High-throughput Analysis of Brugia malayi mRNA 5' ends

马来布鲁吉亚是淋巴丝虫病的病原体，这种疾病影响全球热带和亚热带地区的1.2亿人。6.如装配。马来亚基因组接近完成，一个突出的任务是其基因的全面预测和结构注释。缺乏实验证据和全长cdna限制了基因查找器算法的有效训练。评估基因结构的一个关键标记是确定每个基因的5'转录起始位点。本文提出采用两种新颖的方法，生成马来芽孢杆菌5′末端的综合目录。首先，我们建议使用最近开发的一种技术，反式剪接外显子耦合RNA末端测定（TEC-RED），建立一个连接的5'序列标记库，代表马来马来杆菌中所有SL1反式剪接基因。其次，我们提出了TEC-RED技术的一种新变体，使用改进的寡核苷酸帽盖法来表征5'转录起始位点。在开发这种新方案的过程中，我们建议构建一个寡核苷酸覆盖文库并对其进行测序，该文库将丰富完整的覆盖mRNA序列。马来亚芽孢杆菌mrna的5'序列分析将为马来亚芽孢杆菌基因组的注释提供许多好处，包括确定转录起始位点、替代剪接位点和鉴定先前未识别的基因的转录起始位点。马来芽孢杆菌全长mrna的汇编也将有助于发现新的剪接先导序列。这可能会导致先前未被发现的操作子的发现。马来丝虫。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.