Fourteen BAC clones from an Ae. aegypti genomic library were isolated using PCR primers specific to single-copy genetic markers 9. Shotgun sequences from each BAC were assembled into scaffolds using both the TIGR assembler 2 and Seqman 10. Scaffolds resulting from the different methods of assembly (see Materials and methods) were consistent with the others. Mate-pair inconsistencies were usually from sequences that were in repeat regions of the scaffolds. A small number of single-copy chimeric clones were observed and their elimination, along with other mate-pair inconsistencies, did not change the assembled sequences.

The majority of sequence gaps were filled using primers designed to the unique sequence flanking gaps. Some primers designed to close these gaps did not produce any PCR products and sequencing reactions with these primers using the BAC clones as template terminated at the same region or were unreadable due to polymerase slippage. All remaining gaps in the 14 BACs were flanked by highly repetitive sequence. Assembled BAC sequences were compared with the genome assembly (BLASTN) to see if they assembled in a similar manner. The gaps present in the BAC clone assemblies were either coincident with gaps present in the genome assembly or sequence diverged in the genome assembly when gaps were not present in the same region (as discussed below).

Contigs from each BAC clone were oriented on the basis of end sequences and mate-pairs. Three BACs (BAC4, BAC7 and BAC8) were each assembled into continuous sequences with no gaps. The remaining BAC sequences assembled into sets of oriented scaffolds with gaps (arbitrarily replaced by 100 Ns) (Table 1). The only BAC clones that showed differences with the assembly made at TIGR were BAC8 and BAC9. Assembled contigs seem to have been mixed during their assembly and a careful assembly (using Seqman) separated the two BAC clones into their respective scaffolds. This was verified with PCR spanning gaps and comparison to the genome assembly. The 14 BAC assemblies totaled 1,571,625 bp (approx 0.12% of the 1.3 Gb genome). The average G+C content of all scaffolds was 37.75%. Although all the sequences had a G+C around the average, BAC3 had the lowest at 27% and BAC2 had the highest at 47% (see Table 1).

Repeat masking resulted in the masking of approximately 20% of the sequence. As repeat masking here was based on protein homology, the total sequence consisting of transposon sequence is likely to be higher. Manual annotation and similarity searches with in silico predictions and EST hits with transcribed transposon sequences increased the repeat/transposon content to approximately 35%. The Feilai element 11 was the most common element, comprising approximately 38% of the repeats. Almost all transposons identified were retrotransposons.

In silico gene prediction was performed initially on the raw assembled scaffolds. A preliminary (BLASTX) analysis of these predicted transcripts (data not shown) demonstrated that there was a significant amount of over-prediction, gene-splitting and incorporation of random and transposon-based ORFs into gene models. Masking of repeat sequences before gene prediction reduced the number of gene models and this dataset was used as evidence for manual annotation.

Gene models predicted by Genscan 12 and FGENESH 13 before repeat masking often included exons derived from transposon ORFs. An Aedes gene was often split into two predictions, with the incorporation of unmasked transposon-based and other random ORFs. In addition, the ab initio generated sets of gene models (by Genscan and FGENESH) were different. However, some predicted exons did match Aedes ESTs. Several hundred ESTs were identified from the Aedes database (e < -100) as well as from the Drosophila and Anopheles datasets (e < -50). A preliminary BLAST analysis of Aedes ESTs (e = 0.0) demonstrated that a large portion of them (around 30%) mapped to transposon ORFs.

The 14 BACs were manually annotated in Apollo 14 using various tiers of evidence like ESTs and comparison to other dipteran peptides (see Materials and methods). Transcripts from the Anopheles and Drosophila genomes were used in conjunction with Aedes ESTs to limit the number of exons to those that had similarity to gene models in the other dipteran genomes. Annotations that did not possess similarity to the two dipteran genomes were also analyzed to include ORFs that may be specific to the Aedes genome as well as those that may have diverged significantly from their Anopheles or Drosophila homologs.

