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Pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of Plasmodium vivax in human patients.

Merino EF, Fernandez-Becerra C, Madeira AM, Machado AL, Durham A, Gruber A, Hall N, del Portillo HA - Malar. J. (2003)

Bottom Line: An E-value of <10(-30) was used to define a significant database match.Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them.These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Parasitologia, ICB, Universidade de São Paulo, São Paulo, Brazil. efmerino@yahoo.com

ABSTRACT

Background: Plasmodium vivax is the most widely distributed human malaria, responsible for 70-80 million clinical cases each year and large socio-economical burdens for countries such as Brazil where it is the most prevalent species. Unfortunately, due to the impossibility of growing this parasite in continuous in vitro culture, research on P. vivax remains largely neglected.

Methods: A pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of P. vivax was performed. To do so, 1,184 clones from a cDNA library constructed with parasites obtained from 10 different human patients in the Brazilian Amazon were sequenced. Sequences were automatedly processed to remove contaminants and low quality reads. A total of 806 sequences with an average length of 586 bp met such criteria and their clustering revealed 666 distinct events. The consensus sequence of each cluster and the unique sequences of the singlets were used in similarity searches against different databases that included P. vivax, Plasmodium falciparum, Plasmodium yoelii, Plasmodium knowlesi, Apicomplexa and the GenBank non-redundant database. An E-value of <10(-30) was used to define a significant database match. ESTs were manually assigned a gene ontology (GO) terminology

Results: A total of 769 ESTs could be assigned a putative identity based upon sequence similarity to known proteins in GenBank. Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them.

Conclusion: These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced. Moreover, since the GC-content of the P. vivax genome is strikingly different from that of P. falciparum, these ESTs will help in the validation of gene predictions for P. vivax and to create a gene index of this malaria parasite.

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Related in: MedlinePlus

Overview of the pipeline used in the ESTs identification process. Sequences were automatically assessed for quality and removal of contaminants. BLAST similarity searches against PlasmoDB (PDB), GenBank (GB), and TIGR (preliminary sequence data was obtained from The Institute for Genomic Research through the website at ) data bases were performed after assembling the sequences with CAP3 [10] and masking regions of low complexity with the SEG [11] or DUST (Tatusov & Lipman, unpublished; ) programs. An E-value of < 10-30 for both BLASTN and BLASTX defined a significant database match.
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Figure 1: Overview of the pipeline used in the ESTs identification process. Sequences were automatically assessed for quality and removal of contaminants. BLAST similarity searches against PlasmoDB (PDB), GenBank (GB), and TIGR (preliminary sequence data was obtained from The Institute for Genomic Research through the website at ) data bases were performed after assembling the sequences with CAP3 [10] and masking regions of low complexity with the SEG [11] or DUST (Tatusov & Lipman, unpublished; ) programs. An E-value of < 10-30 for both BLASTN and BLASTX defined a significant database match.

Mentions: Out of 1,184 clones fully sequenced from both ends, a total of 806 were high quality P. vivax ESTs with an average length of 586 bp, which allowed robust similarity analyses. Clustering of the 806 ESTs with CAP3 revealed that they correspond to 571 singlets and 95 contigs representing 666 cluster events. The majority of the contigs (91.48%) comprised 2–3 sequences and the largest one contained 16 sequences. The complete set of 666 cluster events was used in similarity searches against different databases using BLAST (Figure 1). An E-value of <10-30 for both BLASTN and BLASTX was used to define a significant database match. Considering this E-value, 641 cluster events were identified by similarity, whereas 25 did not meet this criterion and were considered unidentified. As expected, the vast majority of the cluster events were similar to P. vivax and more events matched entries in the databases of P. knowlesi than those of P. falciparum or P. yoelii, even though the genomes of the latter two have been completely sequenced [20,21]. These data are in agreement with a closer phylogenetic relationship between P. vivax and monkey malaria parasites than to P. falciparum or rodent malaria parasites [22]. Interestingly, 127 ESTs (corresponding to 103 cluster events) were identified in all plasmodia, probably representing ancestral genes involved in essential functions during the asexual blood stages. Fasta files and results from all analyses are available as supplementary material at the Malaria DataBank of the University of São Paulo .


Pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of Plasmodium vivax in human patients.

Merino EF, Fernandez-Becerra C, Madeira AM, Machado AL, Durham A, Gruber A, Hall N, del Portillo HA - Malar. J. (2003)

Overview of the pipeline used in the ESTs identification process. Sequences were automatically assessed for quality and removal of contaminants. BLAST similarity searches against PlasmoDB (PDB), GenBank (GB), and TIGR (preliminary sequence data was obtained from The Institute for Genomic Research through the website at ) data bases were performed after assembling the sequences with CAP3 [10] and masking regions of low complexity with the SEG [11] or DUST (Tatusov & Lipman, unpublished; ) programs. An E-value of < 10-30 for both BLASTN and BLASTX defined a significant database match.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC183858&req=5

Figure 1: Overview of the pipeline used in the ESTs identification process. Sequences were automatically assessed for quality and removal of contaminants. BLAST similarity searches against PlasmoDB (PDB), GenBank (GB), and TIGR (preliminary sequence data was obtained from The Institute for Genomic Research through the website at ) data bases were performed after assembling the sequences with CAP3 [10] and masking regions of low complexity with the SEG [11] or DUST (Tatusov & Lipman, unpublished; ) programs. An E-value of < 10-30 for both BLASTN and BLASTX defined a significant database match.
Mentions: Out of 1,184 clones fully sequenced from both ends, a total of 806 were high quality P. vivax ESTs with an average length of 586 bp, which allowed robust similarity analyses. Clustering of the 806 ESTs with CAP3 revealed that they correspond to 571 singlets and 95 contigs representing 666 cluster events. The majority of the contigs (91.48%) comprised 2–3 sequences and the largest one contained 16 sequences. The complete set of 666 cluster events was used in similarity searches against different databases using BLAST (Figure 1). An E-value of <10-30 for both BLASTN and BLASTX was used to define a significant database match. Considering this E-value, 641 cluster events were identified by similarity, whereas 25 did not meet this criterion and were considered unidentified. As expected, the vast majority of the cluster events were similar to P. vivax and more events matched entries in the databases of P. knowlesi than those of P. falciparum or P. yoelii, even though the genomes of the latter two have been completely sequenced [20,21]. These data are in agreement with a closer phylogenetic relationship between P. vivax and monkey malaria parasites than to P. falciparum or rodent malaria parasites [22]. Interestingly, 127 ESTs (corresponding to 103 cluster events) were identified in all plasmodia, probably representing ancestral genes involved in essential functions during the asexual blood stages. Fasta files and results from all analyses are available as supplementary material at the Malaria DataBank of the University of São Paulo .

Bottom Line: An E-value of <10(-30) was used to define a significant database match.Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them.These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Parasitologia, ICB, Universidade de São Paulo, São Paulo, Brazil. efmerino@yahoo.com

ABSTRACT

Background: Plasmodium vivax is the most widely distributed human malaria, responsible for 70-80 million clinical cases each year and large socio-economical burdens for countries such as Brazil where it is the most prevalent species. Unfortunately, due to the impossibility of growing this parasite in continuous in vitro culture, research on P. vivax remains largely neglected.

Methods: A pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of P. vivax was performed. To do so, 1,184 clones from a cDNA library constructed with parasites obtained from 10 different human patients in the Brazilian Amazon were sequenced. Sequences were automatedly processed to remove contaminants and low quality reads. A total of 806 sequences with an average length of 586 bp met such criteria and their clustering revealed 666 distinct events. The consensus sequence of each cluster and the unique sequences of the singlets were used in similarity searches against different databases that included P. vivax, Plasmodium falciparum, Plasmodium yoelii, Plasmodium knowlesi, Apicomplexa and the GenBank non-redundant database. An E-value of <10(-30) was used to define a significant database match. ESTs were manually assigned a gene ontology (GO) terminology

Results: A total of 769 ESTs could be assigned a putative identity based upon sequence similarity to known proteins in GenBank. Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them.

Conclusion: These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced. Moreover, since the GC-content of the P. vivax genome is strikingly different from that of P. falciparum, these ESTs will help in the validation of gene predictions for P. vivax and to create a gene index of this malaria parasite.

Show MeSH
Related in: MedlinePlus