Systematic evaluation of signal-to-noise ratio in single cell genome amplification and sequencing

Background: The literature on single cell genomic analysis on the DNA level is conflicting regarding requirements of cell quality, amplification success rates and allelic dropouts, generally lacking comprehensive comparison from multiple cell input down to the single cell. To address these issues we analyzed a leukemic cell line (OCI-AML3) with a known set of genetic aberrations. Methods: By analyzing single and multiple cell replicates (2 to 50 cells) purified by micromanipulation and serial dilution, we have been able to systematically assess the signal-to-noise ratio (SNR), objectively, from single and few cells based on a multiple displacement amplification method and whole exome sequencing. Results: In this setting, known OCI-AML3 mutations could be identified as well as copy number changes. In contrast, at the level of 1–2 cells, we observed that 40–50% of all alleles were not present in the replicate samples, and this allelelic discrepancy displayed a clear exponential function of cell input. Thus, it shows that even under highly optimized conditions, single cell WGA and interpretation must be taken with considerable caution, given that allele dropout is frequent and displays low SNR, with allelic noise rapidly alleviated with increased cell input, and coefficient of variation doubling from 2 to 50 cell assays. Conclusions: In conclusion, we suggest that data on single cell genomic analysis should be accompanied by cell dilution for relative qualitative assessment of individual procedures and inherent SNR, quantitatively.背景：现有关于DNA层面单细胞基因组分析的相关研究，在细胞质量要求、扩增成功率及等位基因脱扣（allelic dropouts）等议题上结论相悖，且普遍缺乏从多细胞输入到单细胞层面的系统性对比研究。为解决上述问题，本研究针对携带已知遗传畸变的白血病细胞系（OCI-AML3）展开分析。 方法：本研究通过显微操作与系列稀释法纯化得到单细胞及多细胞重复样本（2~50个细胞），并基于多重置换扩增（multiple displacement amplification）与全外显子组测序（whole exome sequencing）技术，客观且系统地评估了单细胞及少量细胞的信噪比（signal-to-noise ratio, SNR）。 结果：在本研究的实验体系中，可成功检出OCI-AML3的已知突变与拷贝数变异。与之相对，在1~2个细胞的样本中，我们发现重复样本中缺失了40%~50%的等位基因，且该等位基因差异随细胞输入量呈明显的指数函数变化。由此可见，即便在高度优化的实验条件下，单细胞全基因组扩增（whole genome amplification, WGA）及其结果解读仍需格外谨慎：等位基因脱扣现象频发且信噪比偏低；随着细胞输入量增加，等位基因噪声会快速缓解，但2~50个细胞检测的变异系数（coefficient of variation）却会翻倍。 结论：综上，我们建议在发表单细胞基因组分析相关数据时，应配套开展细胞稀释实验，以相对定性评估各实验流程的表现及内在信噪比，实现定量分析。