Table_2_Dual RNA-seq in filarial nematodes and Wolbachia endosymbionts using RNase H based ribosomal RNA depletion.XLSX

Lymphatic filariasis is caused by parasitic nematodes and is a leading cause of disability worldwide. Many filarial worms contain the bacterium Wolbachia as an obligate endosymbiont. RNA sequencing is a common technique used to study their molecular relationships and to identify potential drug targets against the nematode and bacteria. Ribosomal RNA (rRNA) is the most abundant RNA species, accounting for 80–90% of the RNA in a sample. To reduce sequencing costs, it is necessary to remove ribosomal reads through poly-A enrichment or ribosomal depletion. Bacterial RNA does not contain a poly-A tail, making it difficult to sequence both the nematode and Wolbachia from the same library preparation using standard poly-A selection. Ribosomal depletion can utilize species-specific oligonucleotide probes to remove rRNA through pull-down or degradation methods. While species-specific probes are commercially available for many commonly studied model organisms, there are currently limited depletion options for filarial parasites. Here, we performed total RNA sequencing from Brugia malayi containing the Wolbachia symbiont (wBm) and designed ssDNA depletion probes against their rRNA sequences. We compared the total RNA library to poly-A enriched, Terminator 5′-Phosphate-Dependent Exonuclease treated, NEBNext Human/Bacteria rRNA depleted and our custom nematode probe depleted libraries. The custom nematode depletion library had the lowest percentage of ribosomal reads across all methods, with a 300-fold decrease in rRNA when compared to the total RNA library. The nematode depletion libraries also contained the highest percentage of Wolbachia mRNA reads, resulting in a 16–1,000-fold increase in bacterial reads compared to the other enrichment and depletion methods. Finally, we found that the Brugia malayi depletion probes can remove rRNA from the filarial worm Dirofilaria immitis and the majority of rRNA from the more distantly related free living nematode Caenorhabditis elegans. These custom filarial probes will allow for future dual RNA-seq experiments between nematodes and their bacterial symbionts from a single sequencing library.

淋巴丝虫病（Lymphatic filariasis）由寄生性线虫（parasitic nematodes）引发，是全球范围内导致残疾的主要病因之一。多数丝虫均以沃尔巴克氏体属（Wolbachia）作为专性内共生体（obligate endosymbiont）。RNA测序（RNA sequencing）是解析二者分子互作关系、并针对该线虫与共生细菌筛选潜在药物靶点的常规实验技术。核糖体RNA（ribosomal RNA, rRNA）是样本中丰度最高的RNA类型，占总RNA的80%~90%。为降低测序成本，需通过poly-A富集（poly-A enrichment）或核糖体去除（ribosomal depletion）手段剔除核糖体读段（reads）。细菌RNA不具备poly-A尾，因此采用标准poly-A筛选法时，无法在同一文库制备流程中同时完成线虫与沃尔巴克氏体的测序。核糖体去除技术可借助物种特异性寡核苷酸探针，通过亲和下拉或降解方式移除rRNA。尽管目前市面上已有多款适配多数常见模式生物的商用物种特异性探针，但针对丝虫寄生虫的核糖体去除方案仍较为匮乏。本研究从携带沃尔巴克氏体共生体（wBm）的马来布鲁线虫（Brugia malayi）中提取总RNA并开展测序，同时针对其rRNA序列设计了单链DNA（ssDNA）去除探针。我们将总RNA文库与poly-A富集文库、经Terminator 5′-磷酸依赖性外切酶（Terminator 5′-Phosphate-Dependent Exonuclease）处理的文库、NEBNext 人类/细菌rRNA去除文库，以及本研究定制的线虫探针去除文库进行了对比分析。定制线虫去除文库的核糖体读段占比在所有方法中最低，相较于总RNA文库，其rRNA丰度降低了300倍。同时，线虫去除文库中的沃尔巴克氏体mRNA读段占比最高，与其他富集或去除方法相比，细菌读段的丰度提升了16~1000倍。最后，本研究证实，马来布鲁线虫去除探针不仅可移除犬恶丝虫（Dirofilaria immitis）的rRNA，还能去除亲缘关系更远的自由生活线虫——秀丽隐杆线虫（Caenorhabditis elegans）的大部分rRNA。这套定制丝虫探针将为后续基于单一测序文库开展线虫与其细菌共生体的双RNA测序（dual RNA-seq）实验提供有力支持。