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The reference annotations made for a genome sequence provide the frameworkfor all subsequent analyses of the genome. Correct annotation is particularlyimportant when interpreting the results of RNA-seq experiments where shortsequence reads are mapped against the genome and assigned to genes according tothe annotation. Inconsistencies in annotations between the reference and theexperimental system can lead to incorrect interpretation of the effect on RNAexpression of an experimental treatment or mutation in the system under study.Until recently, the genome-wide annotation of 3-prime untranslated regionsreceived less attention than coding regions and the delineation of intron/exonboundaries. In this paper, data produced for samples in Human, Chicken and A.thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencingtechnology from Helicos Biosciences which locates 3-prime polyadenylation sitesto within +/- 2 nt, were combined with archival EST and RNA-Seq data. Nineexamples are illustrated where this combination of data allowed: (1) gene and3-prime UTR re-annotation (including extension of one 3-prime UTR by 5.9 kb);(2) disentangling of gene expression in complex regions; (3) clearerinterpretation of small RNA expression and (4) identification of novel genes.While the specific examples displayed here may become obsolete as genomesequences and their annotations are refined, the principles laid out in thispaper will be of general use both to those annotating genomes and those seekingto interpret existing publically available annotations in the context of theirown experimental data

Improved annotation of 3-prime untranslated regions and complex loci by combination of strand-specific Direct RNA Sequencing, RNA-seq and ESTs