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Current biology: CB 01/2013; 23(1):R8-9. · 10.99 Impact Factor
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Christelle Godel,
Sujai Kumar,
Georgios Koutsovoulos,
Philipp Ludin,
Daniel Nilsson,
Francesco Comandatore,
Nicola Wrobel,
Marian Thompson,
Christoph D Schmid,
Susumu Goto,
Frédéric Bringaud,
Adrian Wolstenholme,
Claudio Bandi,
Christian Epe,
Ronald Kaminsky, Mark Blaxter,
Pascal Mäser
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ABSTRACT: The heartworm Dirofilaria immitis is an important parasite of dogs. Transmitted by mosquitoes in warmer climatic zones, it is spreading across southern Europe and the Americas at an alarming pace. There is no vaccine, and chemotherapy is prone to complications. To learn more about this parasite, we have sequenced the genomes of D. immitis and its endosymbiont Wolbachia. We predict 10,179 protein coding genes in the 84.2 Mb of the nuclear genome, and 823 genes in the 0.9-Mb Wolbachia genome. The D. immitis genome harbors neither DNA transposons nor active retrotransposons, and there is very little genetic variation between two sequenced isolates from Europe and the United States. The differential presence of anabolic pathways such as heme and nucleotide biosynthesis hints at the intricate metabolic interrelationship between the heartworm and Wolbachia. Comparing the proteome of D. immitis with other nematodes and with mammalian hosts, we identify families of potential drug targets, immune modulators, and vaccine candidates. This genome sequence will support the development of new tools against dirofilariasis and aid efforts to combat related human pathogens, the causative agents of lymphatic filariasis and river blindness.-Godel, C., Kumar, S., Koutsovoulos, G., Ludin, P., Nilsson, D., Comandatore, F., Wrobel, N., Thompson, M., Schmid, C. D., Goto, S., Bringaud, F., Wolstenholme, A., Bandi, C., Epe, C., Kaminsky, R., Blaxter, M., Mäser, P. The genome of the heartworm, Dirofilaria immitis, reveals drug and vaccine targets.
The FASEB Journal 08/2012; · 5.71 Impact Factor
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Katarzyna Kinga Modrzynska,
Alison Creasey,
Laurence Loewe,
Timothee Cezard,
Sofia Trindade Borges,
Axel Martinelli,
Louise Rodrigues,
Pedro Cravo, Mark Blaxter,
Richard Carter,
Paul Hunt
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ABSTRACT: Drug resistance in the malaria parasite Plasmodium falciparum severely compromises the treatment and control of malaria. A knowledge of the critical mutations conferring resistance to particular drugs is important in understanding modes of drug action and mechanisms of resistances. They are required to design better therapies and limit drug resistance.A mutation in the gene (pfcrt) encoding a membrane transporter has been identified as a principal determinant of chloroquine resistance in P. falciparum, but we lack a full account of higher level chloroquine resistance. Furthermore, the determinants of resistance in the other major human malaria parasite, P. vivax, are not known. To address these questions, we investigated the genetic basis of chloroquine resistance in an isogenic lineage of rodent malaria parasite P. chabaudi in which high level resistance to chloroquine has been progressively selected under laboratory conditions.
Loci containing the critical genes were mapped by Linkage Group Selection, using a genetic cross between the high-level chloroquine-resistant mutant and a genetically distinct sensitive strain. A novel high-resolution quantitative whole-genome re-sequencing approach was used to reveal three regions of selection on chr11, chr03 and chr02 that appear progressively at increasing drug doses on three chromosomes. Whole-genome sequencing of the chloroquine-resistant parent identified just four point mutations in different genes on these chromosomes. Three mutations are located at the foci of the selection valleys and are therefore predicted to confer different levels of chloroquine resistance. The critical mutation conferring the first level of chloroquine resistance is found in aat1, a putative aminoacid transporter.
Quantitative trait loci conferring selectable phenotypes, such as drug resistance, can be mapped directly using progressive genome-wide linkage group selection. Quantitative genome-wide short-read genome resequencing can be used to reveal these signatures of drug selection at high resolution. The identities of three genes (and mutations within them) conferring different levels of chloroquine resistance generate insights regarding the genetic architecture and mechanisms of resistance to chloroquine and other drugs. Importantly, their orthologues may now be evaluated for critical or accessory roles in chloroquine resistance in human malarias P. vivax and P. falciparum.
