scRNA-seq 和 scATAC-seq 等单细胞测序方法已成为研究组织组成的广泛且有效的工具。越来越多的变体调用者被应用于这些方法来解决样本的遗传异质性，特别是在检测肿瘤的克隆结构的情况下。通常，传统的批量 DNA 变体识别器应用于单细胞文库的合并读数以检测候选突变。最近，多项研究已将此类调用程序应用于单个细胞的读取，其中一些研究指出能够以更高的灵敏度检测罕见变异。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 的文库可能会给变体调用带来额外的挑战，从而引发对从此类工作流程中获得的变体的有效性的质疑。为了确定 Chromium scRNA-seq 和 scATAC-seq 文库的各种变体调用方法的优点和挑战，我们使用具有匹配的批量全基因组测序的样本文库来评估调用者的性能。我们审查了调用程序的性能，发现应用于池读取的批量调用程序的性能明显优于单个单元方法。我们还评估了 scRNA-seq 和 scATAC-seq 方法独特的变体，发现噪声模式，同时也发现了 RNA 编辑事件的潜在捕获。最后，我们回顾了单细胞水平的变异检出可以检测罕见体细胞变异的概念，提供的经验结果表明在单细胞 Chromium 文库中解决此类变异是不可行的。由冷泉港实验室出版社出版。

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 to existing single-cell methodologies, raising questions for 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.Published by Cold Spring Harbor Laboratory Press.