3'-TARGET-seq: a novel method for high-sensitivity single-cell mutational analysis and parallel high throughput RNA-sequencing [Figures 5 and 6, Figure S7]

We developed 3'-TARGET-seq, a single-cell genotyping and RNA-seq method, which allows accurate detection of multiple mutations within single-cells from genomic and coding DNA in parallel with high throughput 3'-biased whole transcriptome analysis, providing a powerful tool to link transcriptional and genetic tumor heterogeneity. Single cell whole transcriptome sequencing of 2798 Lineage-CD34+ HSPC (Hematopoietic Stem and Progenitor Cells) from patients with JAK2-V617F mutant myelofibrosis and normal controls using 3'-TARGET-seq reveals distinct molecular signatures associated with the presence of somatic mutations in single cells as well as distinct transcriptional profiles of WT cells from patient samples as compared with normal controls. Overall design: We isolated single HSPCs from patients with JAK2V617F mutant myelofibrosis patient samples and aged-matched normal controls and processed them using 3'-TARGET-seq, a novel method for parallel mutational analysis and high throughput whole transcriptome sequencing from the same single cell. We analyzed a cohort of patients with JAK2-V617F mutation and several cooperating mutations in spliceosome components (SF3B1, U2AF1, SRSF2), epigenetic modifiers (TET2, ASXL1) and signalling genes (CBL). We performed differential expression analysis between different genetic subclones from multiple patients as well as cells obtained from normal donors. We also analyzed molecular signatures corresponding to every combination of mutations found in single cells as well as molecular signatures of WT cells from patient samples as compared to WT cells from normal controls.

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Rodriguez-Meira A, Buck G, Clark SA, Povinelli BJ, Alcolea V, Louka E, McGowan S, Hamblin A, Sousos N, Barkas N, Giustacchini A, Psaila B, Jacobsen SEW, Thongjuea S, Mead AJ