Rodolphe Marie, Jonas N. Pedersen, Loic Bærlocher, Kamila Koprowska, Marie Pødenphant, Céline Sabatel, Maksim Zalkovskij, Andrej Mironov, Brian Bilenberg, Neil Ashley, Henrik Flyvbjerg, Walter F. Bodmer, Anders Kristensen, and Kalim U. Mir

PNAS October 30, 2018 115 (44) 11192-11197

GENETICS

Significance

We report optical mapping of DNA from a single cell. Notably, we demonstrate isolation of single cells, DNA extraction, and optical mapping, all within a single integrated micro-/nanofluidic device. Single-cell optical mapping is less complex than sequencing, which we performed after whole-genome amplification of DNA extracted from a single cell isolated on-chip. In some cases, optical mapping was more efficient than sequencing at detecting structural variation. As single-cell analysis can address genomic heterogeneity within a tumor, it may prove useful for the selection of cancer therapies. Thus, optical mapping of the long-range features of single-cell genomes and sequencing of the short-range features may become complementary tools for the analysis of tumors.

Abstract

To elucidate cellular diversity and clonal evolution in tissues and tumors, one must resolve genomic heterogeneity in single cells. To this end, we have developed low-cost, mass-producible micro-/nanofluidic chips for DNA extraction from individual cells. These chips have modules that collect genomic DNA for sequencing or map genomic structure directly, on-chip, with denaturation–renaturation (D-R) optical mapping [Marie R, et al. (2013) Proc Natl Acad Sci USA 110:4893–4898]. Processing of single cells from the LS174T colorectal cancer cell line showed that D-R mapping of single molecules can reveal structural variation (SV) in the genome of single cells. In one experiment, we processed 17 fragments covering 19.8 Mb of the cell’s genome. One megabase-large fragment aligned well to chromosome 19 with half its length, while the other half showed variable alignment. Paired-end single-cell sequencing supported this finding, revealing a region of complexity and a 50-kb deletion. Sequencing struggled, however, to detect a 20-kb gap that D-R mapping showed clearly in a megabase fragment that otherwise mapped well to the reference at the pericentromeric region of chromosome 4. Pericentromeric regions are complex and show substantial sequence homology between different chromosomes, making mapping of sequence reads ambiguous. Thus, D-R mapping directly, from a single molecule, revealed characteristics of the single-cell genome that were challenging for short-read sequencing.

See: http://www.pnas.org/content/115/44/11192

Fig. 1.

Architecture of single-cell processing device and workflow for single-cell D-R mapping and whole-genome sequencing. (A) An all-polymer lab-on-a-chip device with 12 connectors comprises a cell trap (blue), a meandering channel (green), and a flow-stretch device (red). (B) A single cell is captured by hydrodynamic trapping, DNA is extracted and patterned according to AT/GC composition by a heating–cooling cycle, and genomic DNA is stretched and visualized. (C) Workflow using device shown in SI Appendix, Fig. S1 for single-cell trapping and extracting and amplifying DNA before sequencing. (D) Principle of D-R pattern generation: The genomic DNA is homogeneously stained with YOYO-1. During partial denaturation at a temperature Tm