Department of Molecular, Cellular and Developmental Biology, University of California at Santa Cruz, Santa Cruz, CA, USA.
BMC Genomics. 2022 Jul 22;23(1):530. doi: 10.1186/s12864-022-08762-8.
Genome-wide RNA-sequencing technologies are increasingly critical to a wide variety of diagnostic and research applications. RNA-seq users often first enrich for mRNA, with the most popular enrichment method being poly(A) selection. In many applications it is well-known that poly(A) selection biases the view of the transcriptome by selecting for longer tailed mRNA species.
Here, we show that poly(A) selection biases Oxford Nanopore direct RNA sequencing. As expected, poly(A) selection skews sequenced mRNAs toward longer poly(A) tail lengths. Interestingly, we identify a population of mRNAs (> 10% of genes' mRNAs) that are inconsistently captured by poly(A) selection due to highly variable poly(A) tails, and demonstrate this phenomenon in our hands and in published data. Importantly, we show poly(A) selection is dispensable for Oxford Nanopore's direct RNA-seq technique, and demonstrate successful library construction without poly(A) selection, with decreased input, and without loss of quality.
Our work expands the utility of direct RNA-seq by validating the use of total RNA as input, and demonstrates important technical artifacts from poly(A) selection that inconsistently skew mRNA expression and poly(A) tail length measurements.
全基因组 RNA 测序技术对于各种诊断和研究应用越来越重要。RNA-seq 用户通常首先富集 mRNA，最流行的富集方法是 poly(A) 选择。在许多应用中，众所周知，poly(A) 选择通过选择长尾 mRNA 物种来偏向转录组的视图。
在这里，我们表明 poly(A) 选择会影响 Oxford Nanopore 直接 RNA 测序。正如预期的那样，poly(A) 选择会使测序的 mRNAs 偏向更长的 poly(A) 尾巴长度。有趣的是，我们发现由于 poly(A) 尾巴高度可变，有一部分 mRNAs（超过 10%的基因 mRNAs）由于 poly(A) 选择而无法捕获，并且在我们的实验和已发表的数据中证明了这一现象。重要的是，我们表明 poly(A) 选择对于 Oxford Nanopore 的直接 RNA-seq 技术是可有可无的，并且证明了无需 poly(A) 选择、降低输入量且不降低质量即可成功构建文库。
我们的工作通过验证总 RNA 作为输入的用途扩展了直接 RNA-seq 的适用性，并证明了来自 poly(A) 选择的重要技术伪影会不一致地扭曲 mRNA 表达和 poly(A) 尾巴长度测量。
