Genome assembly has a major impact on gene content: a comparison of annotation in two Bos taurus assemblies.

Gene and SNP annotation are among the first and most important steps in analyzing a genome. As the number of sequenced genomes continues to grow, a key question is: how does the quality of the assembled sequence affect the annotations? We compared the gene and SNP annotations for two different Bos taurus genome assemblies built from the same data but with significant improvements in the later assembly. The same annotation software was used for annotating both sequences. While some annotation differences are expected even between high-quality assemblies such as these, we found that a staggering 40% of the genes (>9,500) varied significantly between assemblies, due in part to the availability of new gene evidence but primarily to genome mis-assembly events and local sequence variations. For instance, although the later assembly is generally superior, 660 protein coding genes in the earlier assembly are entirely missing from the later genome's annotation, and approximately 3,600 (15%) of the genes have complex structural differences between the two assemblies. In addition, 12-20% of the predicted proteins in both assemblies have relatively large sequence differences when compared to their RefSeq models, and 6-15% of bovine dbSNP records are unrecoverable in the two assemblies. Our findings highlight the consequences of genome assembly quality on gene and SNP annotation and argue for continued improvements in any draft genome sequence. We also found that tracking a gene between different assemblies of the same genome is surprisingly difficult, due to the numerous changes, both small and large, that occur in some genes. As a side benefit, our analyses helped us identify many specific loci for improvement in the Bos taurus genome assembly.

The ecoresponsive genome of Daphnia pulex.

We describe the draft genome of the microcrustacean Daphnia pulex, which is only 200 megabases and contains at least 30,907 genes. The high gene count is a consequence of an elevated rate of gene duplication resulting in tandem gene clusters. More than a third of Daphnia's genes have no detectable homologs in any other available proteome, and the most amplified gene families are specific to the Daphnia lineage. The coexpansion of gene families interacting within metabolic pathways suggests that the maintenance of duplicated genes is not random, and the analysis of gene expression under different environmental conditions reveals that numerous paralogs acquire divergent expression patterns soon after duplication. Daphnia-specific genes, including many additional loci within sequenced regions that are otherwise devoid of annotations, are the most responsive genes to ecological challenges.

Estimates of positive Darwinian selection are inflated by errors in sequencing, annotation, and alignment.

Published estimates of the proportion of positively selected genes (PSGs) in human vary over three orders of magnitude. In mammals, estimates of the proportion of PSGs cover an even wider range of values. We used 2,980 orthologous protein-coding genes from human, chimpanzee, macaque, dog, cow, rat, and mouse as well as an established phylogenetic topology to infer the fraction of PSGs in all seven terminal branches. The inferred fraction of PSGs ranged from 0.9% in human through 17.5% in macaque to 23.3% in dog. We found three factors that influence the fraction of genes that exhibit telltale signs of positive selection: the quality of the sequence, the degree of misannotation, and ambiguities in the multiple sequence alignment. The inferred fraction of PSGs in sequences that are deficient in all three criteria of coverage, annotation, and alignment is 7.2 times higher than that in genes with high trace sequencing coverage, "known" annotation status, and perfect alignment scores. We conclude that some estimates on the prevalence of positive Darwinian selection in the literature may be inflated and should be treated with caution.

Splign: algorithms for computing spliced alignments with identification of paralogs.

BACKGROUND: The computation of accurate alignments of cDNA sequences against a genome is at the foundation of modern genome annotation pipelines. Several factors such as presence of paralogs, small exons, non-consensus splice signals, sequencing errors and polymorphic sites pose recognized difficulties to existing spliced alignment algorithms. RESULTS: We describe a set of algorithms behind a tool called Splign for computing cDNA-to-Genome alignments. The algorithms include a high-performance preliminary alignment, a compartment identification based on a formally defined model of adjacent duplicated regions, and a refined sequence alignment. In a series of tests, Splign has produced more accurate results than other tools commonly used to compute spliced alignments, in a reasonable amount of time. CONCLUSION: Splign's ability to deal with various issues complicating the spliced alignment problem makes it a helpful tool in eukaryotic genome annotation processes and alternative splicing studies. Its performance is enough to align the largest currently available pools of cDNA data such as the human EST set on a moderate-sized computing cluster in a matter of hours. The duplications identification (compartmentization) algorithm can be used independently in other areas such as the study of pseudogenes.

Microwave tomography for detection/imaging of myocardial infarction. I. Excised canine hearts.

