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PubMed2004

Parallel acquisition techniques in cardiac cine magnetic resonance imaging using TrueFISP sequences: comparison of image quality and artifacts.

PURPOSE: To compare image quality, artifacts, and signal-to-noise ratio (SNR) in cardiac cine TrueFISP magnetic resonance imaging (MRI) with and without parallel acquisition techniques (PAT). MATERIALS AND METHODS: MRI was performed in 16 subjects with a TrueFISP sequence (1.5 T; Magnetom Sonata, Siemens): TR, 3.0 msec; TE, 1.5 msec; flip angle (FA), 60 degrees. Three axes were scanned without PAT (no PAT) and using the generalized autocalibrating partially parallel acquisition (GRAPPA) and modified sensitivity encoding (mSENSE) reconstruction algorithms with an autocalibration mode to reduce scan time. A conventional spine array and a body flex array were used. Artifacts, image noise, and overall image quality were classified on a 4-point scale by an observer blinded to the implemented technique; for quantitative comparison, SNR was measured. RESULTS: With a PAT factor of two, acquisition time could be reduced by 39%. No PAT did not show artifacts, and GRAPPA revealed fewer artifacts than mSENSE. PAT provided inferior-quality scores concerning image noise and overall image quality. In quantitative measurements, GRAPPA and mSENSE (20.1 +/- 6.2 and 15.6 +/- 6.2, respectively) yielded lower SNR than no PAT (30.6 +/- 20.1; P < 0.05) and P < 0.001). CONCLUSION: Time savings in PAT are accompanied by artifacts and an increase in image noise. The GRAPPA algorithm was superior to mSENSE concerning image quality, noise, and SNR.

Adult↗

PubMed2006

Improving the quality of protein structure models by selecting from alignment alternatives.

BACKGROUND: In the area of protein structure prediction, recently a lot of effort has gone into the development of Model Quality Assessment Programs (MQAPs). MQAPs distinguish high quality protein structure models from inferior models. Here, we propose a new method to use an MQAP to improve the quality of models. With a given target sequence and template structure, we construct a number of different alignments and corresponding models for the sequence. The quality of these models is scored with an MQAP and used to choose the most promising model. An SVM-based selection scheme is suggested for combining MQAP partial potentials, in order to optimize for improved model selection. RESULTS: The approach has been tested on a representative set of proteins. The ability of the method to improve models was validated by comparing the MQAP-selected structures to the native structures with the model quality evaluation program TM-score. Using the SVM-based model selection, a significant increase in model quality is obtained (as shown with a Wilcoxon signed rank test yielding p-values below 10(-15)). The average increase in TMscore is 0.016, the maximum observed increase in TM-score is 0.29. CONCLUSION: In template-based protein structure prediction alignment is known to be a bottleneck limiting the overall model quality. Here we show that a combination of systematic alignment variation and modern model scoring functions can significantly improve the quality of alignment-based models.

Computer Simulation↗

PubMed1998

The Genome Sequence DataBase (GSDB): improving data quality and data access.

In 1997 the primary focus of the Genome Sequence DataBase (GSDB; www. ncgr.org/gsdb ) located at the National Center for Genome Resources was to improve data quality and accessibility. Efforts to increase the quality of data within the database included two major projects; one to identify and remove all vector contamination from sequences in the database and one to create premier sequence sets (including both alignments and discontiguous sequences). Data accessibility was improved during the course of the last year in several ways. First, a graphical database sequence viewer was made available to researchers. Second, an update process was implemented for the web-based query tool, Maestro. Third, a web-based tool, Excerpt, was developed to retrieve selected regions of any sequence in the database. And lastly, a GSDB flatfile that contains annotation unique to GSDB (e.g., sequence analysis and alignment data) was developed. Additionally, the GSDB web site provides a tool for the detection of matrix attachment regions (MARs), which can be used to identify regions of high coding potential. The ultimate goal of this work is to make GSDB a more useful resource for genomic comparison studies and gene level studies by improving data quality and by providing data access capabilities that are consistent with the needs of both types of studies.