BMC Genomics 03/2012; 13:106. · 4.07 Impact Factor
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Trevor Tyson,
Georgina O'Mahony Zamora,
Simon Wong,
Máirin Skelton,
Brian Daly,
John T Jones,
Eoin D Mulvihill,
Benjamin Elsworth,
Mark Phillips, Mark Blaxter,
Ann M Burnell
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ABSTRACT: Some organisms can survive extreme desiccation by entering into a state of suspended animation known as anhydrobiosis. Panagrolaimus superbus is a free-living anhydrobiotic nematode that can survive rapid environmental desiccation. The mechanisms that P. superbus uses to combat the potentially lethal effects of cellular dehydration may include the constitutive and inducible expression of protective molecules, along with behavioural and/or morphological adaptations that slow the rate of cellular water loss. In addition, inducible repair and revival programmes may also be required for successful rehydration and recovery from anhydrobiosis.
To identify constitutively expressed candidate anhydrobiotic genes we obtained 9,216 ESTs from an unstressed mixed stage population of P. superbus. We derived 4,009 unigenes from these ESTs. These unigene annotations and sequences can be accessed at http://www.nematodes.org/nembase4/species_info.php?species=PSC. We manually annotated a set of 187 constitutively expressed candidate anhydrobiotic genes from P. superbus. Notable among those is a putative lineage expansion of the lea (late embryogenesis abundant) gene family. The most abundantly expressed sequence was a member of the nematode specific sxp/ral-2 family that is highly expressed in parasitic nematodes and secreted onto the surface of the nematodes' cuticles. There were 2,059 novel unigenes (51.7% of the total), 149 of which are predicted to encode intrinsically disordered proteins lacking a fixed tertiary structure. One unigene may encode an exo-β-1,3-glucanase (GHF5 family), most similar to a sequence from Phytophthora infestans. GHF5 enzymes have been reported from several species of plant parasitic nematodes, with horizontal gene transfer (HGT) from bacteria proposed to explain their evolutionary origin. This P. superbus sequence represents another possible HGT event within the Nematoda. The expression of five of the 19 putative stress response genes tested was upregulated in response to desiccation. These were the antioxidants glutathione peroxidase, dj-1 and 1-Cys peroxiredoxin, an shsp sequence and an lea gene.
P. superbus appears to utilise a strategy of combined constitutive and inducible gene expression in preparation for entry into anhydrobiosis. The apparent lineage expansion of lea genes, together with their constitutive and inducible expression, suggests that LEA3 proteins are important components of the anhydrobiotic protection repertoire of P. superbus.
BMC Research Notes 01/2012; 5:68.
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ABSTRACT: Genome sequencing has been democratized by second-generation technologies, and even small labs can sequence metazoan genomes now. In this article, we describe '959 Nematode Genomes'--a community-curated semantic wiki to coordinate the sequencing efforts of individual labs to collectively sequence 959 genomes spanning the phylum Nematoda. The main goal of the wiki is to track sequencing projects that have been proposed, are in progress, or have been completed. Wiki pages for species and strains are linked to pages for people and organizations, using machine- and human-readable metadata that users can query to see the status of their favourite worm. The site is based on the same platform that runs Wikipedia, with semantic extensions that allow the underlying taxonomy and data storage models to be maintained and updated with ease compared with a conventional database-driven web site. The wiki also provides a way to track and share preliminary data if those data are not polished enough to be submitted to the official sequence repositories. In just over a year, this wiki has already fostered new international collaborations and attracted newcomers to the enthusiastic community of nematode genomicists. www.nematodegenomes.org.
