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首页> 美国卫生研究院文献>PLoS Clinical Trials >Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™
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Removing the needle from the haystack: Enrichment of Wolbachia endosymbiont transcripts from host nematode RNA by Cappable-seq™

机译：从大海捞针中移除针脚：通过Cappable-seq™从宿主线虫RNA中富集Wolbachia内共生体转录本

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Efficient transcriptomic sequencing of microbial mRNA derived from host-microbe associations is often compromised by the much lower relative abundance of microbial RNA in the mixed total RNA sample. One solution to this problem is to perform extensive sequencing until an acceptable level of transcriptome coverage is obtained. More cost-effective methods include use of prokaryotic and/or eukaryotic rRNA depletion strategies, sometimes in conjunction with depletion of polyadenylated eukaryotic mRNA. Here, we report use of Cappable-seq™ to specifically enrich, in a single step, Wolbachia endobacterial mRNA transcripts from total RNA prepared from the parasitic filarial nematode, Brugia malayi. The obligate Wolbachia endosymbiont is a proven drug target for many human filarial infections, yet the precise nature of its symbiosis with the nematode host is poorly understood. Insightful analysis of the expression levels of Wolbachia genes predicted to underpin the mutualistic association and of known drug target genes at different life cycle stages or in response to drug treatments is typically challenged by low transcriptomic coverage. Cappable-seq resulted in up to ~ 5-fold increase in the number of reads mapping to Wolbachia. On average, coverage of Wolbachia transcripts from B. malayi microfilariae was enriched ~40-fold by Cappable-seq. Additionally, this method has an additional benefit of selectively removing abundant prokaryotic ribosomal RNAs.The deeper microbial transcriptome sequencing afforded by Cappable-seq facilitates more detailed characterization of gene expression levels of pathogens and symbionts present in animal tissues.
机译：混合宿主总RNA样品中微生物RNA相对较低的相对丰度通常会损害源自宿主-微生物结合的微生物mRNA的高效转录组测序。解决该问题的一种方法是进行大量测序，直到获得可接受水平的转录组覆盖率。更具成本效益的方法包括使用原核和/或真核rRNA去除策略，有时与多聚腺苷酸化的真核mRNA结合使用。在这里，我们报道了使用Cappable-seq™可以从寄生丝虫线虫马来虫（Brugia malayi）制备的总RNA中一步特异性富集Wolbachia内细菌mRNA转录物。专心的沃尔巴氏菌内共生菌是许多人类丝虫感染的行之有效的药物靶标，但人们对它与线虫宿主共生的确切性质了解甚少。低转录组覆盖率通常会挑战对Wolbachia基因的表达水平进行深入的分析，而Wolbachia基因在不同的生命周期阶段或响应药物治疗时将支持相互关联，并已知药物靶标基因。 Cappable-seq导致映射到Wolbachia的读取数最多增加约5倍。平均而言，来自Cappable-seq的马来亚微丝虫的Wolbachia转录本的覆盖率约为40倍。此外，该方法还具有选择性去除大量原核生物核糖体RNA的额外好处.Cappable-seq提供的更深层的微生物转录组测序有助于更详细地表征动物组织中病原体和共生体的基因表达水平。

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