Base Sequence↗

PubMed2026

Comprehensive evaluation of new sequencer T20 and well-established T7 with 507 human samples.

The DNBSEQ-T20×2 (T20) sequencer, developed by MGI Tech, enables cost-effective human whole-genome sequencing (WGS) at 30× coverage for less than $100 per genome. Here, we evaluate the sequencing performance and data quality of the T20 platform by benchmarking it against the established DNBSEQ-T7 (T7) sequencer using 507 samples derived from blood (N = 75), stool (N = 242), and saliva (N = 190). The T20 exhibited lower sequencing quality metrics compared with the T7, with Q20 scores of 95.76%-95.83% and Q30 scores of 87.25%-87.40%, compared with 97.81%-97.93% and 93.26%-93.60%, respectively, for T7 data. Quality differences were more evident toward the end of reads, and PCR-free libraries sequenced on the T20 showed similar reductions in quality scores. The median empirical base error rate estimated from 102 ZymoBIOMICS samples was 0.33%. The T20 demonstrated comparable coverage uniformity to the T7 and showed high concordance in microbiome composition analysis, with a median Bray-Curtis dissimilarity of 0.02. Variant calling performance was highly consistent between the two platforms. Among variants with non-missing genotype calls on both platforms, 94.92% of SNPs and 87.20% of InDels showed concordant genotypes between T20 and T7. Overall, the T20 delivers reliable sequencing accuracy and reproducibility for large-scale genomic and microbiome studies, providing a cost-effective alternative for high-throughput sequencing applications.

Metagenomics↗

PubMed2004

ORFDB: an information resource linking scientific content to a high-quality Open Reading Frame (ORF) collection.

The ORFDB (http://orf.invitrogen.com/) represents an ongoing effort at Invitrogen Corporation to integrate relevant scientific data with an evolving collection of human and mouse Open Reading Frame (ORF) clones (Ultimate ORF Clones). The ORFDB serves as a central data warehouse enabling researchers to search the ORF collection through its web portal ORFBrowser, allowing researchers to find the Ultimate ORF clones by blast, keyword, GenBank accession, gene symbol, clone ID, Unigene ID, LocusLink ID or through functional relationships by browsing the collection via the Gene Ontology (GO) Browser. As of October 2003, the ORFDB contains 6200 human and 2870 mouse Ultimate ORF clones. All Ultimate ORF clones have been fully sequenced with high quality, and are matched to public reference protein sequences. In addition, the cloned ORFs have been extensively annotated across six categories: Gene, ORF, Clone Format, Protein, SNP and Genomic links, with the information assembled in a format termed the ORFCard. The ORFCard represents an information repository that documents the sequence quality, alignment with respect to public protein sequences, and the latest publicly available information associated with each human and mouse gene represented in the collection.

Animals↗

PubMed2006

The complete genome sequence of a dog: a perspective.

A complete, high-quality reference sequence of a dog genome was recently produced by a team of researchers led by the Broad Institute, achieving another major milestone in deciphering the genomic landscape of mammalian organisms. The genome sequence provides an indispensable resource for comparative analysis and novel insights into dog and human evolution and history. Together with the survey sequence of a poodle previously published in 2003, the two dog genome sequences allowed identification of more than 2.5 million single nucleotide polymorphisms within and between dog breeds, which can be used in evolutionary analysis, behavioral studies and disease gene mapping.(1)

Animals↗

PubMed2026

Translating functional molecular knowledge into crop-breeding success.

