Extraction of mitochondrial DNA mutations from bulk RNA-seq data in CLL.

Assessment of deconvolution performance of synthetic chimeric cell populations in in-silico mixing experiments.

Longitudinal changes of somatic nuclear and mitochondrial DNA mutations in genetically stable CLL.

Co-evolution of somatic nuclear and mitochondrial DNA mutations in AML.

Despite the promise of single-cell transcriptomics for understanding cell states in heterogeneous populations, widely used platforms have limited ability to link transcriptional states to somatic mutations within the same cells. Here, we introduce Genotyping in Fixed Transcriptomes (GIFT) for the simultaneous detection of large numbers of targeted genetic variants with whole transcriptome profiles in single cells. The core innovation of GIFT is a rationally designed gapfilling reaction between adjacent single-stranded DNA (ssDNA) probes that barcodes native transcript sequence to enable highly-specific targeted mutation detection. GIFT achieves greater than 99% genotyping accuracy and flexible capture of hundreds of mutations per cell, including in formalin-fixed, paraffin-embedded (FFPE) tissue, enabling clonal lineage tracing in heterogeneous settings. We demonstrate the unique scalability of GIFT by profiling more than 700,000 cells from 35 donors with myeloproliferative neoplasms (MPN), revealing mutation-dependent hematopoietic responses to systemic inflammation associated with the characteristic JAK2V617 mutation, including an allelic dose gradient of interferon-associated transcriptional programs and priming of hematopoietic stem cells that develop into divergent disease states. The technical advantages of GIFT enable direct resolution of genotype-to-phenotype relationships via clonal tracing with comprehensive cell-state measurements at single-cell resolution.

Identification of cell types and tracking of mitochondrial DNA mutations in immunosuppression tapering (IST) cohort using ASAP-seq.

Identification of high-confidence mitochondrial DNA mutations for Tapestri analysis.

Mutations targeted using single cell DNA sequencing in AML1012.

Mounting evidence implicates herpesvirus reactivation in the etiology of Alzheimer's disease, yet we lack a refined molecular characterization of pathogenesis in neurodegeneration. Here, we mine over 10 petabytes of human sequencing data for viral transcripts, identifying recurrent herpes simplex virus 1 (HSV-1) reactivation in healthy but not pathological post-mortem human brain tissue. Integrative single-nucleus analyses resolve direct evidence of HSV-1 expression in RORB+ glutamatergic neurons, implicating viral reactivation in a neuronal population progressively lost during dementia.

Deconvolution and identification of AML phenotypes in bone marrow using ASAP-seq.

Identification of AML subclones using mitochondrial DNA (mtDNA) mutations.

Blood and bone marrow counts of immunosuppression tapering (IST) cohort from routine clinical measurements.

Differential phenotypes of physiologic (donor-) and malignant hematopoietic (recipient-derived) cells.

Mitochondrial and somatic nuclear DNA mutations identified in CLL using single cell DNA sequencing.

Longitudinal changes of somatic nuclear and mitochondrial DNA mutations in naturally progressing CLL.

Immunosuppression tapering cohort.

Co-segregation of mitochondrial and somatic nuclear DNA mutations in CLL.

Supplementary Figures 1-20 and Supplementary Tables 1-10, Supplementary notes

Participants of ETCTN/CTEP 10026 studied with ASAP-seq.

Innate immune cells constitute the majority of the tumor microenvironment (TME) and mediate anti-tumor immunity and immunotherapy responses. While single-cell T and B cell receptor sequencing have revealed insights into the clonal dynamics of adaptive immunity, the lack of analogous tools has precluded similar analysis of innate immune cells. Here, we describe a method leveraging somatic mitochondrial DNA (mtDNA) mutations to reconstruct clonal lineage relationships between cells in native human tissues. By jointly profiling single-cell chromatin accessibility and mtDNA variants, we resolve clonal dynamics of 218,715 cells from matched tumors, tissues, and blood from patients with lung and ovarian cancers. Clonal tracing reveals that TME-resident myeloid subsets, including macrophages and type 3 dendritic cells (DC3), are clonally related to circulating and tissue-infiltrating monocytes. We further identify distinct DC-biased and macrophage-biased clones, whose circulating monocyte precursors exhibit distinct epigenetic profiles, suggesting intratumoral myeloid differentiation fate may be peripherally programmed before TME infiltration.