Brugia malayi is a causative agent of lymphatic filariasis, a disease which affects 120 million individuals in tropical and subtropical regions of the globe. As the assembly of the 6. malayi genome nears completion, an outstanding task is the full prediction and structural annotation of its genes. A lack of experimental evidence and of full length cDNAs has limited the efficient training of gene finder algorithms. A critical marker in the assessment of gene structure is the determination of the 5' transcriptional start site of each gene. We propose to generate a comprehensive catalog of B. malayi 5' ends by using two novel methods. First, we propose to use a recently developed technique, Trans-spliced Exon Coupled RNA End Determination (TEC-RED), to make a library of concatenated 5' sequence tags representing all SL1 trans-spliced genes in B. malayi. Second, we propose a novel variation of the TEC-RED technique to characterize 5' transcriptional start sites using a modified oligo-capping methodology. In the course of developing this novel protocol we propose to construct and sequence an oligo capped library which will be enriched for complete capped mRNA sequences. The analysis of the 5' sequence of a significant portion of B. malayi mRNAs will provide a number of benefits for the annotation of the B. malayi genome, including determination of transcriptional initiation sites, alternative splice sites and identification of transcriptional start sites of previously unidentified genes. The compilation of B. malayi-full-length mRNAs will also aid in the discovery of novel splice leader sequences. This could potentially lead to the discovery of previously undiscovered operons in 8. malayi. Moreover, the collection of trans-spliced genes will serve as a comparative marker for evolutionary studies in nematodes.