Historical plant breeding, which optimizes phenotypes through selective crossing guided by phenotypic evaluation and molecular markers, is limited by evolutionary constraints that hinder rapid crop improvement. A new paradigm, precision breeding, circumvents these limitations by targeting genetic variants through functional molecular knowledge. To generate this knowledge at scale, sequence-based deep learning leverages high-quality genome sequence data to predict variant effects at base-pair resolution. When linked to agronomically important traits, these predictions enable breeders to prioritize variants for precision selection or editing. Although it is still in the early stages of development, we foresee three key applications for this approach: introgressing genes from distant breeding pools, purging deleterious mutations and designing new plant ideotypes. Looking ahead, refined computational models will facilitate targeted editing and the systematic redesign of complex physiological processes to address emerging breeding goals under shifting environmental conditions.

Crops, Agricultural↗

PubMed2005

Exploring the genome of Trypanosoma vivax through GSS and in silico comparative analysis.

A survey of the Trypanosoma vivax genome was carried out by the genome sequence survey (GSS) approach resulting in 1,086 genomic sequences. A total of 455 high-quality GSS sequences were generated, consisting of 331 non-redundant sequences distributed in 264 singlets and 67 clusters in a total of 135.5 Kb of the T. vivax genome. The estimation of the overall G+C content, and the prediction of the presence of ORFs and putative genes were carried out using the Glimmer and Jemboss packages. Analysis of the obtained sequences was carried out by BLAST programs against 12 different databases and also using the Conserved Domain Database, InterProScan, and tRNAscan-SE. Along with the existing 23 T. vivax entries in the GenBank, the 32 putative genes predicted and the 331 non-redundant GSS sequences reported herein represent new potential markers for the development of PCRbased assays for specific diagnosis and typing of Trypanosoma vivax.

Algorithms↗

PubMed1994

An rRNA variable region has an evolutionarily conserved essential role despite sequence divergence.

Regions extremely variable in size and sequence occur at conserved locations in eukaryotic rRNAs. The functional importance of one such region was determined by gene reconstruction and replacement in Tetrahymena thermophila. Deletion of the D8 region of the large-subunit rRNA inactivates T. thermophila rRNA genes (rDNA): transformants containing only this type of rDNA are unable to grow. Replacement with an unrelated sequence of similar size or a variable region from a different position in the rRNA also inactivated the rDNA. Mutant rRNAs resulting from such constructs were present only in precursor forms, suggesting that these rRNAs are deficient in either processing or stabilization of the mature form. Replacement with D8 regions from three other organisms restored function, even though the sequences are very different. Thus, these D8 regions share an essential functional feature that is not reflected in their primary sequences. Similar tertiary structures may be the quality these sequences share that allows them to function interchangeably.

Animals↗

PubMed1999

Automated system for purification of dye-terminator sequencing products eliminates up-stream purification of templates.

The quality of sequencing results is to a large extent determined by the purity of the template and the purification of the sequencing products. Fragments that can act as unspecific primers and templates are removed before gel analysis, and the background of unspecific signals is highly reduced. Purification of the sequencing products is needed to remove salts, nucleotides, proteins and template DNA that can interfere with the gel separation. We have developed a product, DYNAPURE Dye Terminator Removal, that specifically isolates and purifies the sequencing products in 10 min. The method is based on biotinylated sequencing primers and super-paramagnetic streptavidin beads. A PCR product is sequenced using a biotinylated sequencing primer, and the sequencing products are then bound to streptavidin beads in a 5-min reaction. The bead-DNA complexes are magnetically separated from the rest of the solution, and the remaining buffer constituents are washed away with TE buffer or with 70% ethanol. The whole procedure can be automated on liquid-handling robots fitted with a magnet station. The method eliminates purification of templates before cycle sequencing.

Automation↗

PubMed2006

Should the draft chimpanzee sequence be finished?

Owing to the availability of genome working drafts (WDs), current comparative-sequence studies are frequently performed on a genome-wide scale. In this article, we appraise the utility of WD sequences in the detection of genomic differences in closely related species. We compared human DNA sequences with draft and high-quality versions of the corresponding chimpanzee loci to reveal the overall high quality of the chimp WD sequence. Nevertheless, a significant proportion of the differences between WD and high-quality sequences we observed can be attributed to sequencing errors in the draft. Although we suggest methods to reduce the number of such false positives efficiently, our study emphasizes the benefit expected from finishing the chimpanzee genome sequence.

