Identification and analysis of genes and pseudogenes within duplicated regions in the human and mouse genomes

The identification and classification of genes and pseudogenes in duplicated regions still constitutes a challenge for standard automated genome annotation procedures. Using an integrated homology and orthology analysis independent of current gene annotation, we have identified 9,484 and 9,017 gene duplicates in human and mouse, respectively. On the basis of the integrity of their coding regions, we have classified them into functional and inactive duplicates, allowing us to define the first consistent and comprehensive collection of 1,811 human and 1,581 mouse unprocessed pseudogenes. Furthermore, of the total of 14,172 human and mouse duplicates predicted to be functional genes, as many as 420 are not included in current reference gene databases and therefore correspond to likely novel mammalian genes. Some of these correspond to partial duplicates with less than half of the length of the original source genes, yet they are conserved and syntenic among different mammalian lineages. The genes and unprocessed pseudogenes obtained here will enable further studies on the mechanisms involved in gene duplication as well as of the fate of duplicated genes.     

Avianbase: a community resource for bird genomics

Giving access to sequence and annotation data for genome assemblies is important because, while facilitating research, it places both assembly and annotation quality under scrutiny, resulting in improvements to both. Therefore we announce Avianbase, a resource for bird genomics, which provides access to data released by the Avian Phylogenomics Consortium.Access to complete genome sequences provides the first step towards the understanding of the biology of organisms. It is the template that underpins the phenotypic characteristics of individuals and ultimately separates species due to the accumulation and fixation of mutations over evolutionary timescales. In terms of the available genomic datasets for species, birds, as our more distant relatives, have been historically underrepresented. The high cost of sequencing and annotation in the past led to a bias towards accumulating data for species that are either established model organisms or economically significant (that is, chicken, turkey and duck, representing two sister orders within the Galloanseriformes clade from the large and diverse phylogeny of birds). The recent release of genome assemblies and initial predictions of protein-coding genes [1-4] for 44 bird species, including representatives from all major branches of the bird phylogeny, is, therefore, highly significant.One of the major challenges with the release of this number of newly sequenced genomes and the many more to come [5] is how to make these available to the various research communities in a way that supports basic research. Providing access to the sequences and initial annotations in the format of text files will limit the potential usage of the data as they require significant resources, including bioinformatics personnel and computer infrastructure in place to access and mine - for example, searching for genes belonging to certain protein families or searching for orthologous genes. These overheads pose a serious bottleneck that can hinder research and requires concerted action by the relevant research communities.Once genomes are submitted to public databases, genome-wide annotations are frequently generated and released either via the Ensembl project [6] or by the National Center for Biotechnology Information [7] and sequence and annotation are then made visually available online in integrated views via the Ensembl or the University of California Santa Cruz (UCSC) genome browsers [8]. These systems provide search facilities, sequence alignment tools like BLAT/BLAST and various analysis tools to facilitate subsetting and computational retrieval of the data, including UCSC’s Table Browser or Ensembl’s Perl and REST APIs and BioMart system.While these systems have become almost indispensable for research, not all sequenced genomes are annotated and displayed in genome browsers. Full genome annotation remains time consuming and resource intensive: a full evidence-based Ensembl genebuild takes approximately 4 months. Thus, the list of species represented is currently limited and depends on various factors, including the completeness of the assembled genome sequence and the overall demand in the scientific community for the resources, including whether the species is a model organism (for example, human or mouse), economically important (for example, farmed animals) or of specific phylogenetic interest. Many of the recently sequenced bird genomes do not obviously fall within these categories.     

Assessment of the relationship between signal intensities and transcript concentration for Affymetrix GeneChip®arrays

Background

The availability of both mouse and human draft genomes has marked the beginning of a new era of comparative mammalian genomics. The two available mouse genome assemblies, from the public mouse genome sequencing consortium and Celera Genomics, were obtained using different clone libraries and different assembly methods.

Results

We present here a critical comparison of the two latest mouse genome assemblies. The utility of the combined genomes is further demonstrated by comparing them with the human 'golden path' and through a subsequent analysis of a resulting conserved sequence element (CSE) database, which allows us to identify over 6,000 potential novel genes and to derive independent estimates of the number of human protein-coding genes.

Conclusion

The Celera and public mouse assemblies differ in about 10% of the mouse genome. Each assembly has advantages over the other: Celera has higher accuracy in base-pairs and overall higher coverage of the genome; the public assembly, however, has higher sequence quality in some newly finished bacterial artifical chromosome clone (BAC) regions and the data are freely accessible. Perhaps most important, by combining both assemblies, we can get a better annotation of the human genome; in particular, we can obtain the most complete set of CSEs, one third of which are related to known genes and some others are related to other functional genomic regions. More than half the CSEs are of unknown function. From the CSEs, we estimate the total number of human protein-coding genes to be about 40,000. This searchable publicly available online CSEdb will expedite new discoveries through comparative genomics.     

