Computational identification of protein coding potential of conserved sequence tags through cross-species evolutionary analysis

The identification of conserved sequence tags (CSTs) through comparative genome analysis may reveal important regulatory elements involved in shaping the spatio-temporal expression of genetic information. It is well known that the most significant fraction of CSTs observed in human–mouse comparisons correspond to protein coding exons, due to their strong evolutionary constraints. As we still do not know the complete gene inventory of the human and mouse genomes it is of the utmost importance to establish if detected conserved sequences are genes or not. We propose here a simple algorithm that, based on the observation of the specific evolutionary dynamics of coding sequences, efficiently discriminates between coding and non-coding CSTs. The application of this method may help the validation of predicted genes, the prediction of alternative splicing patterns in known and unknown genes and the definition of a dictionary of non-coding regulatory elements.     

How to usefully compare homologous plant genes and chromosomes as DNA sequences

There are four sequenced and publicly available plant genomes to date. With many more slated for completion, one challenge will be to use comparative genomic methods to detect novel evolutionary patterns in plant genomes. This research requires sequence alignment algorithms to detect regions of similarity within and among genomes. However, different alignment algorithms are optimized for identifying different types of homologous sequences. This review focuses on plant genome evolution and provides a tutorial for using several sequence alignment algorithms and visualization tools to detect useful patterns of conservation: conserved non-coding sequences, false positive noise, subfunctionalization, synteny, annotation errors, inversions and local duplications. Our tutorial encourages the reader to experiment online with the reviewed tools as a companion to the text.     

The random versus fragile breakage models of chromosome evolution: a matter of resolution

Conserved synteny––the sharing of at least one orthologous gene by a pair of chromosomes from two species––can, in the strictest sense, be viewed as sequence conservation between chromosomes of two related species, irrespective of whether coding or non-coding sequence is examined. The recent sequencing of multiple vertebrate genomes indicates that certain chromosomal segments of considerable size are conserved in gene order as well as underlying non-coding sequence across all vertebrates. Some of these segments lost genes or non-coding sequence and/or underwent breakage only in teleost genomes, presumably because evolutionary pressure acting on these regions to remain intact were relaxed after an additional round of whole genome duplication. Random reporter insertions into zebrafish chromosomes combined with computational genome-wide analysis indicate that large chromosomal areas of multiple genes contain long-range regulatory elements, which act on their target genes from several gene distances away. In addition, computational breakpoint analyses suggest that recurrent evolutionary breaks are found in “fragile regions” or “hotspots”, outside of the conserved blocks of synteny. These findings cannot be accommodated by the random breakage model and suggest that this view of genome and chromosomal evolution requires substantial reassessment.     

Syntenator: Multiple gene order alignments with a gene-specific scoring function

Background  

Identification of homologous regions or conserved syntenies across genomes is one crucial step in comparative genomics. This task is usually performed by genome alignment softwares like WABA or blastz. In case of conserved syntenies, such regions are defined as conserved gene orders. On the gene order level, homologous regions can even be found between distantly related genomes, which do not align on the nucleotide sequence level.     

Parametric alignment of Drosophila genomes

The classic algorithms of Needleman-Wunsch and Smith-Waterman find a maximum a posteriori probability alignment for a pair hidden Markov model (PHMM). To process large genomes that have undergone complex genome rearrangements, almost all existing whole genome alignment methods apply fast heuristics to divide genomes into small pieces that are suitable for Needleman-Wunsch alignment. In these alignment methods, it is standard practice to fix the parameters and to produce a single alignment for subsequent analysis by biologists. As the number of alignment programs applied on a whole genome scale continues to increase, so does the disagreement in their results. The alignments produced by different programs vary greatly, especially in non-coding regions of eukaryotic genomes where the biologically correct alignment is hard to find. Parametric alignment is one possible remedy. This methodology resolves the issue of robustness to changes in parameters by finding all optimal alignments for all possible parameters in a PHMM. Our main result is the construction of a whole genome parametric alignment of Drosophila melanogaster and Drosophila pseudoobscura. This alignment draws on existing heuristics for dividing whole genomes into small pieces for alignment, and it relies on advances we have made in computing convex polytopes that allow us to parametrically align non-coding regions using biologically realistic models. We demonstrate the utility of our parametric alignment for biological inference by showing that cis-regulatory elements are more conserved between Drosophila melanogaster and Drosophila pseudoobscura than previously thought. We also show how whole genome parametric alignment can be used to quantitatively assess the dependence of branch length estimates on alignment parameters.     

