A full-length cDNA resource for the pea aphid, Acyrthosiphon pisum

Abstract

Large collections of full-length cDNAs are important resources for genome annotation and functional genomics. We report the creation of a collection of 50 599 full-length cDNA clones from the pea aphid, Acyrthosiphon pisum. Sequencing from 5' and 3' ends of the clones generated 97 828 high-quality expressed sequence tags, representing approximately 9000 genes. These sequences were imported to AphidBase and are shown to play crucial roles in both automatic gene prediction and manual annotation. Our detailed analyses demonstrated that the full-length cDNAs can further improve gene models and can even identify novel genes that are not included in the current version of the official gene set. This full-length cDNA collection can be utilized for a wide variety of functional studies, serving as a community resource for the study of the functional genomics of the pea aphid.

Figure 1. Size distribution of isolated cDNA inserts

Insert size of 96 randomly-selected clones were examined by colony PCR followed by agarose gel electrophoresis. Average insert size was 2.1 kb, with a range of 0.3 to 7.4kb.

Figure 2. Graphical representation of full-length cDNA ESTs on the genome sequence

ESTs from the full-length cDNA library were aligned to the 14-3-3epsilon locus on the genomic scaffold EQ122825. For each clone, the members of the EST pair are linked with a dotted line if they do not overlap. 5’-ESTs and 3’-ESTs are colored dark blue and light blue, respectively. For comparison, RefSeq gene model (CDS: brown, UTR: gray), ESTs of non-full-length cDNA (steel blue), EST contigs generated by sequence-based assembling (green) and Drosophila melanogaster orthologs (orange) are shown. Sequence orientations are indicated by arrows.

Figure 3. Distribution of gene lengths for A. pisum and D. melanogaster

Gene length is defined as the span including exons and introns. The gene length distribution for A. pisum was calculated from 7,432 loci for which one or more EST pairs from full-length cDNA clones were available. For D. melanogaster, all transcripts annotated in FlyBase (Release 5.4) were used for the statistics.

Figure 4. Examples of erroneous gene models detected by the full-length cDNA ESTs

(A) illustrates the locus of PTEN (tumor suppressor gene) as an example in which the gene model was mistakenly split into two different models. The 5’- and 3’-members of the EST pair bridge these mistakenly split gene models. (B) illustrates XM_001952817, a case in which the gene model mistakenly contains two different genes. The ESTs clearly indicate a gene boundary between the 5th and 6th exons. Color coding of gene models and ESTs is the same as Figure 2.