Department of Molecular Biology, The Laboratory for Molecular Infection Medicine Sweden (MIMS), Science for Life Laboratory, Umeå Centre for Microbial Research (UCMR), Umeå University, Umeå, 90187, Sweden.
Department of Medical Biochemistry and Biophysics, Umeå University, Umeå, 90736, Sweden.
BMC Genomics. 2024 Jan 2;25(1):6. doi: 10.1186/s12864-023-09924-y.
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.
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.
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.
微孢子虫是一类具有高度流线型基因组的细胞内病原体，其基因组序列具有极高的差异性和许多物种特异性的适应性。这些独特的因素给基于序列相似性的传统基因组注释方法带来了挑战。因此，迄今为止测序的许多微孢子虫基因组中包含许多未知功能的基因。快速准确的结构预测和比较的最新创新，以及结构数据库中不断增长的数据量，为辅助新测序基因组的功能注释提供了新的机会。
在这项研究中，我们建立了一个工作流程，该流程结合了基于序列和结构的功能基因注释方法，使用了 ChimeraX 插件命名为 ANNOTEX（ChimeraX 的注释扩展），允许进行可视化检查和手动编辑。我们将此工作流程应用于高质量端粒到端粒测序的四倍体 Vairimorpha necatrix 基因组。首先，使用 3080 个预测的编码蛋白 DNA 序列作为输入，其中 89% 用 RNA 测序数据进行了验证。接下来，使用 ColabFold 来创建蛋白质结构预测，然后使用 Foldseek 搜索与 PDB 和 AlphaFold 数据库的结构匹配。随后的基于序列和结构的命中的手动编辑，与仅使用传统注释工具相比，提高了功能基因组注释的准确性和质量。我们的工作流程对 V. necatrix 基因组进行了全面描述，并对最常见的蛋白质组（如蓖麻毒素 B 凝集素家族）进行了结构总结。此外，为了测试我们的工具，我们鉴定了几个以前未表征的 Encephalitozoon cuniculi 基因的功能。
我们为具有差异的生物体提供了一种新的功能注释工具，并将其应用于新测序的高质量微孢子虫基因组，以揭示这种未表征的鳞翅目昆虫的细胞内病原体。基于结构的注释方法的添加可以作为研究其他微孢子虫或类似差异物种的有价值模板。