Nucleic Acids Research 11/2011; 40(Database issue):D1295-300. · 8.03 Impact Factor
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ABSTRACT: The diversity of biology in nematodes is reflected in the diversity of their genomes. Parasitic species in particular have evolved mechanisms to invade and outwit their hosts, and these offer opportunities for the development of control measures. Genomic analyses can reveal the molecular underpinnings of phenotypes such as parasitism and thus, initiate and support research programmes that explore the manipulation of host and parasite physiologies to achieve favourable outcomes. Wide sampling across nematode diversity allows phylogenetically informed formulation of research hypotheses, identification of core features shared by all species or important evolutionary novelties present in isolated clades. Many nematode species have been investigated through the use of the expressed sequence tag approach, which samples from the transcribed genome. Gene catalogues generated in this way can be explored to reveal the patterns of expression associated with parasitism and candidates for testing as drug targets or vaccine components. Analysis environments, such as NEMBASE facilitate exploitation of these data. The development of new high-throughput DNA-sequencing technologies has facilitated transcriptomic and genomic approaches to parasite biology. Whole genome sequencing offers more complete catalogues of genes and assists a systems approach to phenotype dissection. These efforts are being coordinated through the 959 Nematode Genomes initiative.
Parasite Immunology 11/2011; 34(2-3):108-20. · 2.60 Impact Factor
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ABSTRACT: Nematode parasites are of major importance in human health and agriculture, and free-living species deliver essential ecosystem services. The genomics revolution has resulted in the production of many datasets of expressed sequence tags (ESTs) from a phylogenetically wide range of nematode species, but these are not easily compared. NEMBASE4 presents a single portal into extensively functionally annotated, EST-derived transcriptomes from over 60 species of nematodes, including plant and animal parasites and free-living taxa. Using the PartiGene suite of tools, we have assembled the publicly available ESTs for each species into a high-quality set of putative transcripts. These transcripts have been translated to produce a protein sequence resource and each is annotated with functional information derived from comparison with well-studied nematode species such as Caenorhabditis elegans and other non-nematode resources. By cross-comparing the sequences within NEMBASE4, we have also generated a protein family assignment for each translation. The data are presented in an openly accessible, interactive database. To demonstrate the utility of NEMBASE4, we have used the database to examine the uniqueness of the transcriptomes of major clades of parasitic nematodes, identifying lineage-restricted genes that may underpin particular parasitic phenotypes, possible viral pathogens of nematodes, and nematode-unique protein families that may be developed as drug targets.
International journal for parasitology 07/2011; 41(8):881-94. · 3.39 Impact Factor
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ABSTRACT: Increasing numbers of phylogeographic studies make comparative inferences about the histories of co-distributed species. Although the aims of such studies are best achieved by jointly analysing sequences from multiple loci in a model-based framework, such data currently exist for few nonmodel systems. We used existing genomic data and expressed sequence tags (ESTs) for Hymenoptera and other insects to design intron-crossing primers for 40 loci, mainly ribosomal proteins, for chalcidoid parasitoids. Amplification success was scored on a range of taxa associated with two natural communities; oak galls and figs. Taxa were chosen at increasing distance from Nasonia, which was used for primer design, (i) within Pteromalids, (ii) within Chalcidoidea (Eupelmidae, Eulophidae, Eurytomidae, Ormyridae, Torymidae) and (iii) for a selection of distantly related gall and fig wasps (Cynipidae, Agaonidae). To assess the utility of these loci for phylogeographic and population genetic studies, we compared genetic diversity between Western Palaearctic refugia for two species. Our results show that it is feasible to design a large number of exon-primed-intron-crossing (EPIC) loci that may be informative about phylogeographic history within species but amplify across a large taxonomic range.
