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      Draft Genome Sequencing of Giardia intestinalis Assemblage B Isolate GS: Is Human Giardiasis Caused by Two Different Species?

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          Abstract

          Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 16× coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus <0.01% in WB). The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species.

          Author Summary

          Giardia intestinalis is a major contributor to the enormous burden of diarrheal diseases with 250 million symptomatic infections per year, and it is part of the WHO neglected disease initiative. Nonetheless, there is poor insight into how Giardia causes disease; it is not invasive, secretes no known toxin and both the duration and symptoms of giardiasis are highly variable. Currently, there are seven defined variants (assemblages) of G. intestinalis, with only assemblages A and B being known to infect humans. Although assemblage B is the most prevalent worldwide, it is inconclusive whether the various genotypes are associated with different disease outcomes. We have used the 454 sequencing technology to sequence the first assemblage B isolate, and the genome was compared to the earlier sequenced assemblage A isolate. Large genetic differences were detected in genes involved in survival of the parasite during infections. The genomic differences between assemblage A and B can explain some of the observed biological and clinical differences between the two assemblages. Our data suggest that assemblage A and B Giardia can be two different species. The identification of genomic differences between assemblages is indeed very important for further studies of the disease and in the development of new methods for diagnosis and treatment of giardiasis.

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          Most cited references 60

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          The minimum information about a genome sequence (MIGS) specification.

          With the quantity of genomic data increasing at an exponential rate, it is imperative that these data be captured electronically, in a standard format. Standardization activities must proceed within the auspices of open-access and international working bodies. To tackle the issues surrounding the development of better descriptions of genomic investigations, we have formed the Genomic Standards Consortium (GSC). Here, we introduce the minimum information about a genome sequence (MIGS) specification with the intent of promoting participation in its development and discussing the resources that will be required to develop improved mechanisms of metadata capture and exchange. As part of its wider goals, the GSC also supports improving the 'transparency' of the information contained in existing genomic databases.
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            The complete genome of an individual by massively parallel DNA sequencing.

            The association of genetic variation with disease and drug response, and improvements in nucleic acid technologies, have given great optimism for the impact of 'genomic medicine'. However, the formidable size of the diploid human genome, approximately 6 gigabases, has prevented the routine application of sequencing methods to deciphering complete individual human genomes. To realize the full potential of genomics for human health, this limitation must be overcome. Here we report the DNA sequence of a diploid genome of a single individual, James D. Watson, sequenced to 7.4-fold redundancy in two months using massively parallel sequencing in picolitre-size reaction vessels. This sequence was completed in two months at approximately one-hundredth of the cost of traditional capillary electrophoresis methods. Comparison of the sequence to the reference genome led to the identification of 3.3 million single nucleotide polymorphisms, of which 10,654 cause amino-acid substitution within the coding sequence. In addition, we accurately identified small-scale (2-40,000 base pair (bp)) insertion and deletion polymorphism as well as copy number variation resulting in the large-scale gain and loss of chromosomal segments ranging from 26,000 to 1.5 million base pairs. Overall, these results agree well with recent results of sequencing of a single individual by traditional methods. However, in addition to being faster and significantly less expensive, this sequencing technology avoids the arbitrary loss of genomic sequences inherent in random shotgun sequencing by bacterial cloning because it amplifies DNA in a cell-free system. As a result, we further demonstrate the acquisition of novel human sequence, including novel genes not previously identified by traditional genomic sequencing. This is the first genome sequenced by next-generation technologies. Therefore it is a pilot for the future challenges of 'personalized genome sequencing'.
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              The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease.

              Whole-genome sequencing of the protozoan pathogen Trypanosoma cruzi revealed that the diploid genome contains a predicted 22,570 proteins encoded by genes, of which 12,570 represent allelic pairs. Over 50% of the genome consists of repeated sequences, such as retrotransposons and genes for large families of surface molecules, which include trans-sialidases, mucins, gp63s, and a large novel family (>1300 copies) of mucin-associated surface protein (MASP) genes. Analyses of the T. cruzi, T. brucei, and Leishmania major (Tritryp) genomes imply differences from other eukaryotes in DNA repair and initiation of replication and reflect their unusual mitochondrial DNA. Although the Tritryp lack several classes of signaling molecules, their kinomes contain a large and diverse set of protein kinases and phosphatases; their size and diversity imply previously unknown interactions and regulatory processes, which may be targets for intervention.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                August 2009
                August 2009
                21 August 2009
                : 5
                : 8
                Affiliations
                [1 ]Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden
                [2 ]Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, Sweden
                [3 ]Centre for Microbiological Preparedness, Swedish Institute for Infectious Disease Control, Solna, Sweden
                [4 ]The Burnham Institute for Medical Research, La Jolla, California, United States of America
                [5 ]Department of Evolution, Genomics and Systematics, EBC, Uppsala University, Uppsala, Sweden
                University of Virginia Health System, United States of America
                Author notes

                Conceived and designed the experiments: ES JOA BA SGS. Performed the experiments: OF JJH EC JA DSR JOA. Analyzed the data: OF JJH EC ES JA DSR DP JOA BA SGS. Contributed reagents/materials/analysis tools: OF JJH DP JOA. Wrote the paper: OF JJH EC ES JA DSR DP JOA BA SGS.

                Article
                09-PLPA-RA-0523R2
                10.1371/journal.ppat.1000560
                2723961
                19696920
                Franzén et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Page count
                Pages: 14
                Categories
                Research Article
                Genetics and Genomics/Comparative Genomics
                Infectious Diseases/Gastrointestinal Infections
                Infectious Diseases/Neglected Tropical Diseases
                Infectious Diseases/Protozoal Infections

                Infectious disease & Microbiology

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