Single-cell RNA-sequencing (scRNA-seq) has made it possible to profile gene expression in tissues at high resolution. An important preprocessing step prior to performing downstream analyses is to identify and remove cells with poor or degraded sample quality using quality control (QC) metrics. Two widely used QC metrics to identify a ‘low-quality’ cell are (i) if the cell includes a high proportion of reads that map to mitochondrial DNA (mtDNA) encoded genes and (ii) if a small number of genes are detected. Current best practices use these QC metrics independently with either arbitrary, uniform thresholds (e.g. 5%) or biological context-dependent (e.g. species) thresholds, and fail to jointly model these metrics in a data-driven manner. Current practices are often overly stringent and especially untenable on certain types of tissues, such as archived tumor tissues, or tissues associated with mitochondrial function, such as kidney tissue.
Researchers from the University of Pennsylvania Perelman School of Medicine and the Johns Hopkins Bloomberg School of Public Health have developed a data-driven QC metric (miQC) that jointly models both the proportion of reads mapping to mtDNA genes and the number of detected genes with mixture models in a probabilistic framework to predict the low-quality cells in a given dataset. The researchers demonstrate how our QC metric easily adapts to different types of single-cell datasets to remove low-quality cells while preserving high-quality cells that can be used for downstream analyses.
Uniform and data-driven quality control (QC) thresholds for scRNA-seq data
Cells (N = 6618) from one high-grade serous ovarian cancer (HGSOC) tissue sample (Sample ID: 16030X4) with the number of unique genes detected (x-axis) and percent of cell counts mapping to mitochondrial (mtDNA) genes (y-axis). (A) Illustration of removing cells with a uniform QC threshold of greater than 10% cell counts mapping to mtDNA genes (solid black line) and a more data-driven threshold of greater than 3 median absolute deviations (MADs) of the percent of counts mapping to mtDNA genes (dotted black line). (B) Using our data-driven approach (miQC), we fit a finite mixture of standard linear regression models with two lines (black lines) to calculate a posterior probability of being a compromised cell. (C) Cells shaded by their posterior probability of being compromised. (D) Discarding all cells with ≥ 75% probability of being compromised creates a data-driven QC threshold for scRNA-seq data.
Availability – The software package is available at https://bioconductor.org/packages/miQC.