背景:微孢子虫是细胞内病原体的大型分类群,其特征是具有异常高的序列差异和许多物种特异性适应的异常流线型基因组。这些独特的因素对基于序列相似性的传统基因组注释方法提出了挑战。因此,迄今为止测序的许多微孢子虫基因组包含许多功能未知的基因。最近在快速准确的结构预测和比较方面的创新,加上结构数据库中不断增长的数据量,提供新的机会来协助新测序基因组的功能注释。
结果:在这项研究中,我们建立了一个工作流程,结合了基于序列和结构的功能基因注释方法,采用了名为ANNOTEX(ChimeraX注释扩展)的ChimeraX插件,允许目视检查和手动管理。我们将此工作流程应用于高质量的端粒到端粒测序的四倍体基因组。首先,3080个预测的蛋白质编码DNA序列,其中89%被RNA测序数据证实,被用作输入。接下来,ColabFold用于创建蛋白质结构预测,然后进行Foldseek搜索,以便与PDB和AlphaFold数据库进行结构匹配。随后的手动策展,使用基于序列和结构的命中,与仅使用传统注释工具的结果相比,提高了功能基因组注释的准确性和质量。我们的工作流程导致了对V.necatrix基因组的全面描述,以及最普遍的蛋白质组的结构总结,例如蓖麻毒素B凝集素家族。此外,为了测试我们的工具,我们确定了几个以前未表征的cuniculi头孢菌素基因的功能。
结论:我们为不同的生物体提供了一种新的功能注释工具,高质量的微孢子虫基因组揭示了这种未表征的鳞翅目细胞内病原体。添加基于结构的注释方法可以作为研究其他微孢子虫或类似不同物种的有价值的模板。
BACKGROUND: Microsporidia are a large taxon of intracellular pathogens characterized by extraordinarily streamlined genomes with unusually high sequence divergence and many species-specific adaptations. These unique factors pose challenges for traditional genome annotation methods based on sequence similarity. As a result, many of the microsporidian genomes sequenced to date contain numerous genes of unknown function. Recent innovations in rapid and accurate structure prediction and comparison, together with the growing amount of data in structural databases, provide new opportunities to assist in the functional annotation of newly sequenced genomes.
RESULTS: In this study, we established a workflow that combines sequence and structure-based functional gene annotation approaches employing a ChimeraX plugin named ANNOTEX (Annotation Extension for ChimeraX), allowing for visual inspection and manual curation. We employed this workflow on a high-quality telomere-to-telomere sequenced tetraploid genome of Vairimorpha necatrix. First, the 3080 predicted protein-coding DNA sequences, of which 89% were confirmed with RNA sequencing data, were used as input. Next, ColabFold was used to create protein structure predictions, followed by a Foldseek search for structural matching to the PDB and AlphaFold databases. The subsequent manual curation, using sequence and structure-based hits, increased the accuracy and quality of the functional genome annotation compared to results using only traditional annotation tools. Our workflow resulted in a comprehensive description of the V. necatrix genome, along with a structural summary of the most prevalent protein groups, such as the ricin B lectin family. In addition, and to test our tool, we identified the functions of several previously uncharacterized Encephalitozoon cuniculi genes.
CONCLUSIONS: We provide a new functional annotation tool for divergent organisms and employ it on a newly sequenced, high-quality microsporidian genome to shed light on this uncharacterized intracellular pathogen of Lepidoptera. The addition of a structure-based annotation approach can serve as a valuable template for studying other microsporidian or similarly divergent species.