Barai Pallob, Biswas Shishir, Verma Prince, Duncan Elizabeth M
Department of Biology, University of Kentucky, Lexington KY 40506.
bioRxiv. 2024 Jul 21:2024.07.20.604429. doi: 10.1101/2024.07.20.604429.
The overwhelming majority of RNA species isolated from cells or tissues using organic extraction are ribosomal RNAs (rRNA), whereas a relatively small percentage are messenger RNAs (mRNA). For studies that seek to detect mRNA transcripts and measure changes in their expression, this lopsided ratio of desired transcripts to undesired transcripts creates a significant challenge to obtaining sensitive and reproducible results. One method for improving mRNA detection is to selectively amplify polyadenylated (polyA) mRNA molecules when generating RNA-seq libraries, a strategy that is generally very successful in many species. However, this strategy is less effective when starting with total RNA from some species e.g., the planarian species (S.med), as it generates libraries that still contain significant and variable amounts of rRNA reads. Further, commercially available ribodepletion kits do not efficiently deplete rRNAs from these samples because their sequences are divergent from mammalian rRNAs. Here we report a customized, optimized, and economical ribodepletion strategy than allows the generation of comprehensive RNA-seq libraries with less than one percent rRNA contamination. We show that this method improves transcript detection, particularly for those without polyA tails (e.g., core histones) and those that are relatively long (e.g., microtubule motor proteins). Using this custom ribodepletion approach, we also detected many transcripts that are not represented in the most recent set of S.med gene annotations, including a subset that are likely expressed transposable elements (TEs). To facilitate future differential expression analyses of these newly identified loci, we created both an annotation file of the new loci we identified and a bioinformatic pipeline for generating additional annotations from future libraries. As significant recent research shows that TE activation is regulated and functionally important, the resources provided here will provide a starting point for investigating such mechanisms in planarians and other species with less conserved rRNA sequences.
使用有机提取法从细胞或组织中分离得到的绝大多数RNA种类是核糖体RNA（rRNA），而信使RNA（mRNA）的比例相对较小。对于旨在检测mRNA转录本并测量其表达变化的研究而言，所需转录本与非所需转录本的这种不均衡比例给获得灵敏且可重复的结果带来了重大挑战。一种改进mRNA检测的方法是在构建RNA测序文库时选择性扩增多聚腺苷酸化（polyA）的mRNA分子，这一策略在许多物种中通常都非常成功。然而，当从某些物种（如涡虫物种（S.med））的总RNA开始时，该策略效果较差，因为它构建的文库仍包含大量且可变数量的rRNA读数。此外，市售的核糖体去除试剂盒不能有效地从这些样本中去除rRNA，因为它们的序列与哺乳动物rRNA不同。在此，我们报告了一种定制、优化且经济的核糖体去除策略，该策略能够构建rRNA污染低于1%的全面RNA测序文库。我们表明，这种方法改进了转录本检测，特别是对于那些没有polyA尾巴的转录本（如核心组蛋白）和那些相对较长的转录本（如微管运动蛋白）。使用这种定制的核糖体去除方法，我们还检测到了许多在最新的S.med基因注释中未出现的转录本，包括可能表达的转座元件（TE）子集。为了便于对这些新鉴定的基因座进行未来的差异表达分析，我们创建了我们鉴定的新基因座的注释文件以及用于从未来文库生成额外注释的生物信息学管道。由于最近的重要研究表明TE激活受到调控且具有功能重要性，这里提供的资源将为研究涡虫和其他rRNA序列保守性较低的物种中的此类机制提供一个起点。
