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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

Ratio of contigs and reference length, using TBLASTX against An. gambiae, Ae. aegypti and An. funestus, expectation of E = 10−3 and raw score of >45, against average contig coverage (number of reads).Ratios away from unity can be considered hits inside gene families or low scoring false positives, only as the minimum alignment length increases can the validity of the assignment be reasonably certain. Each point is coloured according to reference sequence length, and it can be seen there is a general trend for greater than unity assignments to be to short references and less than unity assignments to longer references. Also included are three lines following three reference cut-off lengths of all, 0.8 to 1.8 K and >1.8 K bp.
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pone-0017418-g004: Ratio of contigs and reference length, using TBLASTX against An. gambiae, Ae. aegypti and An. funestus, expectation of E = 10−3 and raw score of >45, against average contig coverage (number of reads).Ratios away from unity can be considered hits inside gene families or low scoring false positives, only as the minimum alignment length increases can the validity of the assignment be reasonably certain. Each point is coloured according to reference sequence length, and it can be seen there is a general trend for greater than unity assignments to be to short references and less than unity assignments to longer references. Also included are three lines following three reference cut-off lengths of all, 0.8 to 1.8 K and >1.8 K bp.

Mentions: The effect of the coverage depth of contigs in the BLASTX analysis is shown in Figure 4 through the ratio of contig and reference lengths. Contigs were compared to An. gambiae, Ae. aegypti and An. funestus using TBLASTX with an expectation of E = 10−3 and raw score of >45. Average contig coverage is the average number of reads that make up the individual contigs. Ratios well below unity can be considered hits inside gene families or low scoring false positives, only as the minimum alignment length increases can the validity of the assignment be reasonably certain. Each point is coloured according to reference sequence length, and it can be seen there is a general trend for greater than unity assignments to be to short references and less than unity assignments to longer references. The ratio of the length of individual contigs to the length of their reference orthologue coding region (An. gambiae, Ae. aegypti and An. funestus) increased with the depth of the coverage of the contig as observed previously in another 454 de novo assembly [8]. Also included are three lines following three reference cut-off lengths of all, 0.8 to 1.8 K and >1.8 K bp. In all three cases this approaches unity when there are at least 10 contributing reads. It is only the average of all reads that is close to unity. Using the larger minimum read length of 150 bases includes only the high quality alignments, and under these circumstances the three plots have come closer together and become flatter. The remaining contig to-reference assignments are much closer to unity while still being references of approximately 1 kbp or longer.


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)

Ratio of contigs and reference length, using TBLASTX against An. gambiae, Ae. aegypti and An. funestus, expectation of E = 10−3 and raw score of >45, against average contig coverage (number of reads).Ratios away from unity can be considered hits inside gene families or low scoring false positives, only as the minimum alignment length increases can the validity of the assignment be reasonably certain. Each point is coloured according to reference sequence length, and it can be seen there is a general trend for greater than unity assignments to be to short references and less than unity assignments to longer references. Also included are three lines following three reference cut-off lengths of all, 0.8 to 1.8 K and >1.8 K bp.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017418-g004: Ratio of contigs and reference length, using TBLASTX against An. gambiae, Ae. aegypti and An. funestus, expectation of E = 10−3 and raw score of >45, against average contig coverage (number of reads).Ratios away from unity can be considered hits inside gene families or low scoring false positives, only as the minimum alignment length increases can the validity of the assignment be reasonably certain. Each point is coloured according to reference sequence length, and it can be seen there is a general trend for greater than unity assignments to be to short references and less than unity assignments to longer references. Also included are three lines following three reference cut-off lengths of all, 0.8 to 1.8 K and >1.8 K bp.
Mentions: The effect of the coverage depth of contigs in the BLASTX analysis is shown in Figure 4 through the ratio of contig and reference lengths. Contigs were compared to An. gambiae, Ae. aegypti and An. funestus using TBLASTX with an expectation of E = 10−3 and raw score of >45. Average contig coverage is the average number of reads that make up the individual contigs. Ratios well below unity can be considered hits inside gene families or low scoring false positives, only as the minimum alignment length increases can the validity of the assignment be reasonably certain. Each point is coloured according to reference sequence length, and it can be seen there is a general trend for greater than unity assignments to be to short references and less than unity assignments to longer references. The ratio of the length of individual contigs to the length of their reference orthologue coding region (An. gambiae, Ae. aegypti and An. funestus) increased with the depth of the coverage of the contig as observed previously in another 454 de novo assembly [8]. Also included are three lines following three reference cut-off lengths of all, 0.8 to 1.8 K and >1.8 K bp. In all three cases this approaches unity when there are at least 10 contributing reads. It is only the average of all reads that is close to unity. Using the larger minimum read length of 150 bases includes only the high quality alignments, and under these circumstances the three plots have come closer together and become flatter. The remaining contig to-reference assignments are much closer to unity while still being references of approximately 1 kbp or longer.

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