Phylogenetic signal from rearrangements in 18 Anopheles species by joint scaffolding extant and ancestral genomes

Background

Genomes rearrangements carry valuable information for phylogenetic inference or the elucidation of molecular mechanisms of adaptation. However, the detection of genome rearrangements is often hampered by current deficiencies in data and methods: Genomes obtained from short sequence reads have generally very fragmented assemblies, and comparing multiple gene orders generally leads to computationally intractable algorithmic questions.

Results

We present a computational method, ADseq, which, by combining ancestral gene order reconstruction, comparative scaffolding and de novo scaffolding methods, overcomes these two caveats. ADseq provides simultaneously improved assemblies and ancestral genomes, with statistical supports on all local features. Compared to previous comparative methods, it runs in polynomial time, it samples solutions in a probabilistic space, and it can handle a significantly larger gene complement from the considered extant genomes, with complex histories including gene duplications and losses. We use ADseq to provide improved assemblies and a genome history made of duplications, losses, gene translocations, rearrangements, of 18 complete Anopheles genomes, including several important malaria vectors. We also provide additional support for a differentiated mode of evolution of the sex chromosome and of the autosomes in these mosquito genomes.

Conclusions

We demonstrate the method’s ability to improve extant assemblies accurately through a procedure simulating realistic assembly fragmentation. We study a debated issue regarding the phylogeny of the Gambiae complex group of Anopheles genomes in the light of the evolution of chromosomal rearrangements, suggesting that the phylogenetic signal they carry can differ from the phylogenetic signal carried by gene sequences, more prone to introgression.

     

The COG database: an updated version includes eukaryotes

Background

The availability of multiple, essentially complete genome sequences of prokaryotes and eukaryotes spurred both the demand and the opportunity for the construction of an evolutionary classification of genes from these genomes. Such a classification system based on orthologous relationships between genes appears to be a natural framework for comparative genomics and should facilitate both functional annotation of genomes and large-scale evolutionary studies.

Results

We describe here a major update of the previously developed system for delineation of Clusters of Orthologous Groups of proteins (COGs) from the sequenced genomes of prokaryotes and unicellular eukaryotes and the construction of clusters of predicted orthologs for 7 eukaryotic genomes, which we named KOGs after eukaryotic orthologous groups. The COG collection currently consists of 138,458 proteins, which form 4873 COGs and comprise 75% of the 185,505 (predicted) proteins encoded in 66 genomes of unicellular organisms. The eukaryotic orthologous groups (KOGs) include proteins from 7 eukaryotic genomes: three animals (the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster and Homo sapiens), one plant, Arabidopsis thaliana, two fungi (Saccharomyces cerevisiae and Schizosaccharomyces pombe), and the intracellular microsporidian parasite Encephalitozoon cuniculi. The current KOG set consists of 4852 clusters of orthologs, which include 59,838 proteins, or ~54% of the analyzed eukaryotic 110,655 gene products. Compared to the coverage of the prokaryotic genomes with COGs, a considerably smaller fraction of eukaryotic genes could be included into the KOGs; addition of new eukaryotic genomes is expected to result in substantial increase in the coverage of eukaryotic genomes with KOGs. Examination of the phyletic patterns of KOGs reveals a conserved core represented in all analyzed species and consisting of ~20% of the KOG set. This conserved portion of the KOG set is much greater than the ubiquitous portion of the COG set (~1% of the COGs). In part, this difference is probably due to the small number of included eukaryotic genomes, but it could also reflect the relative compactness of eukaryotes as a clade and the greater evolutionary stability of eukaryotic genomes.

Conclusion

The updated collection of orthologous protein sets for prokaryotes and eukaryotes is expected to be a useful platform for functional annotation of newly sequenced genomes, including those of complex eukaryotes, and genome-wide evolutionary studies.     

