cDNA sequences reveal considerable gene prediction inaccuracy in the Plasmodium falciparum genome

Abstract

Background: The completion of the Plasmodium falciparum genome represents a milestone in malaria research. The genome sequence allows for the development of genome-wide approaches such as microarray and proteomics that will greatly facilitate our understanding of the parasite biology and accelerate new drug and vaccine development. Designing and application of these genome-wide assays, however, requires accurate information on gene prediction and genome annotation. Unfortunately, the genes in the parasite genome databases were mostly identified using computer software that could make some erroneous predictions.

Results: We aimed to obtain cDNA sequences to examine the accuracy of gene prediction in silico. We constructed cDNA libraries from mixed blood stages of P. falciparum parasite using the SMART cDNA library construction technique and generated 17332 high-quality expressed sequence tags (EST), including 2198 from primer-walking experiments. Assembly of our sequence tags produced 2548 contigs and 2671 singletons versus 5220 contigs and 5910 singletons when our EST were assembled with EST in public databases. Comparison of all the assembled EST/contigs with predicted CDS and genomic sequences in the PlasmoDB database identified 356 genes with predicted coding sequences fully covered by EST, including 85 genes (23.6%) with introns incorrectly predicted. Careful automatic software and manual alignments found an additional 308 genes that have introns different from those predicted, with 152 new introns discovered and 182 introns with sizes or locations different from those predicted. Alternative spliced and antisense transcripts were also detected. Matching cDNA to predicted genes also revealed silent chromosomal regions, mostly at subtelomere regions.

Conclusion: Our data indicated that approximately 24% of the genes in the current databases were predicted incorrectly, although some of these inaccuracies could represent alternatively spliced transcripts, and that more genes than currently predicted have one or more additional introns. It is therefore necessary to annotate the parasite genome with experimental data, although obtaining complete cDNA sequences from this parasite will be a formidable task due to the high AT nature of the genome. This study provides valuable information for genome annotation that will be critical for functional analyses.

Figure 1

Diagram of the 14 P. falciparum chromosomes showing positions of potentially expressed genes. Expressed sequence tags (EST) from our libraries or from public databases were assembled against predicted coding sequences in PlasmoDB; genes that matched our EST only (green), EST already in public databases (red), or both (yellow) are displayed according to gene order on the chromosomes. Those in white are CDS that were not covered by any EST. Approximately 70% of the 5485 predicted CDS were matched with one or more EST.

Figure 2

PCR products confirming alternatively spliced introns. Oligonucleotide primers flanking selected predicted introns that might be alternatively spliced were amplified from genomic DNA (G lanes), reverse-transcribed mRNA of mixed asexual stages (C lanes), and mRNA controls of mixed asexual stages (without reverse transcriptase, R lanes). Genes with alternatively spliced introns are as marked; M, 100 bp DNA ladder. Note that more than two bands were amplified from PFE1540w, PF13_0220, and PF13_0224.

Figure 3

Diagram of exon/intron structures of predicted gene PFL1420w and cDNA contigs covering the gene. FC (forward contig) is a sense transcript with an intron matching the predicted intron. RC (reverse contig) is an antisense transcript having a smaller intron with GT-AG sites in the opposite direction. The line on top represents plus strand genomic DNA. Dashed lines are introns; heavy lines are predicted exons or ORF.

Figure 4

Functional categories of expressed genes covered by all EST. A total of 3862 genes matched by EST were sorted according to GO molecular functions with P values < 0.0001 on sequence matches. The majority of the genes encode housekeeping proteins involved in DNA/RNA and protein binding, enzyme catalytic activities, transcription, translation, signal transduction, and transport activities.