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A de novo expression profiling of Anopheles funestus, malaria vector in Africa, using 454 pyrosequencing.

Gregory R, Darby AC, Irving H, Coulibaly MB, Hughes M, Koekemoer LL, Coetzee M, Ranson H, Hemingway J, Hall N, Wondji CS - PLoS ONE (2011)

Bottom Line: In total 20.8% of all reads were novel when compared to reference databases.Gene conservation analysis confirmed the close phylogenetic relationship between An. funestus and An. gambiae.This study represents a significant advance for the genetics and genomics of An. funestus since it provides an extensive set of both Expressed Sequence Tags (ESTs) and SNPs which can be readily adopted for the design of new genomic tools such as microarray or SNP platforms.

View Article: PubMed Central - PubMed

Affiliation: University of Liverpool, School of Biological Sciences, Cornwall House, United Kingdom.

ABSTRACT

Background: Anopheles funestus is one of the major malaria vectors in Africa and yet there are few genomic tools available for this species compared to An. gambiae. To start to close this knowledge gap, we sequenced the An. funestus transcriptome using cDNA libraries developed from a pyrethroid resistant laboratory strain and a pyrethroid susceptible field strain from Mali.

Results: Using a pool of life stages (pupae, larvae, adults: females and males) for each strain, 454 sequencing generated 375,619 reads (average length of 182 bp). De novo assembly generated 18,103 contigs with average length of 253 bp. The average depth of coverage of these contigs was 8.3. In total 20.8% of all reads were novel when compared to reference databases. The sequencing of the field strain generated 204,758 reads compared to 170,861 from the insecticide resistant laboratory strain. The contigs most differentially represented in the resistant strain belong to the P450 gene family and cuticular genes which correlates with previous studies implicating both of these gene families in pyrethroid resistance. qPCR carried out on six contigs indicates that these ESTs could be suitable for gene expression studies such as microarray. 31,000 sites were estimated to contain Single Nucleotide Polymorphisms (SNPs) and analysis of SNPs from 20 contigs suggested that most of these SNPs are likely to be true SNPs. Gene conservation analysis confirmed the close phylogenetic relationship between An. funestus and An. gambiae.

Conclusion: This study represents a significant advance for the genetics and genomics of An. funestus since it provides an extensive set of both Expressed Sequence Tags (ESTs) and SNPs which can be readily adopted for the design of new genomic tools such as microarray or SNP platforms.

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Related in: MedlinePlus

Venn diagram showing distribution of similarity search results.Numbers are the sum of unique contigs matching An. gambiae, Ae. aegypti and An. funestus and given with their relative percentage.
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pone-0017418-g001: Venn diagram showing distribution of similarity search results.Numbers are the sum of unique contigs matching An. gambiae, Ae. aegypti and An. funestus and given with their relative percentage.

Mentions: The Roche 454 FLX platform produced 375,619 reads totalling 68,308,429 bp with an average read length of 182 bp similar to the 197 bp observed for the butterfly Meliteae cinxia [8]. One quarter of the reads was between 237 bp and 277 bp in length and 50% of the reads between 185 to 285 bp in length. This dataset was trimmed using in-house tools to 213,410 reads of >30 bp in length and totaling 40,078,792 bp with an average read length of 188 bp. The reads were assembled with Mira 2.9.15 [9] to create 18,103 contigs (average length of 253 bp) using 149,406 reads, of which 1,039 contigs were at least 500 bp in length, similar to that obtained by 454 pyrosequencing in other insects such as M. cinxia [8] and the six-spot burnet moth, Zygaena filipendulae [10]. The average depth of coverage of these contigs (number of reads assembled into a contig) was 8.3 similar to that of M. cinxia [8]. The sequencing reads have been deposited to NCBI's SRA database with the assembly output (Accession number: SRA009034) and contig sequences have been submitted to the Transcriptome Shotgun Assembly sequence database (TSA) of NCBI (accession number: EZ915182 - EZ933284). A comparison of the 18,103 An. funestus 454 contigs to An. gambiae and Aedes aegypti total transcript sets from Vectorbase (with TBLASTX, E = 10−3) as well as previously published An. funestus ESTs from Genbank showed a large degree of overlap between these species (Figure 1). 41.2% (7,471) of the 18,103 contigs match an EST in these three transcriptomes. 65% (1855 ESTs) of the 2,846 already published An. funestus ESTs (using SANGER sequencing) was represented in this 454 transcriptome. As expected, this percentage was lower for An. gambiae (49.7%) and Ae. aegypti (32.2%). The percentage of annotated An. funestus ESTs (41.2%) obtained in this analysis is similar to that observed in other large assembled EST data sets of non-mammalian species suggesting that the un-annotated contigs and even the singletons could represent a substantial portion of An. funestus transcriptome [8], [10].