Microarray meta-analysis database (M2DB): a uniformly pre-processed, quality controlled, and manually curated human clinical microarray database

Background

With the rapid expansion of DNA sequencing databases, it is now feasible to identify relevant information from prior sequencing projects and completed genomes and apply it to de novo sequencing of new organisms. As an example, this paper demonstrates how such extra information can be used to improve de novo assemblies by augmenting the overlapping step. Finding all pairs of overlapping reads is a key task in many genome assemblers, and to this end, highly efficient algorithms have been developed to find alignments in large collections of sequences. It is well known that due to repeated sequences, many aligned pairs of reads nevertheless do not overlap. But no overlapping algorithm to date takes a rigorous approach to separating aligned but non-overlapping read pairs from true overlaps.

Results

We present an approach that extends the Minimus assembler by a data driven step to classify overlaps as true or false prior to contig construction. We trained several different classification models within the Weka framework using various statistics derived from overlaps of reads available from prior sequencing projects. These statistics included percent mismatch and k-mer frequencies within the overlaps as well as a comparative genomics score derived from mapping reads to multiple reference genomes. We show that in real whole-genome sequencing data from the E. coli and S. aureus genomes, by providing a curated set of overlaps to the contigging phase of the assembler, we nearly doubled the median contig length (N50) without sacrificing coverage of the genome or increasing the number of mis-assemblies.

Conclusions

Machine learning methods that use comparative and non-comparative features to classify overlaps as true or false can be used to improve the quality of a sequence assembly.     

Complete reannotation of the Arabidopsis genome: methods, tools, protocols and the final release

Background

Since the initial publication of its complete genome sequence, Arabidopsis thaliana has become more important than ever as a model for plant research. However, the initial genome annotation was submitted by multiple centers using inconsistent methods, making the data difficult to use for many applications.

Results

Over the course of three years, TIGR has completed its effort to standardize the structural and functional annotation of the Arabidopsis genome. Using both manual and automated methods, Arabidopsis gene structures were refined and gene products were renamed and assigned to Gene Ontology categories. We present an overview of the methods employed, tools developed, and protocols followed, summarizing the contents of each data release with special emphasis on our final annotation release (version 5).

Conclusion

Over the entire period, several thousand new genes and pseudogenes were added to the annotation. Approximately one third of the originally annotated gene models were significantly refined yielding improved gene structure annotations, and every protein-coding gene was manually inspected and classified using Gene Ontology terms.     

Anopheles gambiae genome reannotation through synthesis of ab initio and comparative gene prediction algorithms

Background

Complete genome annotation is a necessary tool as Anopheles gambiae researchers probe the biology of this potent malaria vector.

Results

We reannotate the A. gambiae genome by synthesizing comparative and ab initio sets of predicted coding sequences (CDSs) into a single set using an exon-gene-union algorithm followed by an open-reading-frame-selection algorithm. The reannotation predicts 20,970 CDSs supported by at least two lines of evidence, and it lowers the proportion of CDSs lacking start and/or stop codons to only approximately 4%. The reannotated CDS set includes a set of 4,681 novel CDSs not represented in the Ensembl annotation but with EST support, and another set of 4,031 Ensembl-supported genes that undergo major structural and, therefore, probably functional changes in the reannotated set. The quality and accuracy of the reannotation was assessed by comparison with end sequences from 20,249 full-length cDNA clones, and evaluation of mass spectrometry peptide hit rates from an A. gambiae shotgun proteomic dataset confirms that the reannotated CDSs offer a high quality protein database for proteomics. We provide a functional proteomics annotation, ReAnoXcel, obtained by analysis of the new CDSs through the AnoXcel pipeline, which allows functional comparisons of the CDS sets within the same bioinformatic platform. CDS data are available for download.

Conclusion

Comprehensive A. gambiae genome reannotation is achieved through a combination of comparative and ab initio gene prediction algorithms.     