Functional annotations in bacterial genomes based on small RNA signatures

One of the key challenges in computational genomics is annotating coding genes and identification of regulatory RNAs in complete genomes. An attempt is made in this study which uses the regulatory RNA locations and their conserved flanking genes identified within the genomic backbone of template genome to search for similar RNA locations in query genomes. The search is based on recently reported coexistence of small RNAs and their conserved flanking genes in related genomes. Based on our study, 54 additional sRNA locations and functions of 96 uncharacterized genes are predicted in two draft genomes viz., Serratia marcesens Db1 and Yersinia enterocolitica 8081. Although most of the identified additional small RNA regions and their corresponding flanking genes are homologous in nature, the proposed anchoring technique could successfully identify four non-homologous small RNA regions in Y. enterocolitica genome also. The KEGG Orthology (KO) based automated functional predictions confirms the predicted functions of 65 flanking genes having defined KO numbers, out of the total 96 predictions made by this method. This coexistence based method shows more sensitivity than controlled vocabularies in locating orthologous gene pairs even in the absence of defined Orthology numbers. All functional predictions made by this study in Y. enterocolitica 8081 were confirmed by the recently published complete genome sequence and annotations. This study also reports the possible regions of gene rearrangements in these two genomes and further characterization of such RNA regions could shed more light on their possible role in genome evolution.     

Annotation of selection strengths in viral genomes

MOTIVATION: Viral genomes tend to code in overlapping reading frames to maximize informational content. This may result in atypical codon bias and particular evolutionary constraints. Due to the fast mutation rate of viruses, there is additional strong evidence for varying selection between intra- and intergenomic regions. The presence of multiple coding regions complicates the concept of K(a)/K(s) ratio, and thus begs for an alternative approach when investigating selection strengths. Building on the paper by McCauley and Hein, we develop a method for annotating a viral genome coding in overlapping reading frames. We introduce an evolutionary model capable of accounting for varying levels of selection along the genome, and incorporate it into our prior single sequence HMM methodology, extending it now to a phylogenetic HMM. Given an alignment of several homologous viruses to a reference sequence, we may thus achieve an annotation both of coding regions as well as selection strengths, allowing us to investigate different selection patterns and hypotheses. RESULTS: We illustrate our method by applying it to a multiple alignment of four HIV2 sequences, as well as of three Hepatitis B sequences. We obtain an annotation of the coding regions, as well as a posterior probability for each site of the strength of selection acting on it. From this we may deduce the average posterior selection acting on the different genes. Whilst we are encouraged to see in HIV2, that the known to be conserved genes gag and pol are indeed annotated as such, we also discover several sites of less stringent negative selection within the env gene. To the best of our knowledge, we are the first to subsequently provide a full selection annotation of the Hepatitis B genome by explicitly modelling the evolution within overlapping reading frames, and not relying on simple K(a)/K(s) ratios.     

MRD: a microsatellite repeats database for prokaryotic and eukaryotic genomes

MRD is a database system to access the microsatellite repeats information of genomes such as archea, eubacteria, and other eukaryotic genomes whose sequence information is available in public domains. MRD stores information about simple tandemly repeated k-mer sequences where k= 1 to 6, i.e. monomer to hexamer. The web interface allows the users to search for the repeat of their interest and to know about the association of the repeat with genes and genomic regions in the specific organism. The data contains the abundance and distribution of microsatellites in the coding and non-coding regions of the genome. The exact location of repeats with respect to genomic regions of interest (such as UTR, exon, intron or intergenic regions) whichever is applicable to organism is highlighted. MRD is available on the World Wide Web at and/or . The database is designed as an open-ended system to accommodate the microsatellite repeats information of other genomes whose complete sequences will be available in future through public domain.     

