Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, SA, Australia.
Discipline of Medicine, The University of Adelaide, Adelaide, SA, Australia.
Sci Rep. 2020 Sep 3;10(1):14593. doi: 10.1038/s41598-020-71323-0.
The attachment of unique molecular identifiers (UMIs) to RNA molecules prior to PCR amplification and sequencing, makes it possible to amplify libraries to a level that is sufficient to identify rare molecules, whilst simultaneously eliminating PCR bias through the identification of duplicated reads. Accurate de-duplication is dependent upon a sufficiently complex pool of UMIs to allow unique labelling. In applications dealing with complex libraries, such as total RNA-seq, only a limited variety of UMIs are required as the variation in molecules to be sequenced is enormous. However, when sequencing a less complex library, such as small RNAs for which there is a more limited range of possible sequences, we find increased variation in UMIs are required, even beyond that provided in a commercial kit specifically designed for the preparation of small RNA libraries for sequencing. We show that a pool of UMIs randomly varying across eight nucleotides is not of sufficient depth to uniquely tag the microRNAs to be sequenced. This results in over de-duplication of reads and the marked under-estimation of expression of the more abundant microRNAs. Whilst still arguing for the utility of UMIs, this work demonstrates the importance of their considered design to avoid errors in the estimation of gene expression in libraries derived from select regions of the transcriptome or small genomes.
在 PCR 扩增和测序之前，将独特分子标识符 (UMI) 连接到 RNA 分子上，使得可以将文库扩增到足以识别稀有分子的水平，同时通过识别重复读取来消除 PCR 偏倚。准确的去重依赖于足够复杂的 UMI 池，以允许进行独特的标记。在处理复杂文库的应用中，如总 RNA-seq，由于要测序的分子变化巨大，只需要有限种类的 UMI。但是，当测序更简单的文库时，例如可能的序列范围较小的小 RNA，我们发现需要增加 UMI 的变化，甚至超出专门为测序小 RNA 文库制备而设计的商业试剂盒所提供的变化。我们表明，随机变化跨越八个核苷酸的 UMI 池深度不足以唯一标记要测序的 microRNA。这导致读取的过度去重，以及更丰富的 microRNA 的表达明显低估。尽管仍然支持 UMI 的实用性，但这项工作表明需要考虑它们的设计，以避免在从转录组或小基因组的选定区域衍生的文库中估计基因表达时出现错误。