Evidence for abundant transcription of non-coding regions in the Saccharomyces cerevisiae genome

Background

The ChickRH6 whole chicken genome radiation hybrid (RH) panel recently produced has already been used to build radiation hybrid maps for several chromosomes, generating comparative maps with the human and mouse genomes and suggesting improvements to the chicken draft sequence assembly. Here we present the construction of a RH map of chicken chromosome 2. Markers from the genetic map were used for alignment to the existing GGA2 (Gallus gallus chromosome 2) linkage group and EST were used to provide valuable comparative mapping information. Finally, all markers from the RH map were localised on the chicken draft sequence assembly to check for eventual discordances.

Results

Eighty eight microsatellite markers, 10 genes and 219 EST were selected from the genetic map or on the basis of available comparative mapping information. Out of these 317 markers, 270 gave reliable amplifications on the radiation hybrid panel and 198 were effectively assigned to GGA2. The final RH map is 2794 cR₆₀₀₀ long and is composed of 86 framework markers distributed in 5 groups. Conservation of synteny was found between GGA2 and eight human chromosomes, with segments of conserved gene order of varying lengths.

Conclusion

We obtained a radiation hybrid map of chicken chromosome 2. Comparison to the human genome indicated that most of the 8 groups of conserved synteny studied underwent internal rearrangements. The alignment of our RH map to the first draft of the chicken genome sequence assembly revealed a good agreement between both sets of data, indicative of a low error rate.     

Assessing the impact of comparative genomic sequence data on the functional annotation of the Drosophila genome

Background

It is widely accepted that comparative sequence data can aid the functional annotation of genome sequences; however, the most informative species and features of genome evolution for comparison remain to be determined.

Results

We analyzed conservation in eight genomic regions (apterous, even-skipped, fushi tarazu, twist, and Rhodopsins 1, 2, 3 and 4) from four Drosophila species (D. erecta, D. pseudoobscura, D. willistoni, and D. littoralis) covering more than 500 kb of the D. melanogaster genome. All D. melanogaster genes (and 78-82% of coding exons) identified in divergent species such as D. pseudoobscura show evidence of functional constraint. Addition of a third species can reveal functional constraint in otherwise non-significant pairwise exon comparisons. Microsynteny is largely conserved, with rearrangement breakpoints, novel transposable element insertions, and gene transpositions occurring in similar numbers. Rates of amino-acid substitution are higher in uncharacterized genes relative to genes that have previously been studied. Conserved non-coding sequences (CNCSs) tend to be spatially clustered with conserved spacing between CNCSs, and clusters of CNCSs can be used to predict enhancer sequences.

Conclusions

Our results provide the basis for choosing species whose genome sequences would be most useful in aiding the functional annotation of coding and cis-regulatory sequences in Drosophila. Furthermore, this work shows how decoding the spatial organization of conserved sequences, such as the clustering of CNCSs, can complement efforts to annotate eukaryotic genomes on the basis of sequence conservation alone.     

Genome evolution in Reptilia: <Emphasis Type="Italic">in silico</Emphasis> chicken mapping of 12,000 BAC-end sequences from two reptiles and a basal bird

Background

With the publication of the draft chicken genome and the recent production of several BAC clone libraries from non-avian reptiles and birds, it is now possible to undertake more detailed comparative genomic studies in Reptilia. Of interest in particular are the genomic events that transformed the large, repeat-rich genomes of mammals and non-avian reptiles into the minimalist chicken genome. We have used paired BAC end sequences (BESs) from the American alligator (Alligator mississippiensis), painted turtle (Chrysemys picta) and emu (Dromaius novaehollandiae) to investigate patterns of sequence divergence, gene and retroelement content, and microsynteny between these species and chicken.

Results

From a total of 11,967 curated BESs, we successfully mapped 725, 773 and 2597 sequences in alligator, turtle, and emu, respectively, to sites in the draft chicken genome using a stringent BLAST protocol. Most commonly, sequences mapped to a single site in the chicken genome. Of 1675, 1828 and 2936 paired BESs obtained for alligator, turtle, and emu, respectively, a total of 34 (alligator, 2%), 24 (turtle, 1.3%) and 479 (emu, 16.3%) pairs were found to map with high confidence and in the correct orientation and with BAC-sized intermarker distances to single chicken chromosomes, including 25 such paired hits in emu mapping to the chicken Z chromosome. By determining the insert sizes of a subset of BAC clones from these three species, we also found a significant correlation between the intermarker distance in alligator and turtle and in chicken, with slopes as expected on the basis of the ratio of the genome sizes.

