Production of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from Schistosoma mansoni

Abstract

The number of sequences generated by genome projects has increased exponentially, but gene characterization has not followed at the same rate. Sequencing Key words: expressed sequence tags -sequencing -Schistosoma -full-length cDNA While the number of sequences generated by genome projects has increased exponentially, this phenomenon has not been followed by gene characterization at the same rate (Saghatelian & Cravat 2005). Aiming to diminish that gap, several approaches have been used for gene discovery through searches on the genomic sequence or analysis of the transcriptome of the organisms (Okazaki et al. 2002). One such approach is gene discovery using expressed sequence tags (ESTs), short sequences produced by sequencing the ends of cDNA molecules, which represent a snapshot of the gene expression for a given organism at a certain time (Adams et al. 1991). However, a complete picture of the gene products of the organism can only be obtained when the full-length sequence of specific proteins is determined. For that, it is essential to select clones that potentially present the complete coding sequence, up to the initial methionine and proceed to sequencing and characterization of such clones (Das et al. 2001). The initial step in the characterization is to determine the completeness of the cDNAs from which the ESTs were generated, followed by the annotation using the biological description of sequences present in function-oriented databases. Several authors developed systems to attain this goal which use among other tools similarity searches, statistical information and genome mapping (Salamov et al. 1998, Nishikawa et al. 2000, Del Val et al. 2003, Furuno et al. 2003, Hotz-Wagenblatt et al. 2003. Schistosoma mansoni cDNAs have been selected in this work for full-length sequencing using an algorithm based on BLAST searches of parasite ESTs against sequences from the database Eukaryotic Cluster of Orthologous Groups (KOG) (Tatusov et al. 2003). The implementation of the algorithm uses SQL searches to predict the presence of the initial methionine in the parasite sequence, resulting in the selection of ESTs representing clones putatively containing the complete sequence. By this procedure we have been able to select 3988 ESTs representing 398 proteins which have not yet been deposited as S. mansoni complete CDS in any public database. Dedicated 5' and 3' end sequencing of 96 clones has been performed and reads have been assembled using PHRAP. As a final result, 33 full-length sequences have been produced which represent novel S. mansoni proteins. MATERIALS AND METHODS Selection of ESTs representing putative full-length clones -S. mansoniESTs have been selected for full-length sequencing using the algorithm described by Nishikawa et al. (2000) with modifications. The completness of the clones represented by the ESTs was determined by comparison of these to sequence of proteins from the secondary database KOG using tblastn. The positions of start and end of alignment assigned by BLAST in both sequences were...

Identification and isolation of full-length cDNA sequences by sequencing and analysis of expressed sequence tags from guarana (Paullinia cupana)

“…The identification of full-length clones was done according to Faria-Campos et al (2006), with some modifications. The number of codons calculated before the initial methionine was used for all ESTs and was obtained using a MySQL query to apply the search algorithm to the results of a BLAST search (tBLASTn) of protein sequences from the NR database against ESTs of P. cupana, since there are no sequences of proteins for P. cupana stored in public databases.…”

“…According to the analysis, an elevated number of events (46.15%) had a negative number of codons before the initial methionine, a result already expected for ESTs. Faria-Campos et al (2006) demonstrated that these values can vary from 20 to 70% of the ESTs analyzed, depending on the source of organisms of the sequences used in the alignments.…”