De novo reconstruction of the Toxoplasma gondii transcriptome improves on the current genome annotation and reveals alternatively spliced transcripts and putative long non-coding RNAs

Musa A Hassan,Brian Haas,Mariane B Melo,Jeroen P J Saeij,Kirk D C Jensen

doi:10.1186/1471-2164-13-696

Musa A Hassan, Brian Haas + Show 3 more

Open Access

https://doi.org/10.1186/1471-2164-13-696

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Abstract

BackgroundAccurate gene model predictions and annotation of alternative splicing events are imperative for genomic studies in organisms that contain genes with multiple exons. Currently most gene models for the intracellular parasite, Toxoplasma gondii, are based on computer model predictions without cDNA sequence verification. Additionally, the nature and extent of alternative splicing in Toxoplasma gondii is unknown. In this study, we used de novo transcript assembly and the published type II (ME49) genomic sequence to quantify the extent of alternative splicing in Toxoplasma and to improve the current Toxoplasma gene annotations.ResultsWe used high-throughput RNA-sequencing data to assemble full-length transcripts, independently of a reference genome, followed by gene annotation based on the ME49 genome. We assembled 13,533 transcripts overlapping with known ME49 genes in ToxoDB and then used this set to; a) improve the annotation in the untranslated regions of ToxoDB genes, b) identify novel exons within protein-coding ToxoDB genes, and c) report on 50 previously unidentified alternatively spliced transcripts. Additionally, we assembled a set of 2,930 transcripts not overlapping with any known ME49 genes in ToxoDB. From this set, we have identified 118 new ME49 genes, 18 novel Toxoplasma genes, and putative non-coding RNAs.ConclusionRNA-seq data and de novo transcript assembly provide a robust way to update incompletely annotated genomes, like the Toxoplasma genome. We have used RNA-seq to improve the annotation of several Toxoplasma genes, identify alternatively spliced genes, novel genes, novel exons, and putative non-coding RNAs.

Highlights

Accurate gene model predictions and annotation of alternative splicing events are imperative for genomic studies in organisms that contain genes with multiple exons
De novo full-length transcript assembly Approximately 1.2 billion 40 base-pair paired-end RNA-seq reads generated from murine bone-marrow derived macrophages infected with Toxoplasma were used to assemble Toxoplasma full-length transcripts in Trinity [18] and Program to Assemble Spliced Alignment (PASA) [29,31]
Because the parasites used to infect the murine macrophages were grown in human foreskin fibroblasts (HFFs), we initially used the genome alignment tool, Tophat [32,33] to sequentially align the RNAseq reads to the mouse and human reference genomes and a collection of mouse and human splice

Summary

Results

We used high-throughput RNA-sequencing data to assemble full-length transcripts, independently of a reference genome, followed by gene annotation based on the ME49 genome. We assembled 13,533 transcripts overlapping with known ME49 genes in ToxoDB and used this set to; a) improve the annotation in the untranslated regions of ToxoDB genes, b) identify novel exons within protein-coding ToxoDB genes, and c) report on 50 previously unidentified alternatively spliced transcripts. We assembled a set of 2,930 transcripts not overlapping with any known ME49 genes in ToxoDB. From this set, we have identified 118 new ME49 genes, 18 novel Toxoplasma genes, and putative non-coding RNAs

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Background

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