Quality control in scRNA-Seq can discriminate pacemaker cells - the mtDNA bias

Single-cell RNA-Sequencing (scRNA-Seq) provides high-resolution insights into complex tissues. Cardiac tissue, however, poses a major challenge due to the delicate isolation process and the large size of mature cardiomyocytes. Regardless of the experimental technique, captured cells are often impaired and some capture sites may contain multiple or no cells at all. All this refers to “low quality” potentially leading to data misinterpretation. Common standard quality control parameters involve the number of detected genes and transcripts per cell and the fraction of transcripts from mitochondrial genes (%mtDNA). While cutoffs for transcripts and genes per cell are usually user-defined for each experiment or individually calculated, a fixed threshold of 5% mtDNA is standard and often set as default in scRNA-Seq software. However, this parameter is highly dependent on the tissue type. In the heart, mitochondrial transcripts comprise almost 30% of total mRNA due to high energy demands. Here, we demonstrate that a 5%-threshold not only causes an unacceptable exclusion of cardiomyocytes but also introduces a bias that particularly discriminates pacemaker cells. This effect is apparent for our in vitro generated induced-sinoatrial-bodies (iSABs; highly enriched physiologically functional pacemaker cells), and also evident in a public dataset of cells isolated from embryonal murine sinoatrial node tis-sue [2]. Taken together, we recommend omitting this filtering parameter for scRNA-Seq in cardiovascular applications whenever possible. Investigation of subpopulations and their respective features in “induced-sinoatrial-bodies'' (iSABs) assesed by single-cell sequencing using the 10x Genomics system