Editors' ChoiceGenomics

Baring cellular souls

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Science Translational Medicine  15 Feb 2017:
Vol. 9, Issue 377, eaam6064
DOI: 10.1126/scitranslmed.aam6064

Abstract

Coupling chemical approaches for disrupting DNA-protein complexes in intact nuclei with combinatorial barcoding enables high-throughput single-cell DNA sequencing.

A cell’s genetic material provides the underlying blueprint for its phenotype. Variations in single nucleotides (SNVs), copy number (CNVs), or chromosomal abundance (aneuploidy) across individual cells can impact their behaviors in clinically significant ways—determining, for example, which cancerous cells will respond to a therapeutic. The recent emergence of experimental methods for single-cell DNA sequencing now enables researchers to examine SNV and CNV heterogeneity across small sets of single cells, but difficulties in scaling have prevented comprehensive CNV-based characterization of complex samples—for example, determining the different clones within a tumor. Now, Vitak and colleagues have overcome this barrier by coupling a strategy for nucleosome depletion in intact nuclei with two rounds of barcoding.

First and most critically, the authors developed two chemical methods that can dissolve nuclosome DNA-histone complexes without disrupting the nuclear membrane to make almost all DNA within each nucleus freely accessible. Subsequently, the accessible DNA within each nucleus was nearly uniquely barcoded during library generation via a two-step process: first using one of 96 uniquely tagged transposases, and then again via PCR after pooling, mixing, and FACS-sorting 15 to 25 transposed nuclei into each well of a 96-well plate. This strategy provided single-cell resolution (unique barcode sets) for 89 to 94% of all nuclei—a range which can be improved further through use of more barcodes at either stage.

By benchmarking against standard approaches in cell lines and primary monkey tissue, the authors found that although both chemical disruption techniques yielded relatively uniform single-cell DNA coverage, one resulted in fewer false positives. To demonstrate utility, the authors applied their preferred nucleosome depletion method to cells isolated from a primary human pancreatic ductal adenocarcinoma to uncover four distinct constituent subclones with unique CNV profiles. Further improvements will be needed to achieve more uniform genome coverage to improve, for example, the accuracy of CNV detection and finer resolution—currently 250 kilobases—as well as to limit the degree of cross-talk between single-cell libraries. Still, the work by Vitak et al. nicely illustrated how scalable, cost-effective, low-resolution single-cell DNA sequencing can be achieved without specialized microfluidic devices, envisioning several exciting avenues of research.

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