Benchmarking Bulk and Single-cell Variant Calling Approaches on Chromium scRNA-seq and scATAC-seq Libraries

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. Overall design: Single cell ATAC-seq analyes of epithelioid sarcoma

以单细胞RNA测序 (scRNA-seq)、单细胞染色质可及性测序 (scATAC-seq)为代表的单细胞测序技术，现已成为解析组织构成的通用高效工具。越来越多的研究将变异检出工具 (variant caller)应用于这类测序技术，以解析样本的遗传异质性，尤其在肿瘤克隆结构的检测场景中。通常而言，传统批量DNA变异检出工具会被用于单细胞文库的合并测序读段，以识别候选突变位点。近期已有多项研究将这类工具直接应用于单个细胞的测序读段，其中部分研究声称该方案可实现更高灵敏度的罕见变异检测。诸多研究已针对Chromium (10x Genomics)平台的scRNA-seq与scATAC-seq技术，采用上述两种策略开展研究。但相较于现有单细胞测序技术，基于Chromium平台的文库对变异检出会带来额外挑战，这也引发了关于该流程所得变异结果有效性的疑问。为评估不同变异检出策略在Chromium平台scRNA-seq与scATAC-seq文库中的应用价值与挑战，本研究采用配套的批量全基因组测序文库，对各类变异检出工具的性能展开评测。本研究对检出工具的性能进行了评测，结果显示：基于合并读段的批量检出工具，其性能显著优于针对单个细胞的策略。同时，本研究还针对scRNA-seq与scATAC-seq技术特有的变异位点进行了评测，不仅发现了噪声信号特征，还观测到潜在的RNA编辑事件捕获现象。最后，本研究针对“单细胞层面的变异检出可实现罕见体细胞变异检测”这一观点进行了验证，所得实验结果表明：在Chromium平台的单细胞文库中，解析这类罕见变异并不可行。整体实验设计：上皮样肉瘤的单细胞ATAC测序分析。