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 271 records · Page 15Linked to original sources
PubMed2006

Ultra-fast tandem mass spectrometry scanning combined with monolithic column liquid chromatography increases throughput in proteomic analysis.

Liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS) has been used successfully for the characterization of biomolecules in proteomics in the last few years. This methodology relied largely on the use of reversed-phase chromatography, in particular C18-based resins, which are suitable for separation of peptides. Here we show that polymeric [polystyrene divinylbenzene] monolithic columns can be used to separate peptide mixtures faster and at a higher resolution. For 500 fmol bovine serum albumin, up to 68% sequence coverage and Mascot Mowse scores of >2000 were obtained using a 9 min gradient on a monolithic column coupled to an ion trap mass spectrometer with ultra-fast MS/MS scan rates. In order to achieve similar results using C18 columns, it was necessary to extend gradient times to 30 min. In addition, we demonstrate the utility of this approach for the analysis of whole Escherichia coli cell lysates by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) in combination with LC/MS/MS using 4 min gradients on monolithic columns. Our results indicate higher throughput capabilities of monolithic columns (3-fold gain in time or more) for conventional proteomics applications, such as protein identification and high sequence coverage usually required for detection of post-translational modifications (PTMs). Further optimization of sensitivity and quality of sequence information is discussed, in particular when combined with mass spectrometers that have very fast MS-MS/MS switching and scanning capabilities.

Animals↗
PubMed2003

Testing homology with Contact Accepted mutatiOn (CAO): a contact-based Markov model of protein evolution.

Point Accepted Mutation (PAM) is the Markov model of amino acid replacements in proteins introduced by Dayhoff and her co-workers (Dayhoff et al., 1978). The PAM matrices and other matrices based on the PAM model have been widely accepted as the standard scoring system of protein sequence similarity in protein sequence alignment tools. Here, we present Contact Accepted mutatiOn (CAO), a Markov model of protein residue contact mutations. The CAO model simulates the interchanging of structurally defined side-chain contacts, and introduces additional structural information into protein sequence alignments. Therefore, similarities between structurally conserved sequences can be detected even without apparent sequence similarity. CAO has been benchmarked on the HOMSTRAD database and a subset of the CATH database, by comparing sequence alignments with reference alignments derived from structural superposition. CAO yields scores that reflect coherently the structural quality of sequence alignments, which has implications particularly for homology modelling and threading techniques.

Amino Acid Sequence↗
PubMed2003

An Alu transposition model for the origin and expansion of human segmental duplications.

Relative to genomes of other sequenced organisms, the human genome appears particularly enriched for large, highly homologous segmental duplications (> or =90% sequence identity and > or =10 kbp in length). The molecular basis for this enrichment is unknown. We sought to gain insight into the mechanism of origin, by systematically examining sequence features at the junctions of duplications. We analyzed 9,464 junctions within regions of high-quality finished sequence from a genomewide set of 2,366 duplication alignments. We observed a highly significant (P<.0001) enrichment of Alu short interspersed element (SINE) sequences near or within the junction. Twenty-seven percent of all segmental duplications terminated within an Alu repeat. The Alu junction enrichment was most pronounced for interspersed segmental duplications separated by > or =1 Mb of intervening sequence. Alu elements at the junctions showed higher levels of divergence, consistent with Alu-Alu-mediated recombination events. When we classified Alu elements into major subfamilies, younger elements (AluY and AluS) accounted for the enrichment, whereas the oldest primate family (AluJ) showed no enrichment. We propose that the primate-specific burst of Alu retroposition activity (which occurred 35-40 million years ago) sensitized the ancestral human genome for Alu-Alu-mediated recombination events, which, in turn, initiated the expansion of gene-rich segmental duplications and their subsequent role in nonallelic homologous recombination.

Alu Elements↗
PubMed2000

[The influence of some factors on DNA cycle sequencing].

OBJECTIVE: To study the influence of some factors on DNA cycle sequencing. METHODS: The effects of DNA templates, primers, cycle sequencing reaction conditions as well as purification methods were comparatively analyzed. RESULTS: When the DNA concentration was low, the nucleotide curve showed low ratio of signal to noise, even there were no fluorescent signal. While DNA concentration was too high, the readable length of nucleotides was short. Ions in the DNA template could result in bad sequencing reaction. The Tm value, length and G+C content of primers had no obvious influences on sequencing reaction. The alteration of the denaturing, annealing and extension temperature, or the addition of dimethl sulfoxide or glycerin facilitated sequencing some DNA templates. The fluorescent residues in the purified products of the sequence reaction could interfere the automatic reading of the sequencer, but did not influence the manually proofreading of the sequence. CONCLUSION: The purity and concentration of DNA templates are closely related to sequence data quality. The modification of reaction parameters and usage of additives can help to obtain good result of sequencing some DNA with certain structure. The use of 70% ethanol is recommended to precipitate the extension product.