Animals↗

PubMed1998

Analysis of the quality and utility of random shotgun sequencing at low redundancies.

The currently favored approach for sequencing the human genome involves selecting representative large-insert clones (100-200 kb), randomly shearing this DNA to construct shotgun libraries, and then sequencing many different isolates from the library. This method, entitled directed random shotgun sequencing, requires highly redundant sequencing to obtain a complete and accurate finished consensus sequence. Recently it has been suggested that a rapidly generated lower redundancy sequence might be of use to the scientific community. Low-redundancy sequencing has been examined previously using simulated data sets. Here we utilize trace data from a number of projects submitted to GenBank to perform reconstruction experiments that mimic low-redundancy sequencing. These low-redundancy sequences have been examined for the completeness and quality of the consensus product, information content, and usefulness for interspecies comparisons. The data presented here suggest three different sequencing strategies, each with different utilities. (1) Nearly complete sequence data can be obtained by sequencing a random shotgun library at sixfold redundancy. This may therefore represent a good point to switch from a random to directed approach. (2) Sequencing can be performed with as little as twofold redundancy to find most of the information about exons, EST hits, and putative exon similarity matches. (3) To obtain contiguity of coding regions, sequencing at three- to fourfold redundancy would be appropriate. From these results, we suggest that a useful intermediate product for genome sequencing might be obtained by three- to fourfold redundancy. Such a product would allow a large amount of biologically useful data to be extracted while postponing the majority of work involved in producing a high quality consensus sequence.

Animals↗

PubMed2005

Random sheared fosmid library as a new genomic tool to accelerate complete finishing of rice (Oryza sativa spp. Nipponbare) genome sequence: sequencing of gap-specific fosmid clones uncovers new euchromatic portions of the genome.

The International Rice Genome Sequencing Project has recently announced the high-quality finished sequence that covers nearly 95% of the japonica rice genome representing 370 Mbp. Nevertheless, the current physical map of japonica rice contains 62 physical gaps corresponding to approximately 5% of the genome, that have not been identified/represented in the comprehensive array of publicly available BAC, PAC and other genomic library resources. Without finishing these gaps, it is impossible to identify the complete complement of genes encoded by rice genome and will also leave us ignorant of some 5% of the genome and its unknown functions. In this article, we report the construction and characterization of a tenfold redundant, 40 kbp insert fosmid library generated by random mechanical shearing. We demonstrated its utility in refining the physical map of rice by identifying and in silico mapping 22 gap-specific fosmid clones with particular emphasis on chromosomes 1, 2, 6, 7, 8, 9 and 10. Further sequencing of 12 of the gap-specific fosmid clones uncovered unique rice genome sequence that was not previously reported in the finished IRGSP sequence and emphasizes the need to complete finishing of the rice genome.

Base Sequence↗

PubMed2005

Endosperm-preferred expression of maize genes as revealed by transcriptome-wide analysis of expressed sequence tags.

The transcriptome-wide endosperm-preferred expression of maize genes was addressed by analyzing a large database of expressed sequence tags (ESTs). We generated 30,531 high quality sequence-reads from the 5'-ends of cDNA libraries from maize endosperm harvested at 10, 15, and 20 days after pollination. A further 196,900 maize sequence-reads retrieved from public databases were added to this endosperm collection to generate MAIZEST, a database with tools for data storage and analysis. MAIZEST contains 227,431 ESTs, one third of which represents developing endosperm and the remaining two-thirds represent transcripts from 49 cDNA libraries constructed from different organs and tissues. Assembling the MAIZEST ESTs generated 29,206 putative transcripts, of which a set of 4032 assembled sequences was composed exclusively of sequences derived from endosperm cDNA libraries. After sequence analysis using overlapping parameters, a sub-set of 2403 assembled sequences was functionally annotated and revealed a wide variety of putative new genes involved in endosperm development and metabolism.

