School of Biomedical Engineering, University of British Columbia, Vancouver, British Columbia V6T 2B9, Canada.
Department of Cellular & Physiological Sciences, University of British Columbia, Vancouver, British Columbia V6T 2A1, Canada.
Genome Res. 2024 Sep 20;34(8):1196-1210. doi: 10.1101/gr.277066.122.
Single-cell sequencing methodologies such as scRNA-seq and scATAC-seq have become widespread and effective tools to interrogate tissue composition. Increasingly, variant callers are being applied to these methodologies to resolve the genetic heterogeneity of a sample, especially in the case of detecting the clonal architecture of a tumor. Typically, traditional bulk DNA variant callers are applied to the pooled reads of a single-cell library to detect candidate mutations. Recently, multiple studies have applied such callers on reads from individual cells, with some citing the ability to detect rare variants with higher sensitivity. Many studies apply these two approaches to the Chromium (10x Genomics) scRNA-seq and scATAC-seq methodologies. However, Chromium-based libraries may offer additional challenges to variant calling compared with existing single-cell methodologies, raising questions regarding the validity of variants obtained from such a workflow. To determine the merits and challenges of various variant-calling approaches on Chromium scRNA-seq and scATAC-seq libraries, we use sample libraries with matched bulk whole-genome sequencing to evaluate the performance of callers. We review caller performance, finding that bulk callers applied on pooled reads significantly outperform individual-cell approaches. We also evaluate variants unique to scRNA-seq and scATAC-seq methodologies, finding patterns of noise but also potential capture of RNA-editing events. Finally, we review the notion that variant calling at the single-cell level can detect rare somatic variants, providing empirical results that suggest resolving such variants is infeasible in single-cell Chromium libraries.
单细胞测序方法，如 scRNA-seq 和 scATAC-seq，已成为广泛且有效的工具，可用于研究组织组成。越来越多的变体调用程序被应用于这些方法，以解决样本的遗传异质性，特别是在检测肿瘤的克隆结构时。通常，传统的批量 DNA 变体调用程序被应用于单个细胞文库的合并读取，以检测候选突变。最近，多项研究已经将这些调用程序应用于单个细胞的读取，其中一些研究声称具有更高灵敏度检测罕见变体的能力。许多研究将这两种方法应用于 Chromium（10x Genomics） scRNA-seq 和 scATAC-seq 方法。然而，与现有的单细胞方法相比，基于 Chromium 的文库可能会对变体调用提出额外的挑战，这引发了对从这种工作流程中获得的变体的有效性的质疑。为了确定各种变体调用方法在 Chromium scRNA-seq 和 scATAC-seq 文库上的优点和挑战，我们使用具有匹配批量全基因组测序的样本文库来评估调用程序的性能。我们回顾了调用程序的性能，发现合并读取上应用的批量调用程序明显优于单细胞方法。我们还评估了 scRNA-seq 和 scATAC-seq 方法特有的变体，发现了噪声模式，但也有可能捕获 RNA 编辑事件。最后，我们回顾了单细胞水平的变体调用可以检测罕见的体细胞变体的观点，提供了表明在单个细胞 Chromium 文库中解析这些变体是不可行的经验结果。
