在Crimson scRNA-seq和scATAC-seq文库上对比批量和单细胞变异检测方法的性能

Benchmarking bulk and single-cell variant-calling approaches on Chromium scRNA-seq and scATAC-seq libraries

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GENOME RESEARCH

影响因子:5.5

分区:生物学1区 Top / 生物工程与应用微生物1区生化与分子生物学2区遗传学2区

发表日期:2024 Sep 20

作者: Matthew Wiens, Hossein Farahani, R Wilder Scott, T Michael Underhill, Ali Bashashati

DOI: 10.1101/gr.277066.122

摘要

单细胞测序技术如scRNA-seq和scATAC-seq已成为研究组织组成的广泛有效工具。越来越多的变异检测工具被应用于这些技术，以解析样本的遗传异质性，尤其是在检测肿瘤克隆结构方面。通常，传统的批量DNA变异检测器会对单细胞文库的合并reads进行分析，以检测候选突变。近期，多项研究将此类检测器用于单个细胞的reads，有些报告其能以更高灵敏度检测稀有变异。许多研究将这两种方法应用于Chromium（10x Genomics）平台的scRNA-seq和scATAC-seq。然而，基于Chromium的文库相比现有单细胞技术可能面临额外的变异检测挑战，提出了关于此类工作流获得的变异的有效性的问题。为了评估不同变异检测方法在Chromium scRNA-seq和scATAC-seq文库中的优缺点，我们使用匹配的全基因组测序样本库对检测器的性能进行了评估。结果显示，应用于合并reads的批量检测器明显优于单细胞方法。我们还评估了scRNA-seq和scATAC-seq特有的变异，发现噪音模式，但也可能捕获RNA编辑事件。最后，我们提出在单细胞层面进行变异检测可发现稀有体细胞突变的观点，实验证明在单细胞Chromium文库中解析此类变异具有一定的难度。

Abstract

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.