Validation of Gene Predictions Figure 2. Examples of clusters of similar predicted amino acid sequences (red lines) with MS peptide hits (shown as open black blocks on amino acid sequences) and aligned ESTs (blue lines below amino acid sequence clusters), with EST translation directionality indicated. Grey lines indicate areas where sequences differ and are not aligned. Identifiers are listed on the right. a. Cluster of a TigrScan (TgTigrScan_7144) and Release4 (55.m10274) sequence. The TigrScan sequence has additional 59 and 39 regions (exons) compared to the Release4 sequence. The additional regions are verified by MS hits. There are no TwinScan, Glimmer, or known NR sequences in this MS validated coding region. b. Cluster of a Glimmer, TigrScan , Release4, and TwinScan sequence, all validated by MS peptide hits. The Glimmer sequence (TgGlmHmm_2826) has an additional MS validated 59 region. The TigrScan ( TgTigrScan_4753) sequence does not have an exon that is included in the Glimmer, Release4, and TwinScan sequences and that is supported by both MS and EST data. c. Cluster of a Glimmer (TgGlmHmm_2506) sequence, three TwinScan (TgTwinScan) sequences, and three Release4 sequences. The TwinScan and Release4 methods each predict three distinct proteins from this coding region whereas the Glimmer method predicts one. The distribution and location of the MS peptides on the Glimmer sequence indicates that the full length protein is likely to be present in the proteome. However, the shorter splice variants may also be real proteins. An example of the proteomics data confirming the existence of a continuous portion of the sequence is the group of MS peptides indicated by arrows. doi:10.1371/journal.pone.0003899.g002 PLoS ONE | www.plosone.org 5 December 2008 | Volume 3 | Issue 12 | e3899