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. 2007;8(1):R5.
doi: 10.1186/gb-2007-8-1-r5.

Update of the Anopheles Gambiae PEST Genome Assembly

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Free PMC article

Update of the Anopheles Gambiae PEST Genome Assembly

Maria V Sharakhova et al. Genome Biol. .
Free PMC article

Abstract

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.

Figures

Figure 1
Figure 1
Results of in situ hybridization of cDNA clones to the heterochromatic regions on the polytene chromosomes of A. gambiae. Two cDNA clones were labeled with red Cy3 and blue Cy5 dyes and hybridized to the polytene chromosomes: the red signals indicate the beginning and the blue signals show the end of the scaffolds. The location of the scaffolds, (a) AAAB01008973, (b) AAAB01008961 and (c) AAAB01008971, were indicated by in situ hybridization of the cDNA clones: ANP1272B11, ANP1141F09 (a); ANP1302A01, ANP1344A01 (b) and ANP131B08, ANP121D04 (c) on the chromosome X (a), 2 (b) and 3 (c).
Figure 2
Figure 2
Scaffolds located in pericentromeric regions on A. gambiae chromosomes. Black and red lines and arrows on the left side of the picture correspond to the scaffolds previously and newly mapped to the pericentromeric regions of chromosomes (a) X, (b) 2 and (c) 3, respectively; blue arrows indicate newly oriented scaffolds. The dots on the arrows show the beginning of the scaffolds and the arrowheads correspond to the end of the scaffolds. The scaffolds are identified by the last four digits of the scaffold ID. The scale on the left side of the chromosomes indicates divisions and subdivisions in these regions. Black arrows on the right side of the picture show the location of the PCR amplified gene-fragments and BAC and cDNA clones.
Figure 3
Figure 3
Example of joining scaffolds where adjacent ends are alternative assemblies of the same region. (a) Using physical mapping techniques, scaffolds AAAB01008904 and AAAB0108851 are placed adjacent to one another on chromosome arm 2R. In the previous genome assembly, MOZ2, the scaffolds were placed with an arbitrary 10 kbp of gap between them. (b) After alignment of scaffolds using Exonerate and Dotter, it was clear that there was about 64 kbp of sequence overlap between the 3' end of AAAB01008904 and the 5' end of AAAB0108851. Based on BAC coverage of each scaffold and gaps in each of the scaffold sequences, we chose to keep the overlapping region from AAAB01008904 (base-pairs 1102797 to 1759265) and use it for the new chromosome assembly. (c) The corresponding overlapping region from AAAB0108851 (base-pairs 1 to 635373) was deemed to be an alternative assembly segment, with the rest of the scaffold kept as part of the chromosome assembly. The regions retained as parts of chromosome arm 2R were placed adjacent to each other with no inter- scaffold gap.
Figure 4
Figure 4
A comparison of the initial and updated versions of the Anopheles gambiae genome assembly.(a) The scaffolds from the previous and updated versions of the genome are shown by gray and pink bars, respectively. Purple stripes on the scaffolds indicate alternative haplotype scaffolds with sizes bigger than 50 kbp. Black bars correspond to the BAC clones that cross inter-scaffold gaps. (b) The updated status of the A. gambiae genome project. Sectors correspond to the previously mapped scaffolds, additionally physically mapped scaffolds, alternative haplotype scaffolds, Y-specific scaffolds, bacterial contaminant scaffolds and the remaining scaffolds that are not assigned to the chromosomes.

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