https://ror.org/05f950310 Laboratory for Neuroimmunology, Department of Neurosciences, Leuven Brain Institute, KU Leuven, Leuven, Belgium.
https://ror.org/05f950310 Laboratory for Neuroimmunology, Department of Neurosciences, Leuven Brain Institute, KU Leuven, Leuven, Belgium
Life Sci Alliance. 2023 Sep 11;6(11). doi: 10.26508/lsa.202301971. Print 2023 Nov.
As no existing methods within the single-cell RNA sequencing repertoire combine genotyping of specific genomic loci with high throughput, we evaluated a straightforward, targeted sequencing approach as an extension to high-throughput droplet-based single-cell RNA sequencing. Overlaying standard gene expression data with transcript level genotype information provides a strategy to study the impact of genetic variants. Here, we describe this targeted sequencing extension, explain how to process the data and evaluate how technical parameters such as amount of input cDNA, number of amplification rounds, and sequencing depth influence the number of transcripts detected. Finally, we demonstrate how targeted sequencing can be used in two contexts: (1) simultaneous investigation of the presence of a somatic variant and its potential impact on the transcriptome of affected cells and (2) evaluation of allele-specific expression of a germline variant in ad hoc cell subsets. Through these and other comparable applications, our targeted sequencing extension has the potential to improve our understanding of functional effects caused by genetic variation.
由于单细胞 RNA 测序领域中现有的方法都不能将特定基因组位点的基因分型与高通量结合起来，我们评估了一种简单直接的靶向测序方法，作为高通量基于液滴的单细胞 RNA 测序的扩展。将标准基因表达数据与转录水平的基因型信息叠加提供了一种研究遗传变异影响的策略。在这里，我们描述了这种靶向测序的扩展，解释了如何处理数据，并评估了输入 cDNA 的量、扩增轮数和测序深度等技术参数如何影响检测到的转录本数量。最后，我们展示了靶向测序如何在两种情况下使用：（1）同时调查体细胞变异的存在及其对受影响细胞转录组的潜在影响，（2）评估嵌合细胞亚群中种系变异的等位基因特异性表达。通过这些和其他类似的应用，我们的靶向测序扩展有可能提高我们对遗传变异引起的功能影响的理解。