Conclusion

Our results suggest that a large number of small-scale chromosomal rearrangements and deletions in the lineage leading to chicken have drastically reduced the number of detected syntenies observed between the chicken and alligator, turtle, and emu genomes and imply that small deletions occurring widely throughout the genomes of reptilian and avian ancestors led to the ~50% reduction in genome size observed in birds compared to reptiles. We have also mapped and identified likely gene regions in hundreds of new BAC clones from these species.

     

Genome-wide analysis of the role of GlnR in Streptomyces venezuelae provides new insights into global nitrogen regulation in actinomycetes

Background

Knowledge of the origins, distribution, and inheritance of variation in the malaria parasite (Plasmodium falciparum) genome is crucial for understanding its evolution; however the 81% (A+T) genome poses challenges to high-throughput sequencing technologies. We explore the viability of the Roche 454 Genome Sequencer FLX (GS FLX) high throughput sequencing technology for both whole genome sequencing and fine-resolution characterization of genetic exchange in malaria parasites.

Results

We present a scheme to survey recombination in the haploid stage genomes of two sibling parasite clones, using whole genome pyrosequencing that includes a sliding window approach to predict recombination breakpoints. Whole genome shotgun (WGS) sequencing generated approximately 2 million reads, with an average read length of approximately 300 bp. De novo assembly using a combination of WGS and 3 kb paired end libraries resulted in contigs ≤ 34 kb. More than 8,000 of the 24,599 SNP markers identified between parents were genotyped in the progeny, resulting in a marker density of approximately 1 marker/3.3 kb and allowing for the detection of previously unrecognized crossovers (COs) and many non crossover (NCO) gene conversions throughout the genome.

Conclusions

By sequencing the 23 Mb genomes of two haploid progeny clones derived from a genetic cross at more than 30× coverage, we captured high resolution information on COs, NCOs and genetic variation within the progeny genomes. This study is the first to resequence progeny clones to examine fine structure of COs and NCOs in malaria parasites.     

A BAC-based integrated linkage map of the silkworm Bombyx mori

Background

In 2004, draft sequences of the model lepidopteran Bombyx mori were reported using whole-genome shotgun sequencing. Because of relatively shallow genome coverage, the silkworm genome remains fragmented, hampering annotation and comparative genome studies. For a more complete genome analysis, we developed extended scaffolds combining physical maps with improved genetic maps.

Results

We mapped 1,755 single nucleotide polymorphism (SNP) markers from bacterial artificial chromosome (BAC) end sequences onto 28 linkage groups using a recombining male backcross population, yielding an average inter-SNP distance of 0.81 cM (about 270 kilobases). We constructed 6,221 contigs by fingerprinting clones from three BAC libraries digested with different restriction enzymes, and assigned a total of 724 single copy genes to them by BLAST (basic local alignment search tool) search of the BAC end sequences and high-density BAC filter hybridization using expressed sequence tags as probes. We assigned 964 additional expressed sequence tags to linkage groups by restriction fragment length polymorphism analysis of a nonrecombining female backcross population. Altogether, 361.1 megabases of BAC contigs and singletons were integrated with a map containing 1,688 independent genes. A test of synteny using Oxford grid analysis with more than 500 silkworm genes revealed six versus 20 silkworm linkage groups containing eight or more orthologs of Apis versus Tribolium, respectively.

Conclusion

The integrated map contains approximately 10% of predicted silkworm genes and has an estimated 76% genome coverage by BACs. This provides a new resource for improved assembly of whole-genome shotgun data, gene annotation and positional cloning, and will serve as a platform for comparative genomics and gene discovery in Lepidoptera and other insects.     