Identification of common,unique and polymorphic microsatellites among 73 cyanobacterial genomes

Microsatellites also known as Simple Sequence Repeats are short tandem repeats of 1–6 nucleotides. These repeats are found in coding as well as non-coding regions of both prokaryotic and eukaryotic genomes and play a significant role in the study of gene regulation, genetic mapping, DNA fingerprinting and evolutionary studies. The availability of 73 complete genome sequences of cyanobacteria enabled us to mine and statistically analyze microsatellites in these genomes. The cyanobacterial microsatellites identified through bioinformatics analysis were stored in a user-friendly database named CyanoSat, which is an efficient data representation and query system designed using ASP.net. The information in CyanoSat comprises of perfect, imperfect and compound microsatellites found in coding, non-coding and coding-non-coding regions. Moreover, it contains PCR primers with 200 nucleotides long flanking region. The mined cyanobacterial microsatellites can be freely accessed at www.compubio.in/CyanoSat/home.aspx. In addition to this 82 polymorphic, 13,866 unique and 2390 common microsatellites were also detected. These microsatellites will be useful in strain identification and genetic diversity studies of cyanobacteria.     

When Whole-Genome Alignments Just Won't Work: kSNP v2 Software for Alignment-Free SNP Discovery and Phylogenetics of Hundreds of Microbial Genomes

Effective use of rapid and inexpensive whole genome sequencing for microbes requires fast, memory efficient bioinformatics tools for sequence comparison. The kSNP v2 software finds single nucleotide polymorphisms (SNPs) in whole genome data. kSNP v2 has numerous improvements over kSNP v1 including SNP gene annotation; better scaling for draft genomes available as assembled contigs or raw, unassembled reads; a tool to identify the optimal value of k; distribution of packages of executables for Linux and Mac OS X for ease of installation and user-friendly use; and a detailed User Guide. SNP discovery is based on k-mer analysis, and requires no multiple sequence alignment or the selection of a single reference genome. Most target sets with hundreds of genomes complete in minutes to hours. SNP phylogenies are built by maximum likelihood, parsimony, and distance, based on all SNPs, only core SNPs, or SNPs present in some intermediate user-specified fraction of targets. The SNP-based trees that result are consistent with known taxonomy. kSNP v2 can handle many gigabases of sequence in a single run, and if one or more annotated genomes are included in the target set, SNPs are annotated with protein coding and other information (UTRs, etc.) from Genbank file(s). We demonstrate application of kSNP v2 on sets of viral and bacterial genomes, and discuss in detail analysis of a set of 68 finished E. coli and Shigella genomes and a set of the same genomes to which have been added 47 assemblies and four “raw read” genomes of H104:H4 strains from the recent European E. coli outbreak that resulted in both bloody diarrhea and hemolytic uremic syndrome (HUS), and caused at least 50 deaths.     

Investigations of oligonucleotide usage variance within and between prokaryotes

Oligonucleotide usage in archaeal and bacterial genomes can be linked to a number of properties, including codon usage (trinucleotides), DNA base-stacking energy (dinucleotides), and DNA structural conformation (di- to tetranucleotides). We wanted to assess the statistical information potential of different DNA ‘word-sizes’ and explore how oligonucleotide frequencies differ in coding and non-coding regions. In addition, we used oligonucleotide frequencies to investigate DNA composition and how DNA sequence patterns change within and between prokaryotic organisms. Among the results found was that prokaryotic chromosomes can be described by hexanucleotide frequencies, suggesting that prokaryotic DNA is predominantly short range correlated, i.e., information in prokaryotic genomes is encoded in short oligonucleotides. Oligonucleotide usage varied more within AT-rich and host-associated genomes than in GC-rich and free-living genomes, and this variation was mainly located in non-coding regions. Bias (selectional pressure) in tetranucleotide usage correlated with GC content, and coding regions were more biased than non-coding regions. Non-coding regions were also found to be approximately 5.5% more AT-rich than coding regions, on average, in the 402 chromosomes examined. Pronounced DNA compositional differences were found both within and between AT-rich and GC-rich genomes. GC-rich genomes were more similar and biased in terms of tetranucleotide usage in non-coding regions than AT-rich genomes. The differences found between AT-rich and GC-rich genomes may possibly be attributed to lifestyle, since tetranucleotide usage within host-associated bacteria was, on average, more dissimilar and less biased than free-living archaea and bacteria.     

