Gene index analysis of the human genome estimates approximately 120,000 genes.

Although sequencing of the human genome will soon be completed, gene identification and annotation remains a challenge. Early estimates suggested that there might be 60,000-100,000 (ref. 1) human genes, but recent analyses of the available data from EST sequencing projects have estimated as few as 45,000 (ref. 2) or as many as 140, 000 (ref. 3) distinct genes. The Chromosome 22 Sequencing Consortium estimated a minimum of 45,000 genes based on their annotation of the complete chromosome, although their data suggests there may be additional genes. The nearly 2,000,000 human ESTs in dbEST provide an important resource for gene identification and genome annotation, but these single-pass sequences must be carefully analysed to remove contaminating sequences, including those from genomic DNA, spurious transcription, and vector and bacterial sequences. We have developed a highly refined and rigorously tested protocol for cleaning, clustering and assembling EST sequences to produce high-fidelity consensus sequences for the represented genes (F.L. et al., manuscript submitted) and used this to create the TIGR Gene Indices-databases of expressed genes for human, mouse, rat and other species (http://www.tigr.org/tdb/tgi.html). Using highly refined and tested algorithms for EST analysis, we have arrived at two independent estimates indicating the human genome contains approximately 120,000 genes.

Microbial genes in the human genome: lateral transfer or gene loss?

The human genome was analyzed for evidence that genes had been laterally transferred into the genome from prokaryotic organisms. Protein sequence comparisons of the proteomes of human, fruit fly, nematode worm, yeast, mustard weed, eukaryotic parasites, and all completed prokaryote genomes were performed, and all genes shared between human and each of the other groups of organisms were collected. About 40 genes were found to be exclusively shared by humans and bacteria and are candidate examples of horizontal transfer from bacteria to vertebrates. Gene loss combined with sample size effects and evolutionary rate variation provide an alternative, more biologically plausible explanation.

Complete genome sequence of Neisseria meningitidis serogroup B strain MC58.

The 2,272,351-base pair genome of Neisseria meningitidis strain MC58 (serogroup B), a causative agent of meningitis and septicemia, contains 2158 predicted coding regions, 1158 (53.7%) of which were assigned a biological role. Three major islands of horizontal DNA transfer were identified; two of these contain genes encoding proteins involved in pathogenicity, and the third island contains coding sequences only for hypothetical proteins. Insights into the commensal and virulence behavior of N. meningitidis can be gleaned from the genome, in which sequences for structural proteins of the pilus are clustered and several coding regions unique to serogroup B capsular polysaccharide synthesis can be identified. Finally, N. meningitidis contains more genes that undergo phase variation than any pathogen studied to date, a mechanism that controls their expression and contributes to the evasion of the host immune system.

Complete genome sequence of a virulent isolate of Streptococcus pneumoniae.

The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.

KrakenUniq: confident and fast metagenomics classification using unique k-mer counts.

False-positive identifications are a significant problem in metagenomics classification. We present KrakenUniq, a novel metagenomics classifier that combines the fast k-mer-based classification of Kraken with an efficient algorithm for assessing the coverage of unique k-mers found in each species in a dataset. On various test datasets, KrakenUniq gives better recall and precision than other methods and effectively classifies and distinguishes pathogens with low abundance from false positives in infectious disease samples. By using the probabilistic cardinality estimator HyperLogLog, KrakenUniq runs as fast as Kraken and requires little additional memory. KrakenUniq is freely available at https://github.com/fbreitwieser/krakenuniq .

Sequence and analysis of the Arabidopsis genome.

The comprehensive analysis of the genome sequence of the plant Arabidopsis thaliana has been completed recently. The genome sequence and associated analyses provide the foundations for rapid progress in many fields of plant research, such as the exploitation of genetic variation in Arabidopsis ecotypes, the assessment of the transcriptome and proteome, and the association of genome changes at the sequence level with evolutionary processes. Nevertheless, genome sequencing and analysis are only the first steps towards a new plant biology. Much remains to be done to refine the analysis of encoded genes, to define the functions of encoded proteins systematically, and to establish new generations of databases to capture and relate diverse data sets generated in widely distributed laboratories.

