Figure 4.

Validation of ReAnoCDS05 predictions by RT-PCR. Differences between ReAnoCDS05 and Ensembl CDS predictions were experimentally tested by RT-PCR using A. gambiae cDNA or gDNA as templates. The left side of each panel is a map of CDS predictions and supporting lines of evidence, and the right side is a reverse-color image of, from left to right lanes, PhiX/Lambda DNA size standard (Ph), 250 bp DNA ladder (La), and PCR performed on either cDNA (cD), or gDNA template (gD). (a-e) Five cases of potential annotation difference were tested (described in Results); (f) control to test for gDNA contamination of cDNA using primers in two predicted introns to amplify across the intervening exon. In each case except the control, the ReAnoCDS05 and Ensembl annotations made different predictions for the RT-PCR result using cDNA template (in all cases gDNA was the positive control), as follows: (a) ReAnoCDS05 predicted 815 bp, Ensembl predicted no product, RT-PCR estimated 815 bp; (b) ReAnoCDS05 predicted 241 bp, Ensembl predicted no product, RT-PCR estimated 241 bp; (c) ReAnoCDS05 predicted 1,555 bp, Ensembl predicted no product, RT-PCR estimated 1,555 bp; (d) ReAnoCDS05 predicted 1,822 bp, Ensembl predicted no product, RT-PCR estimated 1,822 bp; (e) ReAnoCDS05 predicted 1,600 bp, Ensembl predicted no product, RT-PCR estimated 1,600; (f) both ReAnoCDS05 and Ensembl predicted no product, and no product was present. Left panel key: red bars, CDSs from ReAnoCDS05 re-annotation (numbers are ReAnoCDS05 unique IDs); dark green bars, CDSs from Ensembl (with ENSANGT transcript IDs); dark blue bars, CDS from GENSCAN; light blue bars, CDSs from GeneMark; pink bars, CDSs from SNAP; yellow bars, dbEST contigs; light green bars, ESTs from immune-enriched cDNA library [45]. All bars on map depict CDSs only, except EST and SNAP, which may also contain UTR sequences. Small gray arrowheads indicate the locations of primers used for verification of CDS structure. Ensembl nucleotide coordinates are shown for the indicated chromosomes.