There were a total of 51 manual annotations (Table 2) among the 14 BAC sequences, with BAC2 having no annotated transcripts. Fifty of 51 manual annotations were found in the Ae. aegypti 1.0 Genebuild (AaegL1.1) 4 and 41 of these were identical (see Table 2). The remaining varied in several ways including differences in the 3' or 5' exon (seven transcripts), different intron/exon structure (two transcripts) or the annotation was missing in that region of the genome (one transcript). In all cases, the differences in the manually annotated models were based on Aedes EST comparisons, comparisons to annotations and ESTs in the Drosophila and Anopheles genome as well as confirmation by sequencing of PCR amplicons in a few cases. A number of transcripts differed in the length of the 3' or 5 ' UTRs. These differences were usually 10-20 bp long and not considered discordant with the gene build unless they differed by entire exons. All annotations had nucleotide matches in the Aedes genome and most had hits to Aedes ESTs. The genomic region encompassing BAC11 had two extra transcripts (AAEL03517 and AAEL02535). A protein comparison revealed that both genome-annotated transcripts were exons from a rhabdovirus nucleocapsid protein. These were not included in the list of manual annotations.

To confirm the annotation and expression of a subset of these annotations, primers were designed to all manually annotated transcripts where the prediction lacked necessary evidence. PCR was performed on cDNA obtained from all stages of the mosquito (see Materials and methods). These sequences were utilized to correct or confirm manual annotations when the curator presented multiple possible gene models or splice sites for a particular sequence. All 20 amplicons sequenced were identical to a curated gene model (see Table 2).

Eight of the 14 BAC clones had annotations present more than once in the genome assembly. This was unexpected as these BACs were specifically isolated using validated single-locus genetic markers 9. These replicated transcripts present in AaeL1.1 were virtually identical and usually present along with the same flanking transcripts in different supercontigs. To see if intergenic sequence were also replicated, the assembled BAC scaffolds were compared to the Aedes genome assembly scaffolds containing the identical transcripts. Though replicated transcripts were virtually identical, intergenic/intron sequences were usually identical on one replicate while they varied slightly on the other. These eight blocks of sequence were present in complete or partially replicated segments in different parts of the Aedes genome assembly, with only one replicate possessing identical intergenic sequence and the rest having slightly variable intergenic sequences.

Some replicated blocks were 'hybrids' of the BAC clone and the genomic duplication. This is seen in BAC14, where all five transcripts are found on two supercontigs in the same order and structure. Intergenic sequences from the first two transcripts are identical to that on supercont1.140 while the remaining transcripts have intergenic sequences corresponding to that on supercont1.146. This is also seen with BAC9, where the last transcript and its intergenic sequence are found on one scaffold while the remaining transcripts and their intergenic sequnce correspond to another scaffold - even though all transcripts are found on both scaffolds.

BAC1 was the most complicated with the five transcripts, being found on four supercontigs. All transcripts were seen in supercont1.789 while the remaining usually terminated at the end of a scaffold or had gaps which did not include all transcripts. These three transcripts were also seen with different intergenic sequences on supercont1.1137. The fourth transcript had the 3' end matching up to this scaffold and the 5' end on supercont1.393. The fifth transcript was found on supercont1.1393, whereas a sixth transcript with identical intergenic sequence was not found in the genome, although transcripts matching it but with varying intergenic sequence were found. These replicated regions were usually flanked by highly repetitive DNA and/or gaps or were present at the end of a supercontig.

When compared with the Anopheles and Drosophila gene sets (Table 3), 50 and 43 Aedes transcript annotations had orthologous transcripts in the Anopheles and Drosophila gene sets, respectively. The genes from the two other dipteran genomes that were similar to the manual annotations were almost always orthologs of each other (determined by reciprocal BLASTs) 4. Although most Aedes annotations had a one-to-one relationship in the other genomes, some matches were to genes from multigene families. In some cases, the primary BLAST match was much better than the rest and in these cases, an ortholog was postulated. In cases where a number of transcripts matched the manual annotation with similar e-values, orthologs could not be predicted. A single manual annotation did not have any similarity in either genome, and when compared to other dipteran datasets with less stringent parameters it demonstrated similarity to an Ae. albopictus salivary protein.