GeneSplicer: a new computational method for splice site prediction.

GeneSplicer is a new, flexible system for detecting splice sites in the genomic DNA of various eukaryotes. The system has been tested successfully using DNA from two reference organisms: the model plant Arabidopsis thaliana and human. It was compared to six programs representing the leading splice site detectors for each of these species: NetPlantGene, NetGene2, HSPL, NNSplice, GENIO and SpliceView. In each case GeneSplicer performed comparably to the best alternative, in terms of both accuracy and computational efficiency.

Prediction of operons in microbial genomes.

Operon structure is an important organization feature of bacterial genomes. Many sets of genes occur in the same order on multiple genomes; these conserved gene groupings represent candidate operons. This study describes a computational method to estimate the likelihood that such conserved gene sets form operons. The method was used to analyze 34 bacterial and archaeal genomes, and yielded more than 7600 pairs of genes that are highly likely (P: >/= 0.98) to belong to the same operon. The sensitivity of our method is 30-50% for the Escherichia coli genome. The predicted gene pairs are available from our World Wide Web site http://www.tigr.org/tigr-scripts/operons/operons.cgi.

An optimized protocol for analysis of EST sequences.

The vast body of Expressed Sequence Tag (EST) data in the public databases provide an important resource for comparative and functional genomics studies and an invaluable tool for the annotation of genomic sequences. We have developed a rigorous protocol for reconstructing the sequences of transcribed genes from EST and gene sequence fragments. A key element in developing this protocol has been the evaluation of a number of sequence assembly programs to determine which most faithfully reproduce transcript sequences from EST data. The TIGR Gene Indices constructed using this protocol for human, mouse, rat and a variety of other plant and animal models have demonstrated their utility in a variety of applications and are freely available to the scientific research community.

Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39.

The genome sequences of Chlamydia trachomatis mouse pneumonitis (MoPn) strain Nigg (1 069 412 nt) and Chlamydia pneumoniae strain AR39 (1 229 853 nt) were determined using a random shotgun strategy. The MoPn genome exhibited a general conservation of gene order and content with the previously sequenced C.trachomatis serovar D. Differences between C.trachomatis strains were focused on an approximately 50 kb 'plasticity zone' near the termination origins. In this region MoPn contained three copies of a novel gene encoding a >3000 amino acid toxin homologous to a predicted toxin from Escherichia coli O157:H7 but had apparently lost the tryptophan biosyntheis genes found in serovar D in this region. The C. pneumoniae AR39 chromosome was >99.9% identical to the previously sequenced C.pneumoniae CWL029 genome, however, comparative analysis identified an invertible DNA segment upstream of the uridine kinase gene which was in different orientations in the two genomes. AR39 also contained a novel 4524 nt circular single-stranded (ss)DNA bacteriophage, the first time a virus has been reported infecting C. pneumoniae. Although the chlamydial genomes were highly conserved, there were intriguing differences in key nucleotide salvage pathways: C.pneumoniae has a uridine kinase gene for dUTP production, MoPn has a uracil phosphororibosyl transferase, while C.trachomatis serovar D contains neither gene. Chromosomal comparison revealed that there had been multiple large inversion events since the species divergence of C.trachomatis and C.pneumoniae, apparently oriented around the axis of the origin of replication and the termination region. The striking synteny of the Chlamydia genomes and prevalence of tandemly duplicated genes are evidence of minimal chromosome rearrangement and foreign gene uptake, presumably owing to the ecological isolation of the obligate intracellular parasites. In the absence of genetic analysis, comparative genomics will continue to provide insight into the virulence mechanisms of these important human pathogens.

Genome sequence of Chlamydophila caviae (Chlamydia psittaci GPIC): examining the role of niche-specific genes in the evolution of the Chlamydiaceae.