BEACON: automated tool for Bacterial GEnome Annotation ComparisON

Background

Genome annotation is one way of summarizing the existing knowledge about genomic characteristics of an organism. There has been an increased interest during the last several decades in computer-based structural and functional genome annotation. Many methods for this purpose have been developed for eukaryotes and prokaryotes. Our study focuses on comparison of functional annotations of prokaryotic genomes. To the best of our knowledge there is no fully automated system for detailed comparison of functional genome annotations generated by different annotation methods (AMs).

Results

The presence of many AMs and development of new ones introduce needs to: a/ compare different annotations for a single genome, and b/ generate annotation by combining individual ones. To address these issues we developed an Automated Tool for Bacterial GEnome Annotation ComparisON (BEACON) that benefits both AM developers and annotation analysers. BEACON provides detailed comparison of gene function annotations of prokaryotic genomes obtained by different AMs and generates extended annotations through combination of individual ones. For the illustration of BEACON’s utility, we provide a comparison analysis of multiple different annotations generated for four genomes and show on these examples that the extended annotation can increase the number of genes annotated by putative functions up to 27 %, while the number of genes without any function assignment is reduced.

Conclusions

We developed BEACON, a fast tool for an automated and a systematic comparison of different annotations of single genomes. The extended annotation assigns putative functions to many genes with unknown functions. BEACON is available under GNU General Public License version 3.0 and is accessible at: http://www.cbrc.kaust.edu.sa/BEACON/.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1826-4) contains supplementary material, which is available to authorized users.     

Spectrum of heart diseases in a new cardiac service in Nigeria: An echocardiographic study of 1441 subjects in Abeokuta

Background

The recent determination of the complete nucleotide sequence of several Mycobacterium tuberculosis (MTB) genomes allows the use of comparative genomics as a tool for dissecting the nature and consequence of genetic variability within this species. The multiple alignment of the genomes of clinical strains (CDC1551, F11, Haarlem and C), along with the genomes of laboratory strains (H37Rv and H37Ra), provides new insights on the mechanisms of adaptation of this bacterium to the human host.

Findings

The genetic variation found in six M. tuberculosis strains does not involve significant genomic rearrangements. Most of the variation results from deletion and transposition events preferentially associated with insertion sequences and genes of the PE/PPE family but not with genes implicated in virulence. Using a Perl-based software islandsanalyser, which creates a representation of the genetic variation in the genome, we identified differences in the patterns of distribution and frequency of the polymorphisms across the genome. The identification of genes displaying strain-specific polymorphisms and the extrapolation of the number of strain-specific polymorphisms to an unlimited number of genomes indicates that the different strains contain a limited number of unique polymorphisms.

Conclusion

The comparison of multiple genomes demonstrates that the M. tuberculosis genome is currently undergoing an active process of gene decay, analogous to the adaptation process of obligate bacterial symbionts. This observation opens new perspectives into the evolution and the understanding of the pathogenesis of this bacterium.     

Genome-wide characterization of the biggest grass, bamboo, based on 10,608 putative full-length cDNA sequences

Background

With the availability of rice and sorghum genome sequences and ongoing efforts to sequence genomes of other cereal and energy crops, the grass family (Poaceae) has become a model system for comparative genomics and for better understanding gene and genome evolution that underlies phenotypic and ecological divergence of plants. While the genomic resources have accumulated rapidly for almost all major lineages of grasses, bamboo remains the only large subfamily of Poaceae with little genomic information available in databases, which seriously hampers our ability to take a full advantage of the wealth of grass genomic data for effective comparative studies.

Results

Here we report the cloning and sequencing of 10,608 putative full length cDNAs (FL-cDNAs) primarily from Moso bamboo, Phyllostachys heterocycla cv. pubescens, a large woody bamboo with the highest ecological and economic values of all bamboos. This represents the third largest FL-cDNA collection to date of all plant species, and provides the first insight into the gene and genome structures of bamboos. We developed a Moso bamboo genomic resource database that so far contained the sequences of 10,608 putative FL-cDNAs and nearly 38,000 expressed sequence tags (ESTs) generated in this study.

