Preventing Childhood Obesity: Health in the Balance

Background: The genome of Anopheles gambiae, the major vector of malaria, was sequenced and assembled in 2002. This initial genome assembly and analysis made available to the scientific community was complicated by the presence of assembly issues, such as scaffolds with no chromosomal location, no sequence data for the Y chromosome, haplotype polymorphisms resulting in two different genome assemblies in limited regions and contaminating bacterial DNA. Results: Polytene chromosome in situ hybridization with cDNA clones was used to place 15 unmapped scaffolds (sizes totaling 5.34 Mbp) in the pericentromeric regions of the chromosomes and oriented a further 9 scaffolds. Additional analysis by in situ hybridization of bacterial artificial chromosome (BAC) clones placed 1.32 Mbp (5 scaffolds) in the physical gaps between scaffolds on euchromatic parts of the chromosomes. The Y chromosome sequence information (0.18 Mbp) remains highly incomplete and fragmented among 55 short scaffolds. Analysis of BAC end sequences showed that 22 inter-scaffold gaps were spanned by BAC clones. Unmapped scaffolds were also aligned to the chromosome assemblies in silico, identifying regions totaling 8.18 Mbp (144 scaffolds) that are probably represented in the genome project by two alternative assemblies. An additional 3.53 Mbp of alternative assembly was identified within mapped scaffolds. Scaffolds comprising 1.97 Mbp (679 small scaffolds) were identified as probably derived from contaminating bacterial DNA. In total, about 33% of previously unmapped sequences were placed on the chromosomes. Conclusion: This study has used new approaches to improve the physical map and assembly of the A. gambiae genome. Published: 8 January 2007 Genome Biology 2007, 8:R5 (doi:10.1186/gb-2007-8-1-r5) Received: 26 July 2006 Revised: 24 October 2006 Accepted: 8 January 2007 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2007/8/1/R5 Genome Biology 2007, 8:R5 R5.2 Genome Biology 2007, Volume 8, Issue 1, Article R5 Sharakhova et al. http://genomebiology.com/2007/8/1/R5 Background The genome of Anopheles gambiae, the major vector of malaria in Africa, was sequenced by a whole-genome shotgun approach [1]. Physical mapping of the genome was conducted by in situ hybridization of about 2,000 bacterial artificial chromosome (BAC) clones on ovarian nurse cell polytene chromosomes. As a result, in the first publication of the A. gambiae physical map, 67 scaffolds equivalent to 227 megabase-pairs (Mbp) were assigned to chromosomes. Of these, 52 scaffolds were oriented. However, approximately 18% of the assembled A. gambiae genome was represented in scaffolds that did not have a chromosomal location assigned. About 50 Mbp in the assembly were assigned with arbitrary order and orientation to an unmapped chromosome [2]. In this study, new approaches were used to improve the physical map and assembly of the A. gambiae genome. The most poorly mapped parts of the A. gambiae genome were the pericentromeric regions of the chromosomes. These chromosomal regions are made up of highly and moderately repetitive DNA sequences [3,4] that are extremely depleted of genes [5] and form specific heterochromatic structures on chromosomes [6,7]. Pericentromeric heterochromatin plays an important role in many biological processes, such as cell division [8], meiotic pairing [9], regulation of DNA replication and gene expression [10,11], and is generally associated with gene silencing [12,13]. However, the assembly and physical mapping of these regions is a difficult part of any genome project [14-19]. In Drosophila melanogaster, for example, one-third of the 180 Mbp genome is centric heterochromatin; but in the first genome publication only 2% of the sequence reads contained heterochromatic simple sequence repeats [20], and only 3 scaffolds corresponding to 3.8 Mb were mapped in centromeric areas [14]. According to Cot analysis, 33% (about 86 Mbp) of the A. gambiae genome corresponds to repetitive elements [21]. The highest density of repeats is located in pericentromeric regions and forms the completely heterochromatic Y chromosome [22]. In contrast with Drosophila, short simple repeats are not expanded in the A. gambiae genome; therefore, cloning of the heterochromatic portion of the genome was more successful. However, in the first publication of the A. gambiae genome only 9 scaffolds, with a total size of 3.3 Mbp, were mapped to pericentromeric regions on chromosomes [1]. Mapping is difficult because BAC clones representing pericentromeric regions are likely to map to multiple locations due to their high repeat content. In previous work 27 BAC clones hybridized to all centromeric regions on the chromosomes and 116 BAC clones hybridized to pericentromeric regions and multiple locations on the chromosomes [1]. To determine the genomic location of heterochromatic scaffolds, cDNA clones from the Normalized Anopheles Pool (ANGNAP1) library with sequences matching regions of these scaffolds were mapped to the chromosomes. Additionally, this approach was used to orient scaffolds that were not previously oriented. For some scaffolds, PCR amplified DNA of genes predicted in the scaffolds was used for physical