Molecular Ecology Resources 05/2011; 11(3):521-9. · 3.06 Impact Factor
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Current biology: CB 04/2011; 21(7):R238-40. · 10.99 Impact Factor
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Mark Blaxter
PLoS Biology 04/2011; 9(4):e1001050. · 11.45 Impact Factor
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ABSTRACT: Abstract Background The increasing availability of molecular sequence data means that the accuracy of future phylogenetic studies is likely to by limited by systematic bias and taxon choice rather than by data. In order to take advantage of increasing datasets, user-friendly tools are required to facilitate phylogenetic analyses and to reduce duplication of dataset assembly efforts. Current phylogenetic pipelines are dependency-heavy and have significant technical barriers to use. Results Here we present iPhy, a web application that lets non-technical users assemble, share and analyse DNA sequence datasets for multigene phylogenetic investigations. Built on a simple client-server architecture, iPhy eases the collection of gene sets for analysis, facilitates alignment and reliably generates phylogenetic analysis-ready data files. Phylogenetic trees generated in external programs can be imported and stored, and iPhy integrates with iTol to allow trees to be displayed with rich data annotation. The datasets collated in iPhy can be shared through the client interface. We show how systematic biases can be addressed by using explicit criteria when selecting sequences for analysis from a large dataset. A representative instance of iPhy can be accessed at iphy.bio.ed.ac.uk, but the toolkit can also be deployed on a local server for advanced users. Conclusions iPhy provides an easy-to-use environment for the assembly, analysis and sharing of large phylogenetic datasets, while encouraging best practices in terms of phylogenetic analysis and taxon selection.
BMC Bioinformatics. 01/2011;
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ABSTRACT: DNA barcoding and other DNA sequence-based techniques for investigating and estimating biodiversity require explicit methods for associating individual sequences with taxa, as it is at the taxon level that biodiversity is assessed. For many projects, the bioinformatic analyses required pose problems for laboratories whose prime expertise is not in bioinformatics. User-friendly tools are required for both clustering sequences into molecular operational taxonomic units (MOTU) and for associating these MOTU with known organismal taxonomies.
Here we present jMOTU, a Java program for the analysis of DNA barcode datasets that uses an explicit, determinate algorithm to define MOTU. We demonstrate its usefulness for both individual specimen-based Sanger sequencing surveys and bulk-environment metagenetic surveys using long-read next-generation sequencing data. jMOTU is driven through a graphical user interface, and can analyse tens of thousands of sequences in a short time on a desktop computer. A companion program, Taxonerator, that adds traditional taxonomic annotation to MOTU, is also presented. Clustering and taxonomic annotation data are stored in a relational database, and are thus amenable to subsequent data mining and web presentation.
jMOTU efficiently and robustly identifies the molecular taxa present in survey datasets, and Taxonerator decorates the MOTU with putative identifications. jMOTU and Taxonerator are freely available from http://www.nematodes.org/.
PLoS ONE 01/2011; 6(4):e19259. · 4.09 Impact Factor
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Mark Blaxter
Science 12/2010; 330(6012):1758-9. · 31.20 Impact Factor
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Paul Hunt,
Axel Martinelli,
Katarzyna Modrzynska,
Sofia Borges,
Alison Creasey,
Louise Rodrigues,
Dario Beraldi,
Laurence Loewe,
Richard Fawcett,
Sujai Kumar,
Marian Thomson,
Urmi Trivedi,
Thomas D Otto,
Arnab Pain, Mark Blaxter,
Pedro Cravo
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ABSTRACT: Classical and quantitative linkage analyses of genetic crosses have traditionally been used to map genes of interest, such as those conferring chloroquine or quinine resistance in malaria parasites. Next-generation sequencing technologies now present the possibility of determining genome-wide genetic variation at single base-pair resolution. Here, we combine in vivo experimental evolution, a rapid genetic strategy and whole genome re-sequencing to identify the precise genetic basis of artemisinin resistance in a lineage of the rodent malaria parasite, Plasmodium chabaudi. Such genetic markers will further the investigation of resistance and its control in natural infections of the human malaria, P. falciparum.
A lineage of isogenic in vivo drug-selected mutant P. chabaudi parasites was investigated. By measuring the artemisinin responses of these clones, the appearance of an in vivo artemisinin resistance phenotype within the lineage was defined. The underlying genetic locus was mapped to a region of chromosome 2 by Linkage Group Selection in two different genetic crosses. Whole-genome deep coverage short-read re-sequencing (Illumina Solexa) defined the point mutations, insertions, deletions and copy-number variations arising in the lineage. Eight point mutations arise within the mutant lineage, only one of which appears on chromosome 2. This missense mutation arises contemporaneously with artemisinin resistance and maps to a gene encoding a de-ubiquitinating enzyme.
