High-throughput Analysis of Brugia malayi mRNA 5' ends

马来丝虫是淋巴丝虫病的病原体，淋巴丝虫病影响着全球热带和亚热带地区的1.2亿人。随着6.马来病毒基因组的组装接近完成，一项突出的任务是对其基因进行全面的预测和结构注释。缺乏实验证据和全长cDNA限制了基因搜索器算法的有效训练。评估基因结构的一个重要标志是确定每个基因的5‘转录起始点。我们建议使用两种新的方法来生成一个完整的马来芽胞杆菌5‘末端目录。首先，我们建议使用最近发展的一种技术，反式剪接外显子偶联RNA末端确定(TEC-RED)，以建立代表马来不动杆菌中所有SL1反式剪接基因的串联5‘序列标签文库。其次，我们提出了一种新的TEC-RED技术变体，用于使用改进的寡核苷酸封顶方法来表征5‘转录起始位点。在开发这一新的方案的过程中，我们建议构建一个寡帽文库并对其进行测序，该文库将被丰富为完整的帽状mRNA序列。对马来丝虫mRNAs很大一部分的5‘端序列的分析将为马来丝虫基因组的注释提供许多好处，包括确定转录起始点、选择性剪接位点和鉴定以前未识别的基因的转录起始点。马来芽胞杆菌-全长mRNAs的编辑也将有助于发现新的剪接前导序列。这可能会导致在8.马来发现以前未被发现的操纵子。此外，反式剪接基因的收集将作为线虫进化研究的比较标记。

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.