Evaluation of annotation strategies using an entire genome sequence

MOTIVATION: Genome-wide functional annotation either by manual or automatic means has raised considerable concerns regarding the accuracy of assignments and the reproducibility of methodologies. In addition, a performance evaluation of automated systems that attempt to tackle sequence analyses rapidly and reproducibly is generally missing. In order to quantify the accuracy and reproducibility of function assignments on a genome-wide scale, we have re-annotated the entire genome sequence of Chlamydia trachomatis (serovar D), in a collaborative manner. RESULTS: We have encoded all annotations in a structured format to allow further comparison and data exchange and have used a scale that records the different levels of potential annotation errors according to their propensity to propagate in the database due to transitive function assignments. We conclude that genome annotation may entail a considerable amount of errors, ranging from simple typographical errors to complex sequence analysis problems. The most surprising result of this comparative study is that automatic systems might perform as well as the teams of experts annotating genome sequences.     

PipTools: a computational toolkit to annotate and analyze pairwise comparisons of genomic sequences

Sequence conservation between species is useful both for locating coding regions of genes and for identifying functional noncoding segments. Hence interspecies alignment of genomic sequences is an important computational technique. However, its utility is limited without extensive annotation. We describe a suite of software tools, PipTools, and related programs that facilitate the annotation of genes and putative regulatory elements in pairwise alignments. The alignment server PipMaker uses the output of these tools to display detailed information needed to interpret alignments. These programs are provided in a portable format for use on common desktop computers and both the toolkit and the PipMaker server can be found at our Web site (http://bio.cse.psu.edu/). We illustrate the utility of the toolkit using annotation of a pairwise comparison of the mouse MHC class II and class III regions with orthologous human sequences and subsequently identify conserved, noncoding sequences that are DNase I hypersensitive sites in chromatin of mouse cells.     

Prediction of the coding sequences of mouse homologues of KIAA gene: II. The complete nucleotide sequences of 400 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have accumulated information of the coding sequences of uncharacterized human genes, which are known as KIAA genes, and the number of these genes exceeds 2000 at present. As an extension of this sequencing project, we recently have begun to accumulate mouse KIAA-homologous cDNAs, because it would be useful to prepare a set of human and mouse homologous cDNA pairs for further functional analysis of the KIAA genes. We herein present the entire sequences of 400 mouse KIAA cDNA clones and 4 novel cDNA clones which were incidentally identified during this project. Most of clones entirely sequenced in this study were selected by computer-assisted analysis of terminal sequences of the cDNAs. The average size of the 404 cDNA sequences reached 5.3 kb and that of the deduced amino acid sequences from these cDNAs was 868 amino acid residues. The results of sequence analyses of these clones showed that single mouse KIAA cDNAs bridged two different human KIAA cDNAs in some cases, which indicated that these two human KIAA cDNAs were derived from single genes although they had been supposed to originate from different genes. Furthermore, we successfully mapped all the mouse KIAA cDNAs along the genome using a recently published mouse genome draft sequence.     

Identification and quantification of disease-related gene clusters

MOTIVATION: DNA microarray technology and the completion of human and mouse genome sequencing programs are now offering new avenues for the investigation of complex genetic diseases. In particular, this makes possible the study of the spatial distribution of disease-related genes within the genome. We report on the first systematic search for clustering of genes associated with a polygenic autoimmune disease. RESULTS: Using a set of cDNA microarray chip experiments in two mouse models of rheumatoid arthritis, we have identified approximately 200 genes based on their expression in inflamed joints and mapped them into the genome. We compute the spatial autocorrelation function of the selected genes and find that they tend to cluster over scales of a few megabase pairs. We then identify significant gene clusters using a friends-of-friends algorithm. This approach should aid in discovering functionally related gene clusters in the mammalian genome.     

SNPMeta: SNP annotation and SNP metadata collection without a reference genome

The increase in availability of resequencing data is greatly accelerating SNP discovery and has facilitated the development of SNP genotyping assays. This, in turn, is increasing interest in annotation of individual SNPs. Currently, these data are only available through curation, or comparison to a reference genome. Many species lack a reference genome, but are still important genetic models or are significant species in agricultural production or natural ecosystems. For these species, it is possible to annotate SNPs through comparison with cDNA, or data from well‐annotated genes in public repositories. We present SNPMeta, a tool which gathers information about SNPs by comparison with sequences present in GenBank databases. SNPMeta is able to annotate SNPs from contextual sequence in SNP assay designs, and SNPs discovered through genotyping by sequencing (GBS) approaches. However, SNPs discovered through GBS occur throughout the genome, rather than only in gene space, and therefore do not annotate at high rates. SNPMeta can therefore be used to annotate SNPs in nonmodel species or species that lack a reference genome. Annotations generated by SNPMeta are highly concordant with annotations that would be obtained from a reference genome.     