This integrated approach facilitates the rapid identification of mutations conferring selectable phenotypes, without prior knowledge of biological and molecular mechanisms. For malaria, this model can identify candidate genes before resistant parasites are commonly observed in natural human malaria populations.
BMC Genomics 01/2010; 11:499. · 4.07 Impact Factor
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ABSTRACT: Abstract Background Roche 454 pyrosequencing has become a method of choice for generating transcriptome data from non-model organisms. Once the tens to hundreds of thousands of short (250-450 base) reads have been produced, it is important to correctly assemble these to estimate the sequence of all the transcripts. Most transcriptome assembly projects use only one program for assembling 454 pyrosequencing reads, but there is no evidence that the programs used to date are optimal. We have carried out a systematic comparison of five assemblers (CAP3, MIRA, Newbler, SeqMan and CLC) to establish best practices for transcriptome assemblies, using a new dataset from the parasitic nematode Litomosoides sigmodontis. Results Although no single assembler performed best on all our criteria, Newbler 2.5 gave longer contigs, better alignments to some reference sequences, and was fast and easy to use. SeqMan assemblies performed best on the criterion of recapitulating known transcripts, and had more novel sequence than the other assemblers, but generated an excess of small, redundant contigs. The remaining assemblers all performed almost as well, with the exception of Newbler 2.3 (the version currently used by most assembly projects), which generated assemblies that had significantly lower total length. As different assemblers use different underlying algorithms to generate contigs, we also explored merging of assemblies and found that the merged datasets not only aligned better to reference sequences than individual assemblies, but were also more consistent in the number and size of contigs. Conclusions Transcriptome assemblies are smaller than genome assemblies and thus should be more computationally tractable, but are often harder because individual contigs can have highly variable read coverage. Comparing single assemblers, Newbler 2.5 performed best on our trial data set, but other assemblers were closely comparable. Combining differently optimal assemblies from different programs however gave a more credible final product, and this strategy is recommended.
BMC Genomics. 01/2010;
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ABSTRACT: Ecdysozoa is the recently recognized clade of molting animals that comprises the vast majority of extant animal species and the most important invertebrate model organisms--the fruit fly and the nematode worm. Evolutionary relationships within the ecdysozoans remain, however, unresolved, impairing the correct interpretation of comparative genomic studies. In particular, the affinities of the three Panarthropoda phyla (Arthropoda, Onychophora, and Tardigrada) and the position of Myriapoda within Arthropoda (Mandibulata vs. Myriochelata hypothesis) are among the most contentious issues in animal phylogenetics. To elucidate these relationships, we have determined and analyzed complete or nearly complete mitochondrial genome sequences of two Tardigrada, Hypsibius dujardini and Thulinia sp. (the first genomes to date for this phylum); one Priapulida, Halicryptus spinulosus; and two Onychophora, Peripatoides sp. and Epiperipatus biolleyi; and a partial mitochondrial genome sequence of the Onychophora Euperipatoides kanagrensis. Tardigrada mitochondrial genomes resemble those of the arthropods in term of the gene order and strand asymmetry, whereas Onychophora genomes are characterized by numerous gene order rearrangements and strand asymmetry variations. In addition, Onychophora genomes are extremely enriched in A and T nucleotides, whereas Priapulida and Tardigrada are more balanced. Phylogenetic analyses based on concatenated amino acid coding sequences support a monophyletic origin of the Ecdysozoa and the position of Priapulida as the sister group of a monophyletic Panarthropoda (Tardigrada plus Onychophora plus Arthropoda). The position of Tardigrada is more problematic, most likely because of long branch attraction (LBA). However, experiments designed to reduce LBA suggest that the most likely placement of Tardigrada is as a sister group of Onychophora. The same analyses also recover monophyly of traditionally recognized arthropod lineages such as Arachnida and of the highly debated clade Mandibulata.