Conclusion

Analysis of FL-cDNA sequences show that bamboo diverged from its close relatives such as rice, wheat, and barley through an adaptive radiation. A comparative analysis of the lignin biosynthesis pathway between bamboo and rice suggested that genes encoding caffeoyl-CoA O-methyltransferase may serve as targets for genetic manipulation of lignin content to reduce pollutants generated from bamboo pulping.     

Comparative hybridization reveals extensive genome variation in the AIDS-associated pathogen Cryptococcus neoformans

Background

Genome variability can have a profound influence on the virulence of pathogenic microbes. The availability of genome sequences for two strains of the AIDS-associated fungal pathogen Cryptococcus neoformans presented an opportunity to use comparative genome hybridization (CGH) to examine genome variability between strains of different mating type, molecular subtype, and ploidy.

Results

Initially, CGH was used to compare the approximately 100 kilobase MAT a and MATα mating-type regions in serotype A and D strains to establish the relationship between the Log2 ratios of hybridization signals and sequence identity. Subsequently, we compared the genomes of the environmental isolate NIH433 (MAT a) and the clinical isolate NIH12 (MATα) with a tiling array of the genome of the laboratory strain JEC21 derived from these strains. In this case, CGH identified putative recombination sites and the origins of specific segments of the JEC21 genome. Similarly, CGH analysis revealed marked variability in the genomes of strains representing the VNI, VNII, and VNB molecular subtypes of the A serotype, including disomy for chromosome 13 in two strains. Additionally, CGH identified differences in chromosome content between three strains with the hybrid AD serotype and revealed that chromosome 1 from the serotype A genome is preferentially retained in all three strains.

Conclusion

The genomes of serotypes A, D, and AD strains exhibit extensive variation that spans the range from small differences (such as regions of divergence, deletion, or amplification) to the unexpected disomy for chromosome 13 in haploid strains and preferential retention of specific chromosomes in naturally occurring diploids.     

Genomic diversity of the human intestinal parasite Entamoeba histolytica

Background

Entamoeba histolytica is a significant cause of disease worldwide. However, little is known about the genetic diversity of the parasite. We re-sequenced the genomes of ten laboratory cultured lines of the eukaryotic pathogen Entamoeba histolytica in order to develop a picture of genetic diversity across the genome.

Results

The extreme nucleotide composition bias and repetitiveness of the E. histolytica genome provide a challenge for short-read mapping, yet we were able to define putative single nucleotide polymorphisms in a large portion of the genome. The results suggest a rather low level of single nucleotide diversity, although genes and gene families with putative roles in virulence are among the more polymorphic genes. We did observe large differences in coverage depth among genes, indicating differences in gene copy number between genomes. We found evidence indicating that recombination has occurred in the history of the sequenced genomes, suggesting that E. histolytica may reproduce sexually.

Conclusions

E. histolytica displays a relatively low level of nucleotide diversity across its genome. However, large differences in gene family content and gene copy number are seen among the sequenced genomes. The pattern of polymorphism indicates that E. histolytica reproduces sexually, or has done so in the past, which has previously been suggested but not proven.     

Genomic presence of recombinant porcine endogenous retrovirus in transmitting miniature swine

Background

The Coccolithoviridae is a recently discovered family of viruses that infect the marine coccolithophorid Emiliania huxleyi. Following on from the sequencing of the type strain EhV-86, we have sequenced a second strain, EhV-163.

Results

We have sequenced approximately 80% of the EhV-163 genome, equating to more than 200 full length CDSs. Conserved and variable CDSs and a gene replacement have been identified in the EhV-86 and EhV-163 genomes.

Conclusion

The sequencing of EhV-163 has provided a wealth of information which will aid the re-annotating of the EhV-86 genome and identified a gene insertion in EhV-163.