Sequence Analysis, DNA↗
PubMed2005

Accurate anchoring alignment of divergent sequences.

MOTIVATION: Obtaining high quality alignments of divergent homologous sequences for cross-species sequence comparison remains a challenge. RESULTS: We propose a novel pairwise sequence alignment algorithm, ACANA (ACcurate ANchoring Alignment), for aligning biological sequences at both local and global levels. Like many fast heuristic methods, ACANA uses an anchoring strategy. However, unlike others, ACANA uses a Smith-Waterman-like dynamic programming algorithm to recursively identify near-optimal regions as anchors for a global alignment. Performance evaluations using a simulated benchmark dataset and real promoter sequences suggest that ACANA is accurate and consistent, especially for divergent sequences. Specifically, we use a simulated benchmark dataset to show that ACANA has the highest sensitivity to align constrained functional sites compared to BLASTZ, CHAOS and DIALIGN for local alignment and compared to AVID, ClustalW, DIALIGN and LAGAN for global alignment. Applied to 6007 pairs of human-mouse orthologous promoter sequences, ACANA identified the largest number of conserved regions (defined as over 70% identity over 100 bp) compared to AVID, ClustalW, DIALIGN and LAGAN. In addition, the average length of conserved region identified by ACANA was the longest. Thus, we suggest that ACANA is a useful tool for identifying functional elements in cross-species sequence analysis, such as predicting transcription factor binding sites in non-coding DNA. AVAILABILITY: ACANA software and test sequence data are publicly available at http://BioMedEmpire.org/

Algorithms↗
PubMed1998

Estimation of errors in "raw" DNA sequences: a validation study.

As DNA sequencing is performed more and more in a mass-production-like manner, efficient quality control measures become increasingly important for process control, but so also does the ability to compare different methods and projects. One of the fundamental quality measures in sequencing projects is the position-specific error probability at all bases in each individual sequence. Accurate prediction of base-specific error rates from "raw" sequence data would allow immediate quality control as well as benchmarking different methods and projects while avoiding the inefficiencies and time delays associated with resequencing and assessments after "finishing" a sequence. The program PHRED provides base-specific quality scores that are logarythmically related to error probabilities. This study assessed the accuracy of PHRED's error-rate prediction by analyzing sequencing projects from six different large-scale sequencing laboratories. All projects used four-color fluorescent sequencing, but the sequencing methods used varied widely between the different projects. The results indicate that the error-rate predictions such as those given by PHRED can be highly accurate for a large variety of different sequencing methods as well as over a wide range of sequence quality.

Base Sequence↗
PubMed2005

The PEDANT genome database in 2005.

The PEDANT genome database (http://pedant.gsf.de) contains pre-computed bioinformatics analyses of publicly available genomes. Its main mission is to provide robust automatic annotation of the vast majority of amino acid sequences, which have not been subjected to in-depth manual curation by human experts in high-quality protein sequence databases. By design PEDANT annotation is genome-oriented, making it possible to explore genomic context of gene products, and evaluate functional and structural content of genomes using a category-based query mechanism. At present, the PEDANT database contains exhaustive annotation of over 1,240,000 proteins from 270 eubacterial, 23 archeal and 41 eukaryotic genomes.

Computational Biology↗
PubMed2006

454 sequencing put to the test using the complex genome of barley.

