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

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.

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.

Figure 2

Gene Ontology classification of the P. vivax ESTs Classification of P. vivax ESTs according to "Molecular Function" of the GO system [21]. Figures on "Biological Process" and "Cellular Component" can be obtained from .