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Generation, annotation, and analysis of ESTs from midgut tissue of adult female Anopheles stephensi mosquitoes.

Patil DP, Atanur S, Dhotre DP, Anantharam D, Mahajan VS, Walujkar SA, Chandode RK, Kulkarni GJ, Ghate PS, Srivastav A, Dayananda KM, Gupta N, Bhagwat B, Joshi RR, Mourya DT, Patole MS, Shouche YS - BMC Genomics (2009)

Bottom Line: Statistical comparison of annotated and unannotated ESTs from the two libraries identified 119 differentially regulated genes.Out of 3946 unique transcripts, only 1387 transcripts were mapped on the A. gambiae genome.These also included 189 novel transcripts, which were mapped to the unannotated regions of the genome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lab 3, National Center for Cell Science, Pune - 411007, India. deepakpatilp@gmail.com

ABSTRACT

Background: Malaria is a tropical disease caused by protozoan parasite, Plasmodium, which is transmitted to humans by various species of female anopheline mosquitoes. Anopheles stephensi is one such major malaria vector in urban parts of the Indian subcontinent. Unlike Anopheles gambiae, an African malaria vector, transcriptome of A. stephensi midgut tissue is less explored. We have therefore carried out generation, annotation, and analysis of expressed sequence tags from sugar-fed and Plasmodium yoelii infected blood-fed (post 24 h) adult female A. stephensi midgut tissue.

Results: We obtained 7061 and 8306 ESTs from the sugar-fed and P. yoelii infected mosquito midgut tissue libraries, respectively. ESTs from the combined dataset formed 1319 contigs and 2627 singlets, totaling to 3946 unique transcripts. Putative functions were assigned to 1615 (40.9%) transcripts using BLASTX against UniProtKB database. Amongst unannotated transcripts, we identified 1513 putative novel transcripts and 818 potential untranslated regions (UTRs). Statistical comparison of annotated and unannotated ESTs from the two libraries identified 119 differentially regulated genes. Out of 3946 unique transcripts, only 1387 transcripts were mapped on the A. gambiae genome. These also included 189 novel transcripts, which were mapped to the unannotated regions of the genome. The EST data is available as ESTDB at http://mycompdb.bioinfo-portal.cdac.in/cgi-bin/est/index.cgi.

Conclusion: 3946 unique transcripts were successfully identified from the adult female A. stephensi midgut tissue. These data can be used for microarray development for better understanding of vector-parasite relationship and to study differences or similarities with other malaria vectors. Mapping of putative novel transcripts from A. stephensi on the A. gambiae genome proved fruitful in identification and annotation of several genes. Failure of some novel transcripts to map on the A. gambiae genome indicates existence of substantial genomic dissimilarities between these two potent malaria vectors.

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BLASTX based species distribution of UTs. Graph generated using Blast2GO program shows species distribution of UTs from combined dataset using BLASTX against non-redundant protein database. Top 10 BLAST hits were considered for each transcript.
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Figure 1: BLASTX based species distribution of UTs. Graph generated using Blast2GO program shows species distribution of UTs from combined dataset using BLASTX against non-redundant protein database. Top 10 BLAST hits were considered for each transcript.

Mentions: To assign putative function to ESTs and UTs, we performed BLASTX search against the UniProtKB database. Summary of BLASTX results for SF, BF, and combined UTs are shown in Table 2. BLASTX results for combined UTs are given in Additional file 1. Figure 1 shows BLAST hit distribution across various species of organisms for all UTs from the combined dataset. Putative functions were assigned only to 8946 ESTs (58.2%) and 1615 UTs (40.9%) (E-value ≤ 1e-5). Non-coding EST sequences usually fail to find a homolog in the protein databases during BLASTX search [30]. We therefore, screened unannotated (with no BLAST hits) UTs (2331) for the presence of putative coding region using ESTScan program [31]. 1513 UTs were predicted to contain a coding region, thereby suggesting these as novel genes. The remaining sequences could be potential untranslated regions (UTRs). Table 3 shows ESTScan results of both the libraries and combined dataset.


Generation, annotation, and analysis of ESTs from midgut tissue of adult female Anopheles stephensi mosquitoes.

