Distribution of ESTs and FL-cDNAs within alignment assemblies. The ESTs and FL-cDNAs are non-randomly distributed within alignment assemblies, with relatively small numbers of assemblies containing large numbers of transcript alignments.

Unspliced introns impact on protein products. Unspliced introns have variable effects on translation products. (a) The lack of splicing of the second intron yields a protein of similar length, albeit a different C-terminus. (b) Two different overlapping introns, varying at the donor splice junction, encode an integer number of codons and splicing removes internal segments from the protein. (c) Intron splicing alters the reading frame, providing a different and shorter C-terminus. (d) The lack of splicing truncates the protein sequence due to a stop codon encountered within the unspliced intron sequence.

Examples of annotation updates using alignment assemblies. The types of gene structure updates provided by alignment assemblies are classified into several distinct categories. FL-cDNA containing assemblies are presumed to encode the full-length gene product including UTRs. Existing annotated gene structures can therefore be replaced by gene structures inferred from the FL-cDNA-containing alignment assemblies. Those assemblies lacking FL-cDNAs are presumed to encode only partial gene structures and are stitched into existing annotated gene structures, providing significant alterations to gene structures or simply adding or extending UTR annotations. The protein coding segments of gene structures are shown in red and UTRs are shown in black. Alignment assemblies lacking a FL-cDNA are shown in black, whereas those containing FL-cDNAs are shown in gray. Boundaries consistent with the original gene structure annotation are highlighted in blue.

Five splicing isoforms supported by transcript sequence alignments. The cDNA alignments supporting the five splicing variations identified for the WD-40 repeat gene (At2g32700) are illustrated. For the purpose of comparison, FL-cDNA gi|13605814 is presumed to provide the representative gene structure. EST gi|5842113 contains an unspliced intron within the upstream UTR. EST gi|8688866 provides an alternative AG acceptor splice site within the upstream UTR which extends the spliced transcript length by 3 bp. EST gi|8689273 provides an alternative AG acceptor splice site corresponding to a different upstream UTR exon which removes 3 bp from the spliced transcript length. EST gi|9787494 provides an alternative AG acceptor splice site at a protein coding exon, deleting 6 bp corresponding to two codons of the translated sequence. Only one of the five isoforms encodes a variant protein sequence, while the remainder encode variations restricted to the upstream UTR region.

Pictorial representation of the PASA algorithm. Overlapping Arabidopsis transcript sequence alignments are ordered by their beginning position and assigned indices 0–8 as shown in (a). The matrix shown in (b) provides the ordered alignments along the rows and columns according to their index positions. The predetermined containments (chain links) and incompatibilities (bricks) present obstacles within the matrix, disallowing any direct comparisons between two alignments during L_a or R_a calculations. To compute L_a, each alignment a is compared with all compatible preceding alignments b, generating the value max[C_a, L_b + C_a\b] stored in the upper left of the matrix cell at [row b][column a], with an arrow drawn to the L_b value that yielded the max. L_a is then the maximum upper left value in column a and is shown circled in yellow. After all L_a values are computed, the maximum one is found at [row 7][column 8] and the arrows traced back from it (indicated in red) identify the alignments comprising the maximal assembly (alignments 8, 7, 4 and 0) with their contained alignments (5, 6 and 2). The result is the red assembly 1 shown in (a). An examination of assembly 1 indicates that it lacks alignments 1 and 3. The trace back from the forward scan at L_a where a = 3 provides the maximal assembly containing a originating from the left of a (trace back drawn in blue), but does not identify the alignments to the right of a that are in the a maximal assembly. To find the maximal alignment assembly containing alignment 3, the reverse scan computations were performed, calculating max[C_a, L_b + C_a\b], where b > a, and storing the score in the lower right of cell [row b][column a]. R_a is the maximum lower right value in column a and is shown circled in yellow. The trace forward from R_a, where a = 3, is shown with blue arrows. Combined with the trace back from L_a where a = 3, this yields the maximal assembly containing alignment 3, shown as assembly 2, namely alignments 0–3, 6–8. This assembly is also the maximal assembly containing alignment 1. In general, maximal assemblies for missing alignments are found in order of decreasing L_a + R_a – C_a value until all missing alignments are accounted for within maximal alignment assemblies; this was not done here for brevity. Note that alignment 6 is regarded as contained in alignment 1, even though it extends a few bases into intron 4. PASA has a parameter, called ‘fuzz distance’, which specifies the length of mismatches to discount at transcript ends, where sequence alignment quality is often poor.