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Background    Anopheles funestus  is one of the primary vectors of human malaria, which causes a million deaths each year in sub-Saharan Africa. Few scientific resources are available to facilitate studies of this mosquito species and relatively little is known about its basic biology and evolution, making development and implementation of novel disease control efforts more difficult. The  An. funestus  genome has not been sequenced, so in order to facilitate genome-scale experimental biology, we have sequenced the adult female transcriptome of  An. funestus  from a newly founded colony in Burkina Faso, West Africa, using the Illumina GAIIx next generation sequencing platform.    Methodology/Principal Findings  We assembled short Illumina reads  de novo  using a novel approach involving iterative  de novo  assemblies and “target-based” contig clustering. We then selected a conservative set of 15,527 contigs through comparisons to four Dipteran transcriptomes as well as multiple functional and conserved protein domain databases. Comparison to the  Anopheles gambiae  immune system identified 339 contigs as putative immune genes, thus identifying a large portion of the immune system that can form the basis for subsequent studies of this important malaria vector. We identified 5,434 1∶1 orthologues between  An. funestus  and  An. gambiae  and found that among these 1∶1 orthologues, the protein sequence of those with putative immune function were significantly more diverged than the transcriptome as a whole. Short read alignments to the contig set revealed almost 367,000 genetic polymorphisms segregating in the  An. funestus  colony and demonstrated the utility of the assembled transcriptome for use in RNA-seq based measurements of gene expression.    Conclusions/Significance  We developed a pipeline that makes  de novo  transcriptome sequencing possible in virtually any organism at a very reasonable cost ($6,300 in sequencing costs in our case). We anticipate that our approach could be used to develop genomic resources in a diversity of systems for which full genome sequence is currently unavailable. Our  An. funestus  contig set and analytical results provide a valuable resource for future studies in this non-model, but epidemiologically critical, vector insect.

De Novo Transcriptome Sequencing in Anopheles funestus Using Illumina RNA-Seq Technology