Patil DP, Atanur S, Dhotre DP, Anantharam D, Mahajan VS, Walujkar SA, Chandode RK, Kulkarni GJ, Ghate PS, Srivastav A, Dayananda KM, Gupta N, Bhagwat B, Joshi RR, Mourya DT, Patole MS, Shouche YS - BMC Genomics (2009)

BLASTX based species distribution of UTs. Graph generated using Blast2GO program shows species distribution of UTs from combined dataset using BLASTX against non-redundant protein database. Top 10 BLAST hits were considered for each transcript.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743715&req=5

Figure 1: BLASTX based species distribution of UTs. Graph generated using Blast2GO program shows species distribution of UTs from combined dataset using BLASTX against non-redundant protein database. Top 10 BLAST hits were considered for each transcript.
Mentions: To assign putative function to ESTs and UTs, we performed BLASTX search against the UniProtKB database. Summary of BLASTX results for SF, BF, and combined UTs are shown in Table 2. BLASTX results for combined UTs are given in Additional file 1. Figure 1 shows BLAST hit distribution across various species of organisms for all UTs from the combined dataset. Putative functions were assigned only to 8946 ESTs (58.2%) and 1615 UTs (40.9%) (E-value ≤ 1e-5). Non-coding EST sequences usually fail to find a homolog in the protein databases during BLASTX search [30]. We therefore, screened unannotated (with no BLAST hits) UTs (2331) for the presence of putative coding region using ESTScan program [31]. 1513 UTs were predicted to contain a coding region, thereby suggesting these as novel genes. The remaining sequences could be potential untranslated regions (UTRs). Table 3 shows ESTScan results of both the libraries and combined dataset.

Bottom Line: Statistical comparison of annotated and unannotated ESTs from the two libraries identified 119 differentially regulated genes.Out of 3946 unique transcripts, only 1387 transcripts were mapped on the A. gambiae genome.These also included 189 novel transcripts, which were mapped to the unannotated regions of the genome.

View Article: PubMed Central - HTML - PubMed

Affiliation: Lab 3, National Center for Cell Science, Pune - 411007, India. deepakpatilp@gmail.com

ABSTRACT

Background: Malaria is a tropical disease caused by protozoan parasite, Plasmodium, which is transmitted to humans by various species of female anopheline mosquitoes. Anopheles stephensi is one such major malaria vector in urban parts of the Indian subcontinent. Unlike Anopheles gambiae, an African malaria vector, transcriptome of A. stephensi midgut tissue is less explored. We have therefore carried out generation, annotation, and analysis of expressed sequence tags from sugar-fed and Plasmodium yoelii infected blood-fed (post 24 h) adult female A. stephensi midgut tissue.

Results: We obtained 7061 and 8306 ESTs from the sugar-fed and P. yoelii infected mosquito midgut tissue libraries, respectively. ESTs from the combined dataset formed 1319 contigs and 2627 singlets, totaling to 3946 unique transcripts. Putative functions were assigned to 1615 (40.9%) transcripts using BLASTX against UniProtKB database. Amongst unannotated transcripts, we identified 1513 putative novel transcripts and 818 potential untranslated regions (UTRs). Statistical comparison of annotated and unannotated ESTs from the two libraries identified 119 differentially regulated genes. Out of 3946 unique transcripts, only 1387 transcripts were mapped on the A. gambiae genome. These also included 189 novel transcripts, which were mapped to the unannotated regions of the genome. The EST data is available as ESTDB at http://mycompdb.bioinfo-portal.cdac.in/cgi-bin/est/index.cgi.

Conclusion: 3946 unique transcripts were successfully identified from the adult female A. stephensi midgut tissue. These data can be used for microarray development for better understanding of vector-parasite relationship and to study differences or similarities with other malaria vectors. Mapping of putative novel transcripts from A. stephensi on the A. gambiae genome proved fruitful in identification and annotation of several genes. Failure of some novel transcripts to map on the A. gambiae genome indicates existence of substantial genomic dissimilarities between these two potent malaria vectors.

Show MeSH
Related in: MedlinePlus