Shenzhen Key Laboratory of Gene Regulation and Systems Biology, Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, 518055, China.
BMC Genomics. 2022 Jul 4;23(1):487. doi: 10.1186/s12864-022-08717-z.
Investigating the functions and activities of genes requires proper annotation of the transcribed units. However, transcript assembly efforts have produced a surprisingly large variation in the number of transcripts, and especially so for noncoding transcripts. This heterogeneity in assembled transcript sets might be partially explained by sequencing depth. Here, we used real and simulated short-read sequencing data as well as long-read data to systematically investigate the impact of sequencing depths on the accuracy of assembled transcripts. We assembled and analyzed transcripts from 671 human short-read data sets and four long-read data sets. At the first level, there is a positive correlation between the number of reads and the number of recovered transcripts. However, the effect of the sequencing depth varied based on cell or tissue type, the type of read and the nature and expression levels of the transcripts. The detection of coding transcripts saturated rapidly with both short and long-reads, however, there was no sign of early saturation for noncoding transcripts at any sequencing depth. Increasing long-read sequencing depth specifically benefited transcripts containing transposable elements. Finally, we show how single-cell RNA-seq can be guided by transcripts assembled from bulk long-read samples, and demonstrate that noncoding transcripts are expressed at similar levels to coding transcripts but are expressed in fewer cells. This study highlights the impact of sequencing depth on transcript assembly.
研究基因的功能和活动需要对转录单位进行适当的注释。然而，转录本组装工作产生了转录本数量惊人的变化，尤其是非编码转录本。组装转录本集中的这种异质性部分可以通过测序深度来解释。在这里，我们使用真实和模拟的短读测序数据以及长读数据系统地研究了测序深度对组装转录本准确性的影响。我们从 671 个人类短读数据集和四个长读数据集组装和分析了转录本。在第一级，读取次数与恢复转录本的数量之间存在正相关。然而，测序深度的影响因细胞或组织类型、读取类型以及转录本的性质和表达水平而异。无论是短读还是长读，编码转录本的检测都迅速达到饱和，但是在任何测序深度下，非编码转录本都没有出现早期饱和的迹象。增加长读测序深度特别有利于包含转座元件的转录本。最后，我们展示了如何根据批量长读样本组装的转录本来指导单细胞 RNA-seq，并证明非编码转录本的表达水平与编码转录本相似，但在较少的细胞中表达。这项研究强调了测序深度对转录本组装的影响。
