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 .

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.

TigrScan and GlimmerHMM: two open source ab initio eukaryotic gene-finders.

UNLABELLED: We describe two new Generalized Hidden Markov Model implementations for ab initio eukaryotic gene prediction. The C/C++ source code for both is available as open source and is highly reusable due to their modular and extensible architectures. Unlike most of the currently available gene-finders, the programs are re-trainable by the end user. They are also re-configurable and include several types of probabilistic submodels which can be independently combined, such as Maximal Dependence Decomposition trees and interpolated Markov models. Both programs have been used at TIGR for the annotation of the Aspergillus fumigatus and Toxoplasma gondii genomes. AVAILABILITY: Source code and documentation are available under the open source Artistic License from http://www.tigr.org/software/pirate

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.

Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains.

Virulence and immunity are poorly understood in Mycobacterium tuberculosis. We sequenced the complete genome of the M. tuberculosis clinical strain CDC1551 and performed a whole-genome comparison with the laboratory strain H37Rv in order to identify polymorphic sequences with potential relevance to disease pathogenesis, immunity, and evolution. We found large-sequence and single-nucleotide polymorphisms in numerous genes. Polymorphic loci included a phospholipase C, a membrane lipoprotein, members of an adenylate cyclase gene family, and members of the PE/PPE gene family, some of which have been implicated in virulence or the host immune response. Several gene families, including the PE/PPE gene family, also had significantly higher synonymous and nonsynonymous substitution frequencies compared to the genome as a whole. We tested a large sample of M. tuberculosis clinical isolates for a subset of the large-sequence and single-nucleotide polymorphisms and found widespread genetic variability at many of these loci. We performed phylogenetic and epidemiological analysis to investigate the evolutionary relationships among isolates and the origins of specific polymorphic loci. A number of these polymorphisms appear to have occurred multiple times as independent events, suggesting that these changes may be under selective pressure. Together, these results demonstrate that polymorphisms among M. tuberculosis strains are more extensive than initially anticipated, and genetic variation may have an important role in disease pathogenesis and immunity.

A probabilistic method for identifying start codons in bacterial genomes.

As the pace of genome sequencing has accelerated, the need for highly accurate gene prediction systems has grown. Computational systems for identifying genes in prokaryotic genomes have sensitivities of 98-99% or higher (Delcher et al., Nucleic Acids Res., 27, 4636-4641, 1999). These accuracy figures are calculated by comparing the locations of verified stop codons to the predictions. Determining the accuracy of start codon prediction is more problematic, however, due to the relatively small number of start sites that have been confirmed by independent, non-computational methods. Nonetheless, the accuracy of gene finders at predicting the exact gene boundaries at both the 5' and 3' ends of genes is of critical importance for microbial genome annotation, especially in light of the important signaling information that is sometimes found on the 5' end of a protein coding region. In this paper we propose a probabilistic method to improve the accuracy of gene identification systems at finding precise translation start sites. The new system, RBSfinder, is tested on a validated set of genes from Escherichia coli, for which it improves the accuracy of start site locations predicted by computational gene finding systems from the range 67-77% to 90% correct.

Understanding the adaptation of Halobacterium species NRC-1 to its extreme environment through computational analysis of its genome sequence.

The genome of the halophilic archaeon Halobacterium sp. NRC-1 and predicted proteome have been analyzed by computational methods and reveal characteristics relevant to life in an extreme environment distinguished by hypersalinity and high solar radiation: (1) The proteome is highly acidic, with a median pI of 4.9 and mostly lacking basic proteins. This characteristic correlates with high surface negative charge, determined through homology modeling, as the major adaptive mechanism of halophilic proteins to function in nearly saturating salinity. (2) Codon usage displays the expected GC bias in the wobble position and is consistent with a highly acidic proteome. (3) Distinct genomic domains of NRC-1 with bacterial character are apparent by whole proteome BLAST analysis, including two gene clusters coding for a bacterial-type aerobic respiratory chain. This result indicates that the capacity of halophiles for aerobic respiration may have been acquired through lateral gene transfer. (4) Two regions of the large chromosome were found with relatively lower GC composition and overrepresentation of IS elements, similar to the minichromosomes. These IS-element-rich regions of the genome may serve to exchange DNA between the three replicons and promote genome evolution. (5) GC-skew analysis showed evidence for the existence of two replication origins in the large chromosome. This finding and the occurrence of multiple chromosomes indicate a dynamic genome organization with eukaryotic character.