BACKGROUND: During the past decade, Sanger sequencing has been used to completely sequence hundreds of microbial and a few higher eukaryote genomes. In recent years, a number of alternative technologies became available, among them adaptations of the pyrosequencing procedure (i.e. "454 sequencing"), promising an approximately 100-fold increase in throughput over Sanger technology--an advancement which is needed to make large and complex genomes more amenable to full genome sequencing at affordable costs. Although several studies have demonstrated its potential usefulness for sequencing small and compact microbial genomes, it was unclear how the new technology would perform in large and highly repetitive genomes such as those of wheat or barley. RESULTS: To study its performance in complex genomes, we used 454 technology to sequence four barley Bacterial Artificial Chromosome (BAC) clones and compared the results to those from ABI-Sanger sequencing. All gene containing regions were covered efficiently and at high quality with 454 sequencing whereas repetitive sequences were more problematic with 454 sequencing than with ABI-Sanger sequencing. 454 sequencing provided a much more even coverage of the BAC clones than ABI-Sanger sequencing, resulting in almost complete assembly of all genic sequences even at only 9 to 10-fold coverage. To obtain highly advanced working draft sequences for the BACs, we developed a strategy to assemble large parts of the BAC sequences by combining comparative genomics, detailed repeat analysis and use of low-quality reads from 454 sequencing. Additionally, we describe an approach of including small numbers of ABI-Sanger sequences to produce hybrid assemblies to partly compensate the short read length of 454 sequences. CONCLUSION: Our data indicate that 454 pyrosequencing allows rapid and cost-effective sequencing of the gene-containing portions of large and complex genomes and that its combination with ABI-Sanger sequencing and targeted sequence analysis can result in large regions of high-quality finished genomic sequences.

Base Pairing↗
PubMed2004

Centrically reordered inversion recovery half-Fourier single-shot turbo spin-echo sequence: improvement of the image quality of oxygen-enhanced MRI.

PURPOSE: The purpose of the study presented here was to determine the improvement in image quality of oxygen-enhanced magnetic resonance (MR) subtraction imaging obtained with a centrically reordered inversion recovery half-Fourier single-shot turbo spin-echo (c-IR-HASTE) sequence compared with that obtained with a conventional sequentially reordered inversion recovery single-shot HASTE (s-IR-HASTE) sequence for pulmonary imaging. MATERIALS AND METHODS: Oxygen-enhanced MR imaging using a 1.5 T whole body scanner was performed on 12 healthy, non-smoking volunteers. Oxygen-enhanced MR images were obtained with the coronal two-dimensional (2D) c-IR-HASTE sequence and 2D s-IR-HASTE sequence combined with respiratory triggering. For a 256x256 matrix, 132 phase-encoding steps were acquired including four steps for phase correction. Inter-echo spacing for each sequence was 4.0 ms. The effective echo time (TE) for c-IR-HASTE was 4.0 ms, and 16 ms for s-IR-HASTE. The inversion time (TI) was 900 ms. To determine the improvement in oxygen-enhanced MR subtraction imaging by c-IR-HASTE, CNRs of subtraction image, overall image quality, and image degradation of the c-IR-HASTE and s-IR-HASTE techniques were statistically compared. RESULTS: CNR, overall image quality, and image degradation of c-IR-HASTE images showed significant improvement compared to those s-IR-HASTE images (P<0.05). CONCLUSION: Centrically reordered inversion recovery half-Fourier single-shot turbo spin-echo (c-IR-HASTE) sequence enhanced the signal from the lung and improved the image quality of oxygen-enhanced MR subtraction imaging.

Adult↗
PubMed2004

Gene cataloging and expression profiling in human gastric cancer cells by expressed sequence tags.

To understand the molecular mechanism associated with gastric carcinogenesis, we identified genes expressed in gastric cancer cell lines and tissues. Of 97,609 high-quality ESTs sequenced from 36 cDNA libraries, 92,545 were coalesced into 10,418 human Unigene clusters (Build 151). The gene expression profile was produced by counting the cluster frequencies in each library. Although the profiles of highly expressed genes varied greatly from library to library, those genes related to cell structure formation, heat shock proteins, the glycolysis pathway, and the signaling pathway were highly represented in human gastric cancer cell lines and in primary tumors. Conversely, the genes encoding immunoglobulins, ribosomal proteins, and digestive proteins were down-regulated in gastric cancer cell lines and tissues compared to normal tissues. The transcription levels of some of these genes were confirmed by RT-PCR. We found that genes related to cell adhesion, apoptosis, and cytoskeleton formation were particularly up-regulated in the gastric cancer cell lines established from malignant ascites compared to those from primary tumors. This comprehensive molecular profiling of human gastric cancer should be useful for elucidating the genetic events associated with human gastric cancer.

Base Sequence↗
PubMed2005

Identification of endopeptidase genes from the genomic sequence of Lactobacillus helveticus CNRZ32 and the role of these genes in hydrolysis of model bitter peptides.

Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5alpha derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, beta-casein (beta-CN) (f193-209) and alpha(S1)-casein (alpha(S1)-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10 degrees C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards alpha(S1)-CN (f1-9) and beta-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze beta-CN (f193-209) and alpha(S1)-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides alpha(S1)-CN (f1-9), alpha(S1)-CN (f1-13), and alpha(S1)-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with beta-CN (f193-209) and alpha(S1)-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.