The genome of Chlamydophila caviae (formerly Chlamydia psittaci, GPIC isolate) (1 173 390 nt with a plasmid of 7966 nt) was determined, representing the fourth species with a complete genome sequence from the Chlamydiaceae family of obligate intracellular bacterial pathogens. Of 1009 annotated genes, 798 were conserved in all three other completed Chlamydiaceae genomes. The C.caviae genome contains 68 genes that lack orthologs in any other completed chlamydial genomes, including tryptophan and thiamine biosynthesis determinants and a ribose-phosphate pyrophosphokinase, the product of the prsA gene. Notable amongst these was a novel member of the virulence-associated invasin/intimin family (IIF) of Gram-negative bacteria. Intriguingly, two authentic frameshift mutations in the ORF indicate that this gene is not functional. Many of the unique genes are found in the replication termination region (RTR or plasticity zone), an area of frequent symmetrical inversion events around the replication terminus shown to be a hotspot for genome variation in previous genome sequencing studies. In C.caviae, the RTR includes several loci of particular interest including a large toxin gene and evidence of ancestral insertion(s) of a bacteriophage. This toxin gene, not present in Chlamydia pneumoniae, is a member of the YopT effector family of type III-secreted cysteine proteases. One gene cluster (guaBA-add) in the RTR is much more similar to orthologs in Chlamydia muridarum than those in the phylogenetically closest species C.pneumoniae, suggesting the possibility of horizontal transfer of genes between the rodent-associated Chlamydiae. With most genes observed in the other chlamydial genomes represented, C.caviae provides a good model for the Chlamydiaceae and a point of comparison against the human atherosclerosis-associated C.pneumoniae. This crucial addition to the set of completed Chlamydiaceae genome sequences is enabling dissection of the roles played by niche-specific genes in these important bacterial pathogens.

Prediction of transcription terminators in bacterial genomes.

This study describes an algorithm that finds rho-independent transcription terminators in bacterial genomes and evaluates the accuracy of its predictions. The algorithm identifies terminators by searching for a common mRNA motif: a hairpin structure followed by a short uracil-rich region. For each terminator, an energy-scoring function that reflects hairpin stability, and a tail-scoring function based on the number of U nucleotides and their proximity to the stem, are computed. A confidence value can be assigned to each terminator by analyzing candidate terminators found both within and between genes, and taking into account the energy and tail scores. The confidence is an empirical estimate of the probability that the sequence is a true terminator. The algorithm was used to conduct a comprehensive analysis of 12 bacterial genomes to identify likely candidates for rho-independent transcription terminators. Four of these genomes (Deinococcus radiodurans, Escherichia coli, Haemophilus influenzae and Vibrio cholerae) were found to have large numbers of rho-independent terminators. Among the other genomes, most appear to have no transcription terminators of this type, with the exception of Thermotoga maritima. A set of 131 experimentally determined E. coli terminators was used to evaluate the sensitivity of the method, which ranges from 89 % to 98 %, with corresponding false positive rates of 2 % and 18 %.

Skewed oligomers and origins of replication.

The putative origin of replication in prokaryotic genomes can be located by a new method that finds short oligomers whose orientation is preferentially skewed around the origin. The skewed oligomer method is shown to work for all bacterial genomes and one of three archaeal genomes sequences to date, confirming known or predicted origins in most cases and in three cases (H. pylori, M. thermoautotrophicum, and Synechocystis sp.), suggesting origins that were previously unknown. In many cases, the presence of conserved genes and nucleotide motifs confirms the predictions. An algorithm for finding these skewed seven-base and eight-base sequences is described, along with a method for combining evidence from multiple skewed oligomers to accurately locate the replication origin. Possible explanations for the phenomenon of skewed oligomers are discussed. Explanations are presented for why some bacterial genomes contain hundreds of highly skewed oligomers, whereas others contain only a handful.

DNA uptake signal sequences in naturally transformable bacteria.

The naturally transformable bacterium Haemophilus influenzae Rd contains 1471 copies of the DNA uptake signal sequence (USS) 5'-AAGTGCGGT in its genome. Neisseria meningitidis contains 1891 copies of the USS sequence 5'-GCCGTCTGAA. The USSs are often found in the base paired stem of transcription terminators.