A clustering method for repeat analysis in DNA sequences.

BACKGROUND: A computational system for analysis of the repetitive structure of genomic sequences is described. The method uses suffix trees to organize and search the input sequences; this data structure has been used previously for efficient computation of exact and degenerate repeats. RESULTS: The resulting software tool collects all repeat classes and outputs summary statistics as well as a file containing multiple sequences (multi fasta), that can be used as the target of searches. Its use is demonstrated here on several complete microbial genomes, the entire Arabidopsis thaliana genome, and a large collection of rice bacterial artificial chromosome end sequences. CONCLUSIONS: We propose a new clustering method for analysis of the repeat data captured in suffix trees. This method has been incorporated into a system that can find repeats in individual genome sequences or sets of sequences, and that can organize those repeats into classes. It quickly and accurately creates repeat databases from small and large genomes. The associated software (RepeatFinder), should prove helpful in the analysis of repeat structure for both complete and partial genome sequences.

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.

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.

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.

Complete genome sequence of Caulobacter crescentus.

The complete genome sequence of Caulobacter crescentus was determined to be 4,016,942 base pairs in a single circular chromosome encoding 3,767 genes. This organism, which grows in a dilute aquatic environment, coordinates the cell division cycle and multiple cell differentiation events. With the annotated genome sequence, a full description of the genetic network that controls bacterial differentiation, cell growth, and cell cycle progression is within reach. Two-component signal transduction proteins are known to play a significant role in cell cycle progression. Genome analysis revealed that the C. crescentus genome encodes a significantly higher number of these signaling proteins (105) than any bacterial genome sequenced thus far. Another regulatory mechanism involved in cell cycle progression is DNA methylation. The occurrence of the recognition sequence for an essential DNA methylating enzyme that is required for cell cycle regulation is severely limited and shows a bias to intergenic regions. The genome contains multiple clusters of genes encoding proteins essential for survival in a nutrient poor habitat. Included are those involved in chemotaxis, outer membrane channel function, degradation of aromatic ring compounds, and the breakdown of plant-derived carbon sources, in addition to many extracytoplasmic function sigma factors, providing the organism with the ability to respond to a wide range of environmental fluctuations. C. crescentus is, to our knowledge, the first free-living alpha-class proteobacterium to be sequenced and will serve as a foundation for exploring the biology of this group of bacteria, which includes the obligate endosymbiont and human pathogen Rickettsia prowazekii, the plant pathogen Agrobacterium tumefaciens, and the bovine and human pathogen Brucella abortus.

Rice bioinformatics. analysis of rice sequence data and leveraging the data to other plant species.

Rice (Oryza sativa) is a model species for monocotyledonous plants, especially for members in the grass family. Several attributes such as small genome size, diploid nature, transformability, and establishment of genetic and molecular resources make it a tractable organism for plant biologists. With an estimated genome size of 430 Mb (Arumuganathan and Earle, 1991), it is feasible to obtain the complete genome sequence of rice using current technologies. An international effort has been established and is in the process of sequencing O. sativa spp. japonica var "Nipponbare" using a bacterial artificial chromosome/P1 artificial chromosome shotgun sequencing strategy. Annotation of the rice genome is performed using prediction-based and homology-based searches to identify genes. Annotation tools such as optimized gene prediction programs are being developed for rice to improve the quality of annotation. Resources are also being developed to leverage the rice genome sequence to partial genome projects such as expressed sequence tag projects, thereby maximizing the output from the rice genome project. To provide a low level of annotation for rice genomic sequences, we have aligned all rice bacterial artificial chromosome/P1 artificial chromosome sequences with The Institute of Genomic Research Gene Indices that are a set of nonredundant transcripts that are generated from nine public plant expressed sequence tag projects (rice, wheat, sorghum, maize, barley, Arabidopsis, tomato, potato, and barrel medic). In addition, we have used data from The Institute of Genomic Research Gene Indices and the Arabidopsis and Rice Genome Projects to identify putative orthologues and paralogues among these nine genomes.

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.

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.