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Molecular tools for studying the major malaria vector Anopheles funestus: improving the utility of the genome using a comparative poly(A) and Ribo-Zero RNAseq analysis

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Weedall, Gareth ORCID: https://orcid.org/0000-0002-8927-1063, Irving, Helen, Hughes, Margaret A. and Wondji, Charles ORCID: https://orcid.org/0000-0003-0791-3673 (2015) 'Molecular tools for studying the major malaria vector Anopheles funestus: improving the utility of the genome using a comparative poly(A) and Ribo-Zero RNAseq analysis'. BMC Genomics, Vol 16, p. 931.

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Abstract

Background
Next-generation sequencing (NGS) offers great opportunities for studying the biology of insect vectors of disease. Prerequisites for successful analyses include high quality annotated genome assemblies and that techniques designed for use with model organisms be tested and optimised for use with these insects. We aimed to test and improve genomic tools for studying the major malaria vector Anopheles funestus.

Results
To guide future RNAseq transcriptomic studies of An. funestus, we compared two methods for enrichment of non-ribosomal RNA for analysis: enrichment of polyadenylated RNA and ribosomal RNA depletion using a kit designed to deplete human/rat/mouse rRNA. We found large differences between the two methods in the resulting transcriptomes, some of which is due to differential representation of polyadenylated and non-polyadenylated transcripts. We used the RNAseq data for validation and targeted manual editing of the draft An. funestus genome annotation, validating 62 % of annotated introns, manually improving the annotation of seven gene families involved in the detoxification of xenobiotics and integrated two published transcriptomic datasets with the recently published genome assembly.

Conclusions
The mRNA enrichment method makes a substantial, replicable difference to the transcriptome composition, at least partly due to the representation of non-polyadenylated transcripts in the final transcriptome. Therefore, great care should be taken in comparing gene expression data among studies. Ribosomal RNA depletion of total RNA using a kit designed to deplete human/rat/mouse rRNA works in mosquitoes and, we argue, results in a truer representation of the transcriptome than poly(A) selection. The An. funestus genome annotation can be considerably improved with the help of these new RNAseq data and further guided manual gene editing efforts will be of great benefit to the Anopheles research community for studies of this insect’s genome and transcriptome.

Item Type: Article
Subjects: QU Biochemistry > Genetics > QU 500 Genetic phenomena
QU Biochemistry > Proteins. Amino Acids. Peptides > QU 58.7 RNA
QX Parasitology > Insects. Other Parasites > QX 515 Anopheles
Faculty: Department: Biological Sciences > Vector Biology Department
Digital Object Identifer (DOI): https://doi.org/10.1186/s12864-015-2114-z
Depositing User: Samantha Sheldrake
Date Deposited: 16 Dec 2015 09:59
Last Modified: 06 Feb 2018 13:11
URI: http://archive.lstmed.ac.uk/id/eprint/5439

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