A de novo expression profiling of Anopheles funestus, malaria vector in Africa, using 454 pyrosequencing.

Gregory R, Darby AC, Irving H, Coulibaly MB, Hughes M, Koekemoer LL, Coetzee M, Ranson H, Hemingway J, Hall N, Wondji CS - PLoS ONE (2011)

Venn diagram showing distribution of similarity search results.Numbers are the sum of unique contigs matching An. gambiae, Ae. aegypti and An. funestus and given with their relative percentage.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045460&req=5

pone-0017418-g001: Venn diagram showing distribution of similarity search results.Numbers are the sum of unique contigs matching An. gambiae, Ae. aegypti and An. funestus and given with their relative percentage.
Mentions: The Roche 454 FLX platform produced 375,619 reads totalling 68,308,429 bp with an average read length of 182 bp similar to the 197 bp observed for the butterfly Meliteae cinxia [8]. One quarter of the reads was between 237 bp and 277 bp in length and 50% of the reads between 185 to 285 bp in length. This dataset was trimmed using in-house tools to 213,410 reads of >30 bp in length and totaling 40,078,792 bp with an average read length of 188 bp. The reads were assembled with Mira 2.9.15 [9] to create 18,103 contigs (average length of 253 bp) using 149,406 reads, of which 1,039 contigs were at least 500 bp in length, similar to that obtained by 454 pyrosequencing in other insects such as M. cinxia [8] and the six-spot burnet moth, Zygaena filipendulae [10]. The average depth of coverage of these contigs (number of reads assembled into a contig) was 8.3 similar to that of M. cinxia [8]. The sequencing reads have been deposited to NCBI's SRA database with the assembly output (Accession number: SRA009034) and contig sequences have been submitted to the Transcriptome Shotgun Assembly sequence database (TSA) of NCBI (accession number: EZ915182 - EZ933284). A comparison of the 18,103 An. funestus 454 contigs to An. gambiae and Aedes aegypti total transcript sets from Vectorbase (with TBLASTX, E = 10−3) as well as previously published An. funestus ESTs from Genbank showed a large degree of overlap between these species (Figure 1). 41.2% (7,471) of the 18,103 contigs match an EST in these three transcriptomes. 65% (1855 ESTs) of the 2,846 already published An. funestus ESTs (using SANGER sequencing) was represented in this 454 transcriptome. As expected, this percentage was lower for An. gambiae (49.7%) and Ae. aegypti (32.2%). The percentage of annotated An. funestus ESTs (41.2%) obtained in this analysis is similar to that observed in other large assembled EST data sets of non-mammalian species suggesting that the un-annotated contigs and even the singletons could represent a substantial portion of An. funestus transcriptome [8], [10].

Bottom Line: In total 20.8% of all reads were novel when compared to reference databases.Gene conservation analysis confirmed the close phylogenetic relationship between An. funestus and An. gambiae.This study represents a significant advance for the genetics and genomics of An. funestus since it provides an extensive set of both Expressed Sequence Tags (ESTs) and SNPs which can be readily adopted for the design of new genomic tools such as microarray or SNP platforms.

View Article: PubMed Central - PubMed

Affiliation: University of Liverpool, School of Biological Sciences, Cornwall House, United Kingdom.

ABSTRACT

Background: Anopheles funestus is one of the major malaria vectors in Africa and yet there are few genomic tools available for this species compared to An. gambiae. To start to close this knowledge gap, we sequenced the An. funestus transcriptome using cDNA libraries developed from a pyrethroid resistant laboratory strain and a pyrethroid susceptible field strain from Mali.

Results: Using a pool of life stages (pupae, larvae, adults: females and males) for each strain, 454 sequencing generated 375,619 reads (average length of 182 bp). De novo assembly generated 18,103 contigs with average length of 253 bp. The average depth of coverage of these contigs was 8.3. In total 20.8% of all reads were novel when compared to reference databases. The sequencing of the field strain generated 204,758 reads compared to 170,861 from the insecticide resistant laboratory strain. The contigs most differentially represented in the resistant strain belong to the P450 gene family and cuticular genes which correlates with previous studies implicating both of these gene families in pyrethroid resistance. qPCR carried out on six contigs indicates that these ESTs could be suitable for gene expression studies such as microarray. 31,000 sites were estimated to contain Single Nucleotide Polymorphisms (SNPs) and analysis of SNPs from 20 contigs suggested that most of these SNPs are likely to be true SNPs. Gene conservation analysis confirmed the close phylogenetic relationship between An. funestus and An. gambiae.

Conclusion: This study represents a significant advance for the genetics and genomics of An. funestus since it provides an extensive set of both Expressed Sequence Tags (ESTs) and SNPs which can be readily adopted for the design of new genomic tools such as microarray or SNP platforms.

Show MeSH
Related in: MedlinePlus