Genome Biology and Evolution 01/2010; 2:425-40. · 4.62 Impact Factor
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ABSTRACT: BACKGROUND: Branchiopod crustaceans in the genus Daphnia are key model organisms for investigating interactions between genes and the environment. One major theme of research on Daphnia species has been the evolution of resistance to pathogens and parasites, but lack of knowledge of the Daphnia immune system has limited the study of immune responses. Here we provide a survey of the immune-related genome of D. pulex, derived from the newly completed genome sequence. Genes likely to be involved in innate immune responses were identified by comparison to homologues from other arthropods. For each candidate, the gene model was refined, and we conducted an analysis of sequence divergence from homologues from other taxa. RESULTS AND CONCLUSION: We found that some immune pathways, in particular the TOLL pathway, are fairly well conserved between insects and Daphnia, while other elements, in particular antimicrobial peptides, could not be recovered from the genome sequence. We also found considerable variation in gene family copy number when comparing Daphnia to insects and present phylogenetic analyses to shed light on the evolution of a range of conserved immune gene families.
BMC Genomics 05/2009; 10:175. · 4.07 Impact Factor
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ABSTRACT: We inferred the rate and properties of new spontaneous mutations in Drosophila melanogaster by carrying out whole-genome shotgun sequencing-by-synthesis of three mutation accumulation (MA) lines that had been maintained by close inbreeding for an average of 262 generations. We tested for the presence of new mutations by generating alignments of each MA line to the D. melanogaster reference genome sequence and then compared these alignments base by base. We determined empirically that at least five reads at a site within each line are required for accurate single nucleotide mutation calling. We mapped a total of 174 single-nucleotide mutations, giving a single nucleotide mutation rate of 3.5 x 10(-9) per site per generation. There were no false positives in a random sample of 40 of these mutations checked by Sanger sequencing. Variation in the numbers of mutations among the MA lines was small and nonsignificant. Numbers of transition and transversion mutations were 86 and 88, respectively, implying that transition mutation rate is close to 2x the transversion rate. We observed 1.5x as many G or C --> A or T as A or T --> G or C mutations, implying that the G or C --> A or T mutation rate is close to 2x the A or T --> G or C mutation rate. The base composition of the genome is therefore not at an equilibrium determined solely by mutation. The predicted G + C content at mutational equilibrium (33%) is similar to that observed in transposable element remnants. Nearest-neighbor mutational context dependencies are nonsignificant, suggesting that this is a weak phenomenon in Drosophila. We also saw nonsignificant differences in the mutation rate between transcribed and untranscribed regions, implying that any transcription-coupled repair process is weak. Of seven short indel mutations confirmed, six were deletions, consistent with the deletion bias that is thought to exist in Drosophila.
Genome Research 05/2009; 19(7):1195-201. · 13.61 Impact Factor
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ABSTRACT: Expressed sequence tags (ESTs) are fragments of mRNA sequences derived through single sequencing reactions performed on randomly selected clones from cDNA libraries. To date, over 45 million ESTs have been generated from over 1400 different species of eukaryotes. For the most part, EST projects are used to either complement existing genome projects or serve as low-cost alternatives for purposes of gene discovery. However, with improvements in accuracy and coverage, they are beginning to find application in fields such as phylogenetics, transcript profiling and proteomics. This volume provides practical details on the generation and analysis of ESTs. Chapters are presented which cover creation of cDNA libraries; generation and processing of sequence data; bioinformatics analysis of ESTs; and their application to phylogenetics and transcript profiling.
Methods in molecular biology (Clifton, N.J.) 02/2009; 533:1-12.
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ABSTRACT: A common task in EST projects is the conversion of sequence chromatograms originating from gel-based or capillary sequencers into annotated sequence objects. Here we describe the usage of a software pipeline (available from http://www.nematodes.org/bioinformatics/ ), which has been developed to make the most of EST datasets. This modular software solution is targeted toward small- to medium-sized EST projects and comprises a series of Perl scripts. The software design is based on our experience during EST projects for parasitic nematodes and other species. The trace2dbest module processes sequence trace files and prepares the text files necessary for the submission of the sequences to the public repository dbEST. PartiGene provides facilities for clustering and assembling the ESTs into putative gene objects or unigenes and organizes the data in a relational database. Additional tools are available for annotation and for making the data accessible via the World Wide Web.
Methods in molecular biology (Clifton, N.J.) 02/2009; 533:189-220.
