Reconstructing lineage relationships in complex tissues can reveal mechanisms underlying development and disease. Recent methods combine single-cell transcriptomics with mitochondrial DNA variant detection to establish lineage relationships in primary human cells, but are not scalable to interrogate complex tissues. To overcome this limitation, here we develop a technology for highconfidence detection of mitochondrial mutations from high-throughput single-cell RNA-sequencing. We use the new method to identify skewed immune cell expansions in primary human clonal hematopoiesis. Main textSingle-cell RNA-sequencing (scRNA-seq) enables the unbiased assessment of cell states in health and disease 1,2 . Combined acquisition of cell state and genetic information can provide additional insight, such as targeted enrichment of cancer driver mutations from single-cell transcriptomes 3,4 . Separately, combining scRNA-seq with genetic cell barcodes is a powerful method to reveal clonal relationships and evolutionary dynamics of cells within organisms 5,6 . However, this has largely been limited to experimental model systems that can be genetically manipulated to insert cell barcodes. To infer clonal dynamics in primary human cells, recent methods have detected and utilized mitochondrial DNA (mtDNA) mutations as naturally occurring genetic cell barcodes [7][8][9] . The combination of scRNA-seq with mtDNA mutation detection can inform clonal relationships with high confidence, but is currently restricted to expensive, low-throughput, full-length transcript sequencing technologies like SmartSeq2 7,10 . To enable the reconstruction of clonal relationships in complex human tissues, we developed a method that captures genetic variants from high-throughput scRNA-seq platforms: MAESTER, or Mitochondrial Alteration Enrichment from Singlecell Transcriptomes to Establish Relatedness (Figure 1A). MAESTER is compatible with the most common high-throughput scRNA-seq platforms, including 10x Genomics 3' protocols, Seq-Well S 3 , and Drop-seq (Supplemental Figures 1-3) 11,12 . An intermediate step in each of these platforms yields full length cDNA transcripts, from which we enrich all 15 mitochondrial transcripts using pools of primers, while maintaining cell-identifying barcodes (Figure 1B, Supplemental Figure 4). Standard next-generation sequencing with 250 bp reads is then used to obtain the sequence of the amplified mitochondrial transcripts (Figure 1A). We developed a computational toolkit to call mtDNA variants from MAESTER data, the Mitochondrial Alteration Enrichment and Genome Analysis Toolkit (maegatk, Supplemental Figure 5, Methods). Building on previous tools that we developed 8 for mtDNA variant detection from single-cell ATAC or SmartSeq2, maegatk specifically handles technical biases implicit in high-throughput transcriptomic libraries. Critically, maegatk leverages unique molecular identifiers (UMIs) to collapse multiple sequencing reads of the same starting transcript, creating a consensus call for every

The blood system is often represented as a tree-like structure with stem cells that give rise to mature blood cell types through a series of demarcated steps. Although this representation has served as a model of hierarchical tissue organization for decades, single-cell technologies are shedding new light on the abundance of cell type intermediates and the molecular mechanisms that ensure balanced replenishment of differentiated cells. In this Brief Review, we exemplify new insights into blood cell differentiation generated by single-cell RNA sequencing, summarize considerations for the application of this technology, and highlight innovations that are leading the way to understand hematopoiesis at the resolution of single cells.