Amino Acid Sequence↗
PubMed2006

NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins.

NCBI's reference sequence (RefSeq) database (http://www.ncbi.nlm.nih.gov/RefSeq/) is a curated non-redundant collection of sequences representing genomes, transcripts and proteins. The database includes 3774 organisms spanning prokaryotes, eukaryotes and viruses, and has records for 2,879,860 proteins (RefSeq release 19). RefSeq records integrate information from multiple sources, when additional data are available from those sources and therefore represent a current description of the sequence and its features. Annotations include coding regions, conserved domains, tRNAs, sequence tagged sites (STS), variation, references, gene and protein product names, and database cross-references. Sequence is reviewed and features are added using a combined approach of collaboration and other input from the scientific community, prediction, propagation from GenBank and curation by NCBI staff. The format of all RefSeq records is validated, and an increasing number of tests are being applied to evaluate the quality of sequence and annotation, especially in the context of complete genomic sequence.

Amino Acid Sequence↗
PubMed2001

Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species.

The use of the 16S rRNA gene for identification of nontuberculous mycobacteria (NTM) provides a faster and better ability to accurately identify them in addition to contributing significantly in the discovery of new species. Despite their associated problems, many rely on the use of public sequence databases for sequence comparisons. To best evaluate the taxonomic status of NTM species submitted to our reference laboratory, we have created a 16S rRNA sequence database by sequencing 121 American Type Culture Collection strains encompassing 92 species of mycobacteria, and have also included chosen unique mycobacterial sequences from public sequence repositories. In addition, the Ribosomal Differentiation of Medical Microorganisms (RIDOM) service has made freely available on the Internet mycobacterial identification by 16S rRNA analysis. We have evaluated 122 clinical NTM species using our database, comparing >1,400 bp of the 16S gene, and the RIDOM database, comparing approximately 440 bp. The breakdown of analysis was as follows: 61 strains had a sequence with 100% similarity to the type strain of an established species, 19 strains showed a 1- to 5-bp divergence from an established species, 11 strains had sequences corresponding to uncharacterized strain sequences in public databases, and 31 strains represented unique sequences. Our experience with analysis of the 16S rRNA gene of patient strains has shown that clear-cut results are not the rule. As many clinical, research, and environmental laboratories currently employ 16S-based identification of bacteria, including mycobacteria, a freely available quality-controlled database such as that provided by RIDOM is essential to accurately identify species or detect true sequence variations leading to the discovery of new species.

Databases, Nucleic Acid↗
PubMed1998

Short-insert libraries as a method of problem solving in genome sequencing.

As the Human Genome Project moves into its sequencing phase, a serious problem has arisen. The same problem has been increasingly vexing in the closing phase of the Caenorhabditis elegans project. The difficulty lies in sequencing efficiently through certain regions in which the templates (DNA substrates for the sequencing process) form complex folded secondary structures that are inaccessible to the enzymes. The solution, however, is simply to break them up. Specifically, the offending fragments are sonicated heavily and recloned, as much smaller fragments, into pUC vector. The sequences obtained from the resulting library can subsequently be assembled, free from the effects of secondary structure, to produce high-quality, complete sequence. Because of the success and simplicity of this procedure, we have begun to use it for the sequencing of all regions in which standard primer walking has been at all difficult.

Animals↗
PubMed2003

The genome sequence of the filamentous fungus Neurospora crassa.

Neurospora crassa is a central organism in the history of twentieth-century genetics, biochemistry and molecular biology. Here, we report a high-quality draft sequence of the N. crassa genome. The approximately 40-megabase genome encodes about 10,000 protein-coding genes--more than twice as many as in the fission yeast Schizosaccharomyces pombe and only about 25% fewer than in the fruitfly Drosophila melanogaster. Analysis of the gene set yields insights into unexpected aspects of Neurospora biology including the identification of genes potentially associated with red light photobiology, genes implicated in secondary metabolism, and important differences in Ca2+ signalling as compared with plants and animals. Neurospora possesses the widest array of genome defence mechanisms known for any eukaryotic organism, including a process unique to fungi called repeat-induced point mutation (RIP). Genome analysis suggests that RIP has had a profound impact on genome evolution, greatly slowing the creation of new genes through genomic duplication and resulting in a genome with an unusually low proportion of closely related genes.

Calcium Signaling↗
PubMed2004

Integrated web service for improving alignment quality based on segments comparison.

