The former case is particularly problematic because RNA-seq alignersometimeses prefer direct alignments to spliced alignments, causing spuriously aligned reads to accumulate on processed pseudogenes.
The genome annotation tool PASA (Program to Assemble Spliced Alignments) (40 ) clusters aligned sequences, generates gene models and compares these to the existing gene sets.
The PASA-filtered transcripts were then anchored to the genome scaffolds by assembling overlapped transcript alignments into maximal alignment assemblies in order to remove multiple spliced isoforms.
Most genes use alternative splicing to generate multiple spliced transcripts.
Exon-first methods begin by mapping whole reads to the genome using unspliced read aligners, and then search for spliced alignments with the remaining reads.
The spliced alignments are used to identify putative exons, splice junctions and transcripts that explain the alignments.
In addition to that EST data and full-length cDNA, data are being aligned with exonerate or PASA (Program to Assemble Spliced Alignments) (40).
Accuracy of spliced alignments is crucial in such areas as studies of alternative splicing and regulatory elements.
The algorithm reports all of the spliced alignments it finds, and then builds a set of non-redundant splice junctions using these alignments.
Spliced alignments were only considered if 75% (score >0.75) or more bases could be aligned between the genomic sequence and a probe set.