Expressed Sequence Tags↗

PubMed2002

The genome sequence and structure of rice chromosome 1.

The rice species Oryza sativa is considered to be a model plant because of its small genome size, extensive genetic map, relative ease of transformation and synteny with other cereal crops. Here we report the essentially complete sequence of chromosome 1, the longest chromosome in the rice genome. We summarize characteristics of the chromosome structure and the biological insight gained from the sequence. The analysis of 43.3 megabases (Mb) of non-overlapping sequence reveals 6,756 protein coding genes, of which 3,161 show homology to proteins of Arabidopsis thaliana, another model plant. About 30% (2,073) of the genes have been functionally categorized. Rice chromosome 1 is (G + C)-rich, especially in its coding regions, and is characterized by several gene families that are dispersed or arranged in tandem repeats. Comparison with a draft sequence indicates the importance of a high-quality finished sequence.

Arabidopsis↗

PubMed1995

DNAView: a quality assessment tool for the visualization of large sequenced regions.

This communication describes DNAView, a graphical tool for the visualization and printing of large nucleic acid sequences. DNAView uses color coding to compactly display genomic segments of up to 100 kb on a single printed page. The specific color schemes integrated into DNAView can highlight 'local aggregate' properties of large segments of DNA. We have also incorporated a confidence expression for the assigned sequence. This is represented by base color intensity that is proportional to the number of times that base was sequenced. Areas of interest, such as exons, introns, repetitive elements and splice sites, can be emphasized using overlays. The colored image can be saved in a standard TIFF image file format that may be imported and annotated by other application software.

Base Sequence↗

PubMed2006

MUMMALS: multiple sequence alignment improved by using hidden Markov models with local structural information.

We have developed MUMMALS, a program to construct multiple protein sequence alignment using probabilistic consistency. MUMMALS improves alignment quality by using pairwise alignment hidden Markov models (HMMs) with multiple match states that describe local structural information without exploiting explicit structure predictions. Parameters for such models have been estimated from a large library of structure-based alignments. We show that (i) on remote homologs, MUMMALS achieves statistically best accuracy among several leading aligners, such as ProbCons, MAFFT and MUSCLE, albeit the average improvement is small, in the order of several percent; (ii) a large collection (>10 000) of automatically computed pairwise structure alignments of divergent protein domains is superior to smaller but carefully curated datasets for estimation of alignment parameters and performance tests; (iii) reference-independent evaluation of alignment quality using sequence alignment-dependent structure superpositions correlates well with reference-dependent evaluation that compares sequence-based alignments to structure-based reference alignments.

Markov Chains↗

PubMed2006

Quality assessment of DNA sequence data: autopsy of a mis-sequenced mtDNA population sample.

Published DNA data sets constitute a body of sequencing results resting in silico that are supposed to reflect the variation of (once) living cells. In cases where the DNA variation reported is suspected to be fraught with artefacts, an autopsy of the full body of data is needed to clarify the amount and causes of mis-sequencing. In this paper we elaborate on strategies that allow a clear-cut identification of the problems in severely flawed mtDNA data. This approach is applied, by way of example, to a data set of HVS-I sequences from the Caucasus, published by Nasidze & Stoneking in 2001. These data bear numerous ambiguous nucleotide positions and suffer from an even higher number of phantom mutations, indicating that severe biochemical problems adversely influenced those sequencing results at the time. Furthermore, systematic omission of sequences with a long C-stretch (incurred by a transition at position 16189) must have severely biased the data set. Since no complete correction of these data has appeared to date, this example of mis-sequencing necessitates circumstantial evidence that is bullet-proof.

Artifacts↗