双翅目昆虫线粒体基因组结构特点及其测序通用引物的设计和应用

研究双翅目昆虫线粒体基因组的结构特点, 并设计其测序的通用引物, 为今后双翅目昆虫线粒体基因组的研究提供参考和依据。利用比较基因组学和生物信息学方法, 分析了已经完全测序的26个双翅目昆虫线粒体基因组的结构特点、 碱基组成和保守区, 并据此设计了双翅目昆虫基因组测序的通用引物。结果表明： 双翅目昆虫线粒体基因组长14 503~19 517 bp, 其结构保守, 含有37个编码基因, 包括13个蛋白质编码基因, 22个tRNA编码基因和2个rRNA编码基因, 此外还包含一段长度差异很大的非编码区(AT富含区)。基因组内基因排列次序稳定, 除个别基因外, 其余都与黑腹果蝇Drosophila melanogaster基因排列次序一致。基因组的碱基组成不均衡, AT含量在72.59%~85.15%之间, 碱基使用存在偏向性, 偏好使用AC碱基。全基因组的核苷酸和氨基酸序列保守, 共鉴定了11个保守区。在保守区内共设计了26对双翅目线粒体基因组测序通用引物, 扩增的目标片段都在1 200 bp以内。将该套通用引物用于葱蝇Delia antiqua线粒体全基因组测序, 结果证明其高效、 合用。     

Multi-Platform Next-Generation Sequencing of the Domestic Turkey (Meleagris gallopavo): Genome Assembly and Analysis

A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (∼1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.     

Complete Chloroplast Genome Sequence of Poisonous and Medicinal Plant Datura stramonium: Organizations and Implications for Genetic Engineering

Datura stramonium is a widely used poisonous plant with great medicinal and economic value. Its chloroplast (cp) genome is 155,871 bp in length with a typical quadripartite structure of the large (LSC, 86,302 bp) and small (SSC, 18,367 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,601 bp). The genome contains 113 unique genes, including 80 protein-coding genes, 29 tRNAs and four rRNAs. A total of 11 forward, 9 palindromic and 13 tandem repeats were detected in the D. stramonium cp genome. Most simple sequence repeats (SSR) are AT-rich and are less abundant in coding regions than in non-coding regions. Both SSRs and GC content were unevenly distributed in the entire cp genome. All preferred synonymous codons were found to use A/T ending codons. The difference in GC contents of entire genomes and of the three-codon positions suggests that the D. stramonium cp genome might possess different genomic organization, in part due to different mutational pressures. The five most divergent coding regions and four non-coding regions (trnH-psbA, rps4-trnS, ndhD-ccsA, and ndhI-ndhG) were identified using whole plastome alignment, which can be used to develop molecular markers for phylogenetics and barcoding studies within the Solanaceae. Phylogenetic analysis based on 68 protein-coding genes supported Datura as a sister to Solanum. This study provides valuable information for phylogenetic and cp genetic engineering studies of this poisonous and medicinal plant.     

Chimeric mitochondrial minichromosomes of the human body louse, Pediculus humanus: evidence for homologous and non-homologous recombination

The mitochondrial (mt) genome of the human body louse, Pediculus humanus, consists of 18 minichromosomes. Each minichromosome is 3 to 4 kb long and has 1 to 3 genes. There is unequivocal evidence for recombination between different mt minichromosomes in P. humanus. It is not known, however, how these minichromosomes recombine. Here, we report the discovery of eight chimeric mt minichromosomes in P. humanus. We classify these chimeric mt minichromosomes into two groups: Group I and Group II. Group I chimeric minichromosomes contain parts of two different protein-coding genes that are from different minichromosomes. The two parts of protein-coding genes in each Group I chimeric minichromosome are joined at a microhomologous nucleotide sequence; microhomologous nucleotide sequences are hallmarks of non-homologous recombination. Group II chimeric minichromosomes contain all of the genes and the non-coding regions of two different minichromosomes. The conserved sequence blocks in the non-coding regions of Group II chimeric minichromosomes resemble the "recombination repeats" in the non-coding regions of the mt genomes of higher plants. These repeats are essential to homologous recombination in higher plants. Our analyses of the nucleotide sequences of chimeric mt minichromosomes indicate both homologous and non-homologous recombination between minichromosomes in the mitochondria of the human body louse.