Full-length messenger RNA sequences greatly improve genome annotation

Abstract

Background: Annotation of eukaryotic genomes is a complex endeavor that requires the integration of evidence from multiple, often contradictory, sources. With the ever-increasing amount of genome sequence data now available, methods for accurate identification of large numbers of genes have become urgently needed. In an effort to create a set of very high-quality gene models, we used the sequence of 5,000 full-length gene transcripts from Arabidopsis to re-annotate its genome. We have mapped these transcripts to their exact chromosomal locations and, using alignment programs, have created gene models that provide a reference set for this organism.

Results: Approximately 35% of the transcripts indicated that previously annotated genes needed modification, and 5% of the transcripts represented newly discovered genes. We also discovered that multiple transcription initiation sites appear to be much more common than previously known, and we report numerous cases of alternative mRNA splicing. We include a comparison of different alignment software and an analysis of how the transcript data improved the previously published annotation.

Conclusions: Our results demonstrate that sequencing of large numbers of full-length transcripts followed by computational mapping greatly improves identification of the complete exon structures of eukaryotic genes. In addition, we are able to find numerous introns in the untranslated regions of the genes.

Figure 1

An example showing how a micro-exon improves a cDNA alignment. (a) Alignment showing the boundaries of the fourth and fifth exons from the sim4 alignment of cDNA Ceres:20761 to chromosome 4. (b) The improvement resulting from insertion of a micro-exon; all three exons now align with 100% identity to the cDNA sequence. Intron positions are shown by '>' in the alignment.

Figure 2

Alternative splice variants discovered by cDNA alignments. Red bars indicate the protein-coding portion of each exon. Black bars indicate noncoding exons and the UTR portions of the initial and terminal exons. Exon boundaries that line up exactly between two or more cDNAs are highlighted in blue. Thin lines connecting the exons represent introns. The genes involved are: (a) auxin-regulated protein, At2g20820, chromosome (chr) 2; (b) SKP1-interacting partner 5 (SKIP5), At3g54480, chr 3; (c) acidic ribosomal protein, At1g01100, chr 1; (d) auxin-regulated protein, At5g53860, chr 5; (e) unknown expressed protein, At2g45740, chr 2.

Figure 3

Comparison of the lengths of the 941 cDNAs from the clones that are contained in both the Ceres and RIKEN collections. (a) Comparison of the 5'-end difference between Ceres and RIKEN clones; (b) comparison of the 3'-end difference between Ceres and RIKEN clones. Peak height indicates the percentage of sequences with a length difference as indicated along the horizontal axis. Positive values on the horizontal axis correspond to longer Ceres clones, while negative values correspond to longer RIKEN clones.