BACKGROUND: Defining blocks forming the global protein structure on the basis of local structural regularity is a very fruitful idea, extensively used in description, and prediction of structure from only sequence information. Over many years the secondary structure elements were used as available building blocks with great success. Specially prepared sets of possible structural motifs can be used to describe similarity between very distant, non-homologous proteins. The reason for utilizing the structural information in the description of proteins is straightforward. Structural comparison is able to detect approximately twice as many distant relationships as sequence comparison at the same error rate. RESULTS: Here we provide a new fragment library for Local Structure Segment (LSS) prediction called FRAGlib which is integrated with a previously described segment alignment algorithm SEA. A joined FRAGlib/SEA server provides easy access to both algorithms, allowing a one stop alignment service using a novel approach to protein sequence alignment based on a network matching approach. The FRAGlib used as secondary structure prediction achieves only 73% accuracy in Q3 measure, but when combined with the SEA alignment, it achieves a significant improvement in pairwise sequence alignment quality, as compared to previous SEA implementation and other public alignment algorithms. The FRAGlib algorithm takes approximately 2 min. to search over FRAGlib database for a typical query protein with 500 residues. The SEA service align two typical proteins within circa approximately 5 min. All supplementary materials (detailed results of all the benchmarks, the list of test proteins and the whole fragments library) are available for download on-line at http://ffas.ljcrf.edu/darman/results/. CONCLUSIONS: The joined FRAGlib/SEA server will be a valuable tool both for molecular biologists working on protein sequence analysis and for bioinformaticians developing computational methods of structure prediction and alignment of proteins.

Computational Biology↗
PubMed2005

Drosophila melanogaster: a case study of a model genomic sequence and its consequences.

The sequencing and annotation of the Drosophila melanogaster genome, first published in 2000 through collaboration between Celera Genomics and the Drosophila Genome Projects, has provided a number of important contributions to genome research. By demonstrating the utility of methods such as whole-genome shotgun sequencing and genome annotation by a community "jamboree," the Drosophila genome established the precedents for the current paradigm used by most genome projects. Subsequent releases of the initial genome sequence have been improved by the Berkeley Drosophila Genome Project and annotated by FlyBase, the Drosophila community database, providing one of the highest-quality genome sequences and annotations for any organism. We discuss the impact of the growing number of genome sequences now available in the genus on current Drosophila research, and some of the biological questions that these resources will enable to be solved in the future.

Animals↗
PubMed2003

Evaluation of a multicapillary electrophoresis instrument for mitochondrial DNA typing.

Laser-induced detection of fluorescent labeled PCR products and multi-wavelength detection (i.e., multicolor analysis) enables rapid generation of mtDNA sequencing profiles. Traditionally, polyacrylamide slab gels have been used as the electrophoretic medium for mtDNA sequencing in forensic analyses. Replacement of slab gel electrophoresis with capillary electrophoresis (CE) can facilitate automation of the analytical process. Automation and high throughput can be further enhanced by using multicapillary electrophoretic systems. The use of the ABI Prism 3100 Genetic Analyzer (ABI 3100, Applied Biosystems, Foster City, CA) as well as the ABI Prism 310 Genetic Analyzer (ABI 310, Applied Biosystems, Foster City, CA) were evaluated for mtDNA sequencing capabilities and compared with sequencing results obtained on the platform currently in use in the FBI Laboratory (the ABI Prism 377 DNA Sequencer, ABI 377, Applied Biosystems, Foster City, CA). Various studies were performed to assess the utility of the ABI 3100, as well as the ABI 310 for mtDNA sequencing. The tests included: comparisons of results obtained among the ABI 3100, the ABI 310 and the ABI 377 instruments; comparisons of results obtained within and between capillary arrays; evaluation of capillary length; evaluation of sample injection time; evaluation of the resolution of mixtures/heteroplasmic samples; and evaluation of the sensitivity of detection of a minor component with reduced template on the ABI 3100. In addition, other studies were performed to improve sample preparation; these included: comparison of template suppression reagent (TSR, Applied Biosystems, Foster City, CA) versus formamide; the use of Performa DTR Gel Filtration Cartridges (Edge BioSystems Inc., Gaithersburg, MD) versus Centri-Sep Spin Columns (Princeton Separations, Adelphia, NJ) for product purification after cycle sequencing; and sample stability after denaturation. The data support that valid and reliable results can be obtained using either capillary electrophoresis instrument, and the quality of sequencing results are comparable to or better than those obtained from the ABI 377 instrument.

DNA, Mitochondrial↗