Strategies for Integrating Single-Cell RNA Sequencing Results With Multiple Species

Single-cell RNA sequencing (scRNAseq) is a robust technology for parsing gene expression in individual cells from a tissue or other complex source. One application involves experiments where cells from multiple species are recovered from a single sample, such as when human cells are transplanted into an animal model. We transplanted microglial precursor cells into newborn mouse brain and then recovered unenriched cortical tissue six months later. Dissociated cells were assessed by scRNAseq. The default method for analyzing these results begins by aligning sequencing reads with a mixture of both mouse and human reference genomes. While this clearly identifies the human cells as a distinct cluster, the clustering is artificially driven by expression from non-comparable gene identifiers from different species. We devised a method for translating expression counts from human to mouse and evaluated four algorithms for parsing mixed-species scRNAseq data. Our optimal approach split raw sequencing reads according to the best alignment score in each genome, and then re-aligned reads only with the appropriate genome. After gene symbol translation, pooled results indicate that cell types are more appropriately clustered and that differential expression analysis identifies species-specific patterns. This method should be applicable to any mixed-species scRNAseq experiment.Summary of optimal strategyMixed-species scRNAseq data are aligned with mixture of mouse and human reference genomesThe BAM file is scanned to find the best alignment score for each sequencing read identifier; these are used to split the paired FASTQ files into two sets of filesEach set of species-specific, paired FASTQ files is re-aligned with only the appropriate reference genomeRaw counts imported into SeuratThe human counts table is translated to mouse gene symbols using a custom HomoloGene translation tableResults are merged and analyzed