The perils of gene patents.

I argue here that gene patents, and patented genetic tests based on them, are a very bad idea. First, I discuss whether genes can reasonably be the subject of patents in the first place; I maintain that the answer is no. Second, I explain how gene patents interfere with scientific progress, slowing down the development of new cures and treatments for genetic diseases.

Analysis of Carica papaya Telomeres and Telomere-Associated Proteins: Insights into the Evolution of Telomere Maintenance in Brassicales.

Telomeres are terminal regions of linear eukaryotic chromosomes that are critical for genome stability and continued cell proliferation. The draft assembly of the papaya genome provides an opportunity to analyze and compare the evolution of telomeric DNA sequence composition and telomere maintenance machinery in this and other organisms of the Brassicales Order, which includes Arabidopsis. Here we investigate telomere size and sequence variation at papaya chromosome ends. As with most other plant species, papaya telomeres consist of TTTAGGG repeats. However, in contrast to members of the closely related Brassicaceae family, telomeres in papaya are ~10-fold longer. Sequence analysis reveals that many centromereproximal telomere repeats in papaya harbor nucleotide substitutions and insertions of Gs and Ts. In contrast, we found very few N-to-C substitutions, and even fewer instances of nucleotide deletion, suggesting that a six-nucleotide telomere repeat is not well tolerated. The papaya genome encodes single-copy sequence homologues of several genes involved in telomere maintenance and chromosome end protection, including the Telomerase Reverse Transcriptase (TERT) and Protection Of Telomeres (POT1). Notably, unlike Arabidopsis, which encodes six Telomere Repeat binding Factor-like (TRFL) proteins that bind double-stranded telomere DNA, papaya appears to encode only two such proteins. Thus, the more streamlined genome of papaya will provide an excellent resource for comparative and functional analysis of telomeres in plants.

Efficient implementation of a generalized pair hidden Markov model for comparative gene finding.

MOTIVATION: The increased availability of genome sequences of closely related organisms has generated much interest in utilizing homology to improve the accuracy of gene prediction programs. Generalized pair hidden Markov models (GPHMMs) have been proposed as one means to address this need. However, all GPHMM implementations currently available are either closed-source or the details of their operation are not fully described in the literature, leaving a significant hurdle for others wishing to advance the state of the art in GPHMM design. RESULTS: We have developed an open-source GPHMM gene finder, TWAIN, which performs very well on two related Aspergillus species, A.fumigatus and A.nidulans, finding 89% of the exons and predicting 74% of the gene models exactly correctly in a test set of 147 conserved gene pairs. We describe the implementation of this GPHMM and we explicitly address the assumptions and limitations of the system. We suggest possible ways of relaxing those assumptions to improve the utility of the system without sacrificing efficiency beyond what is practical. AVAILABILITY: Available at http://www.tigr.org/software/pirate/twain/twain.html under the open-source Artistic License.

A method for identifying splice sites and translational start sites in eukaryotic mRNA.

This paper describes a new method for determining the consensus sequences that signal the start of translation and the boundaries between exons and introns (donor and acceptor sites) in eukaryotic mRNA. The method takes into account the dependencies between adjacent bases, in contrast to the usual technique of considering each position independently. When coupled with a dynamic program to compute the most likely sequence, new consensus sequences emerge. The consensus sequence information is summarized in conditional probability matrices which, when used to locate signals in uncharacterized genomic DNA, have greater sensitivity and specificity than conventional matrices. Species-specific versions of these matrices are especially effective at distinguishing true and false sites.

Microbial genome sequencing.

Complete genome sequences of 30 microbial species have been determined during the past five years, and work in progress indicates that the complete sequences of more than 100 further microbial species will be available in the next two to four years. These results have revealed a tremendous amount of information on the physiology and evolution of microbial species, and should provide novel approaches to the diagnosis and treatment of infectious disease.