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Abstract
SummarySingle-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for resolving transcriptional heterogeneity. However, its application to studying cancerous tissues is currently hampered by the lack of coverage across key mutation hotspots in the vast majority of cells; this lack of coverage prevents the correlation of genetic and transcriptional readouts from the same single cell. To overcome this, we developed TARGET-seq, a method for the high-sensitivity detection of multiple mutations within single cells from both genomic and coding DNA, in parallel with unbiased whole-transcriptome analysis. Applying TARGET-seq to 4,559 single cells, we demonstrate how this technique uniquely resolves transcriptional and genetic tumor heterogeneity in myeloproliferative neoplasms (MPN) stem and progenitor cells, providing insights into deregulated pathways of mutant and non-mutant cells. TARGET-seq is a powerful tool for resolving the molecular signatures of genetically distinct subclones of cancer cells.
Highlights
Resolving intratumoral heterogeneity (ITH) is critical for our understanding of tumor evolution and resistance to therapies; this understanding, in turn, is required for the development of effective cancer treatments and biomarkers for precision medicine (McGranahan and Swanton, 2017)
Target-specific primers for coding DNA (cDNA) and genomic DNA (gDNA) were added to the reverse transcription (RT) and PCR-amplification steps (Table S2), which used modified enzymes (Table S1) that provided more efficient amplification (Figure 1A)
We used an aliquot of the pre-amplified gDNA and cDNA libraries for targeted next-generation sequencing (NGS) of specific cDNA and gDNA amplicons and another aliquot for whole-transcriptome library preparation
Summary
Resolving intratumoral heterogeneity (ITH) is critical for our understanding of tumor evolution and resistance to therapies; this understanding, in turn, is required for the development of effective cancer treatments and biomarkers for precision medicine (McGranahan and Swanton, 2017). The best-characterized source of ITH has been at the genetic level; this heterogeneity has been identified through advances in next-generation sequencing (NGS) techniques at the bulk and single-cell levels (Vogelstein et al, 2013). Some tumors are hierarchically organized and contain cancer stem cells (CSCs), which propagate disease relapse. The genetic events underlying tumor evolution originate in CSCs, which in some tumors are rare within the total tumor bulk population (Clevers, 2011; Magee et al, 2012; Woll et al, 2014). Resolving ITH requires methods that allow these multiple layers of heterogeneity to be teased apart
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