Annotation of Two Large Contiguous Regions from the Haemonchus contortus Genome Using RNA-seq and Comparative Analysis with Caenorhabditis elegans

The genomes of numerous parasitic nematodes are currently being sequenced, but their complexity and size, together with high levels of intra-specific sequence variation and a lack of reference genomes, makes their assembly and annotation a challenging task. Haemonchus contortus is an economically significant parasite of livestock that is widely used for basic research as well as for vaccine development and drug discovery. It is one of many medically and economically important parasites within the strongylid nematode group. This group of parasites has the closest phylogenetic relationship with the model organism Caenorhabditis elegans, making comparative analysis a potentially powerful tool for genome annotation and functional studies. To investigate this hypothesis, we sequenced two contiguous fragments from the H. contortus genome and undertook detailed annotation and comparative analysis with C. elegans. The adult H. contortus transcriptome was sequenced using an Illumina platform and RNA-seq was used to annotate a 409 kb overlapping BAC tiling path relating to the X chromosome and a 181 kb BAC insert relating to chromosome I. In total, 40 genes and 12 putative transposable elements were identified. 97.5% of the annotated genes had detectable homologues in C. elegans of which 60% had putative orthologues, significantly higher than previous analyses based on EST analysis. Gene density appears to be less in H. contortus than in C. elegans, with annotated H. contortus genes being an average of two-to-three times larger than their putative C. elegans orthologues due to a greater intron number and size. Synteny appears high but gene order is generally poorly conserved, although areas of conserved microsynteny are apparent. C. elegans operons appear to be partially conserved in H. contortus. Our findings suggest that a combination of RNA-seq and comparative analysis with C. elegans is a powerful approach for the annotation and analysis of strongylid nematode genomes.

目前诸多寄生线虫的基因组正处于测序进程中，但受限于其基因组复杂度与规模较高、存在高水平的种内序列变异，且缺乏参考基因组，导致其基因组组装与注释工作极具挑战性。捻转血矛线虫（Haemonchus contortus）是一类具有重要经济危害的家畜寄生虫，被广泛应用于基础研究、疫苗开发与药物研发领域。它隶属于圆线虫类群（strongylid nematode group）中诸多兼具医学与经济重要性的寄生虫之列。该类群寄生虫与模式生物秀丽隐杆线虫（Caenorhabditis elegans）的系统发育关系最为密切，这使得比较分析成为基因组注释与功能研究中极具潜力的有效手段。为验证这一假说，本研究对捻转血矛线虫基因组中的两段连续片段进行了测序，并针对其开展了精细注释，同时与秀丽隐杆线虫进行了比较分析。本研究采用Illumina测序平台对成年捻转血矛线虫的转录组进行了测序，并借助RNA测序（RNA-seq）完成了两项注释任务：一是针对与X染色体相关的409 kb细菌人工染色体（BAC）叠连群重叠路径，二是针对与I号染色体相关的181 kb BAC插入片段。本次研究共鉴定出40个基因与12个推定转座因子。其中，97.5%的注释基因可在秀丽隐杆线虫中检测到同源基因，且60%的同源基因为推定直系同源基因，这一比例显著高于此前基于表达序列标签（EST）分析得到的结果。捻转血矛线虫的基因密度似乎低于秀丽隐杆线虫，其注释基因的平均长度为秀丽隐杆线虫对应推定直系同源基因的2至3倍，这一差异源于前者基因拥有更多且更长的内含子。二者基因组的共线性（synteny）水平整体较高，但基因的排列顺序通常保守性较差，不过仍存在部分保守的微共线性区域。此外，秀丽隐杆线虫的操纵子在捻转血矛线虫中呈现出部分保守性。本研究结果表明，将RNA测序与秀丽隐杆线虫的比较分析相结合，是开展圆线虫类群基因组注释与分析的高效策略。