Upstream - news in genomics

Since our last issue, several important genomes have been completely or 'almost completely' sequenced. The debate over the number of human genes has flared up once more, with one computational and one experimental study into the annotation of the human genome. The mouse genome project has a clone fingerprint map to aid their sequencing effort. The SAGE technique has been applied to Drosophila and the US National Science Foundation announced increased spending on plant genome research.     

A database for G proteins and their interaction with GPCRs

Background

The explosion in biological information creates the need for databases that are easy to develop, easy to maintain and can be easily manipulated by annotators who are most likely to be biologists. However, deployment of scalable and extensible databases is not an easy task and generally requires substantial expertise in database development.

Results

BioBuilder is a Zope-based software tool that was developed to facilitate intuitive creation of protein databases. Protein data can be entered and annotated through web forms along with the flexibility to add customized annotation features to protein entries. A built-in review system permits a global team of scientists to coordinate their annotation efforts. We have already used BioBuilder to develop Human Protein Reference Database http://www.hprd.org, a comprehensive annotated repository of the human proteome. The data can be exported in the extensible markup language (XML) format, which is rapidly becoming as the standard format for data exchange.

Conclusions

As the proteomic data for several organisms begins to accumulate, BioBuilder will prove to be an invaluable platform for functional annotation and development of customizable protein centric databases. BioBuilder is open source and is available under the terms of LGPL.     

Expressed peptide tags: an additional layer of data for genome annotation

While genome sequencing is becoming ever more routine, genome annotation remains a challenging process. Identification of the coding sequences within the genomic milieu presents a tremendous challenge, especially for eukaryotes with their complex gene architectures. Here, we present a method to assist the annotation process through the use of proteomic data and bioinformatics. Mass spectra of digested protein preparations of the organism of interest were acquired and searched against a protein database created by a six-frame translation of the genome. The identified peptides were mapped back to the genome, compared to the current annotation, and then categorized as supporting or extending the current genome annotation. We named the classified peptides Expressed Peptide Tags (EPTs). The well-annotated bacterium Rhodopseudomonas palustris was used as a control for the method and showed a high degree of correlation between EPT mapping and the current annotation, with 86% of the EPTs confirming existing gene calls and less than 1% of the EPTs expanding on the current annotation. The eukaryotic plant pathogens Phytophthora ramorum and Phytophthora sojae, whose genomes have been recently sequenced and are much less well-annotated, were also subjected to this method. A series of algorithmic steps were taken to increase the confidence of EPT identification for these organisms, including generation of smaller subdatabases to be searched against, and definition of EPT criteria that accommodates the more complex eukaryotic gene architecture. As expected, the analysis of the Phytophthora species showed less correlation between EPT mapping and their current annotation. While approximately 76% of Phytophthora EPTs supported the current annotation, a portion of them (7.7% and 12.9% for P. ramorum and P. sojae, respectively) suggested modification to current gene calls or identified novel genes that were missed by the current genome annotation of these organisms.     

Genome Information Broker (GIB): data retrieval and comparative analysis system for completed microbial genomes and more

Genome Information Broker (GIB) is a powerful tool for the study of comparative genomics. GIB allows users to retrieve and display partial and/or whole genome sequences together with the relevant biological annotation. GIB has accumulated all the completed microbial genome and has recently been expanded to include Arabidopsis thaliana genome data from DDBJ/EMBL/GenBank. In the near future, hundreds of genome sequences will be determined. In order to handle such huge data, we have enhanced the GIB architecture by using XML, CORBA and distributed RDBs. We introduce the new GIB here. GIB is freely accessible at http://gib.genes.nig.ac.jp/.     

Integrating 'omic' information: a bridge between genomics and systems biology

The availability of genome sequences for several organisms, including humans, and the resulting first-approximation lists of genes, have allowed a transition from molecular biology to 'modular biology'. In modular biology, biological processes of interest, or modules, are studied as complex systems of functionally interacting macromolecules. Functional genomic and proteomic ('omic') approaches can be helpful to accelerate the identification of the genes and gene products involved in particular modules, and to describe the functional relationships between them. However, the data emerging from individual omic approaches should be viewed with caution because of the occurrence of false-negative and false-positive results and because single annotations are not sufficient for an understanding of gene function. To increase the reliability of gene function annotation, multiple independent datasets need to be integrated. Here, we review the recent development of strategies for such integration and we argue that these will be important for a systems approach to modular biology.     

An extensible application for assembling annotation for genomic data

SUMMARY: AnnBuilder is an R package for assembling genomic annotation data. The system currently provides parsers to process annotation data from LocusLink, Gene Ontology Consortium, and Human Gene Project and can be extended to new data sources via user defined parsers. AnnBuilder differs from other existing systems in that it provides users with unlimited ability to assemble data from user selected sources. The products of AnnBuilder are files in XML format that can be easily used by different systems. AVAILABILITY: (http://www.bioconductor.org). Open source.