We have demonstrated previously that the dielectric properties of myocardium at microwave spectrum are a sensitive indicator of its blood content, ischemia, and infarction. The purpose of this study is to validate the feasibility of microwave tomography for detection of myocardial infarction based on the differences in dielectric properties between normal and infarcted tissues. Excised canine heats with two weeks myocardial infarction were imaged. Tomographic imaging experiments were conducted using a three-dimensional (3D) microwave tomographic system operating at a frequency of 1.0 GHz. To obtain the images, we used 3D reconstruction algorithms. Images of excised canine hearts with myocardial infarction were obtained at a frequency of 1 GHz, applicable for whole body imaging. Microwave tomographic images were compared with anatomical slices. The comparison confirms that microwave tomography is capable of detection of myocardial infarction.

The relationship of protein conservation and sequence length.

BACKGROUND: In general, the length of a protein sequence is determined by its function and the wide variance in the lengths of an organism's proteins reflects the diversity of specific functional roles for these proteins. However, additional evolutionary forces that affect the length of a protein may be revealed by studying the length distributions of proteins evolving under weaker functional constraints. RESULTS: We performed sequence comparisons to distinguish highly conserved and poorly conserved proteins from the bacterium Escherichia coli, the archaeon Archaeoglobus fulgidus, and the eukaryotes Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens. For all organisms studied, the conserved and nonconserved proteins have strikingly different length distributions. The conserved proteins are, on average, longer than the poorly conserved ones, and the length distributions for the poorly conserved proteins have a relatively narrow peak, in contrast to the conserved proteins whose lengths spread over a wider range of values. For the two prokaryotes studied, the poorly conserved proteins approximate the minimal length distribution expected for a diverse range of structural folds. CONCLUSIONS: There is a relationship between protein conservation and sequence length. For all the organisms studied, there seems to be a significant evolutionary trend favoring shorter proteins in the absence of other, more specific functional constraints.

Genomic BLAST: custom-defined virtual databases for complete and unfinished genomes.

BLAST (Basic Local Alignment Search Tool) searches against DNA and protein sequence databases have become an indispensable tool for biomedical research. The proliferation of the genome sequencing projects is steadily increasing the fraction of genome-derived sequences in the public databases and their importance as a public resource. We report here the availability of Genomic BLAST, a novel graphical tool for simplifying BLAST searches against complete and unfinished genome sequences. This tool allows the user to compare the query sequence against a virtual database of DNA and/or protein sequences from a selected group of organisms with finished or unfinished genomes. The organisms for such a database can be selected using either a graphic taxonomy-based tree or an alphabetical list of organism-specific sequences. The first option is designed to help explore the evolutionary relationships among organisms within a certain taxonomy group when performing BLAST searches. The use of an alphabetical list allows the user to perform a more elaborate set of selections, assembling any given number of organism-specific databases from unfinished or complete genomes. This tool, available at the NCBI web site http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/genom_table_cgi, currently provides access to over 170 bacterial and archaeal genomes and over 40 eukaryotic genomes.

Dielectrical spectroscopy of canine myocardium during acute ischemia and hypoxia at frequency spectrum from 100 kHz to 6 GHz.

We studied dielectrical properties of canine myocardium during acute ischemia and hypoxia using dielectrical spectroscopy method at frequency spectrum from 100 kHz to 6 GHz. This study was conducted on a group of six canines with acute ischemia and seven canines with hypoxia. Hypoxia (10% for 30 min) decreases myocardial resistance (rho), while the dielectrical permittivity (epsilon') of the myocardial tissue remains statistically unchanged. Acute ischemia for 2 hr causes significant frequency-dependent changes in both epsilon' and rho of myocardial tissue. Myocardial resistance increases, while the sign and amplitude of changes in the myocardial epsilon' are frequency and time dependent. These observations open up an opportunity for assessing the properties of myocardial tissue using dielectrical spectroscopy as well as noninvasively with the help of imaging methods based on electrical impedance and microwave tomography.

Three-dimensional microwave tomography: initial experimental imaging of animals.

The purpose of this study was to construct a microwave tomographic system capable of conducting experiments with whole scale biological objects and to demonstrate the feasibility of microwave tomography for imaging such objects using a canine model. Experiments were conducted using a three-dimensional (3-D) microwave tomographic system with working chamber dimensions of 120 cm in diameter and 135 cm in height. The operating frequency was 0.9 GHz. The object under study was located in the central area of the tomographic chamber filled with a salt solution. Experimentally measured attenuation of the electromagnetic field through the thorax was about -120 dB. To obtain images, we used various two-dimensional and 3-D reconstruction schemes. Images of the canine were obtained. In spite of imperfections, the images represent a significant milestone in the development of microwave tomography for whole body imaging and demonstrate its feasibility.