颜林林 (2022-07-06 00:02):
#paper doi:10.1186/s12864-022-08717-z BMC Genomics, 2022, The effects of sequencing depth on the assembly of coding and noncoding transcripts in the human genome. 众所周知,测序深度会影响其数据的分析结果。然而,到底影响多大,怎么影响的,往往视研究目的和研究对象而定,得具体分析,也值得研究。这篇文章,就是在系统研究测序深度对转录组数据的转录本组装的影响。文章纳入了来自150个人类干细胞样本的不同细胞组织的RNA-seq数据,除了短读长平台外,还包括四个PacBio平台的长读长数据。其中有两个样本还测了高达200M reads的NGS数据量,于是可以用它们来抽取不同比例数据,以模拟不同的测序数据量。分析结果表明,编码转录本与非编码转录本之间存在差异,前者随着测序深度增加而迅速进入饱和,后者在所分析的数据中则几乎始终未达到饱和。这可能与两者的组装难度有关。此外,长读长信息有助于含有转座元件的转录本组装。比较有意思的是单细胞RNA-seq(scRNA-seq),其非编码转录本的表达水平低,是由于表达细胞较少,而在表达的细胞中,非编码转录本的表达水平其实与编码转录本相似,这个现象的发现得益于长读长测序平台,因此文章得出结论是长读长测序更适合scRNA-seq。但我个人多少还是怀疑这些结论很可能与分析评估方法有关,也许值得重复下这篇文章的分析过程。
IF:3.500Q2 BMC genomics, 2022-Jul-04. DOI: 10.1186/s12864-022-08717-z PMID: 35787153
The effects of sequencing depth on the assembly of coding and noncoding transcripts in the human genome
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Abstract:
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.
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