indexes for oligonucleotide arrays.

MOTIVATION: Oligonucleotide expression arrays exhibit systematic and reproducible variation produced by the multiple distinct probes used to represent a gene. Recently, a gene expression index has been proposed that explicitly models probe effects, and provides improved fits of hybridization intensity for arrays containing perfect match (PM) and mismatch (MM) probe pairs. RESULTS: Here we use a combination of analytical arguments and empirical data to show directly that the estimates provided by model-based expression indexes are superior to those provided by commercial software. The improvement is greatest for genes in which probe effects vary substantially, and modeling the PM and MM intensities separately is superior to using the PM-MM differences. To empirically compare expression indexes, we designed a mixing experiment involving three groups of human fibroblast cells (serum starved, serum stimulated, and a 50:50 mixture of starved/stimulated), with six replicate HuGeneFL arrays in each group. Careful spiking of control genes provides evidence that 88-98% of the genes on the array are detectably transcribed, and that the model-based estimates can accurately detect the presence versus absence of a gene. The use of extensive replication from single RNA sources enables exploration of the technical variability of the array.

Artifacts↗
PubMed1999

Pfam 3.1: 1313 multiple alignments and profile HMMs match the majority of proteins.

Pfam is a collection of multiple alignments and profile hidden Markov models of protein domain families. Release 3.1 is a major update of the Pfam database and contains 1313 families which are available on the World Wide Web in Europe at http://www.sanger.ac.uk/Software/Pfam/ and http://www.cgr.ki.se/Pfam/, and in the US at http://pfam.wustl.edu/. Over 54% of proteins in SWISS-PROT-35 and SP-TrEMBL-5 match a Pfam family. The primary changes of Pfam since release 2.1 are that we now use the more advanced version 2 of the HMMER software, which is more sensitive and provides expectation values for matches, and that it now includes proteins from both SP-TrEMBL and SWISS-PROT.

Algorithms↗
PubMed2004

Recent improvements to the PROSITE database.

The PROSITE database consists of a large collection of biologically meaningful signatures that are described as patterns or profiles. Each signature is linked to documentation that provides useful biological information on the protein family, domain or functional site identified by the signature. The PROSITE web page has been redesigned and several tools have been implemented to help the user discover new conserved regions in their own proteins and to visualize domain arrangements. We also introduced the facility to search PDB with a PROSITE entry or a user's pattern and visualize matched positions on 3D structures. The latest version of PROSITE (release 18.17 of November 30, 2003) contains 1676 entries. The database is accessible at http://www.expasy.org/prosite/.

Amino Acid Motifs↗
PubMed2005

PISCES: recent improvements to a PDB sequence culling server.

PISCES is a database server for producing lists of sequences from the Protein Data Bank (PDB) using a number of entry- and chain-specific criteria and mutual sequence identity. Our goal in culling the PDB is to provide the longest list possible of the highest resolution structures that fulfill the sequence identity and structural quality cut-offs. The new PISCES server uses a combination of PSI-BLAST and structure-based alignments to determine sequence identities. Structure alignment produces more complete alignments and therefore more accurate sequence identities than PSI-BLAST. PISCES now allows a user to cull the PDB by-entry in addition to the standard culling by individual chains. In this scenario, a list will contain only entries that do not have a chain that has a sequence identity to any chain in any other entry in the list over the sequence identity cut-off. PISCES also provides fully annotated sequences including gene name and species. The server allows a user to cull an input list of entries or chains, so that other criteria, such as function, can be used. Results from a search on the re-engineered RCSB's site for the PDB can be entered into the PISCES server by a single click, combining the powerful searching abilities of the PDB with PISCES's utilities for sequence culling. The server's data are updated weekly. The server is available at http://dunbrack.fccc.edu/pisces.

Databases, Protein↗