Single-molecule DNA-mapping and whole-genome sequencing of individual cells

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, Kalim U. Mir

*Corresponding author for this work

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    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.
    Original languageEnglish
    JournalProceedings of the National Academy of Sciences of the United States of America
    Volume115
    Issue number44
    Pages (from-to)11192-11197
    ISSN0027-8424
    DOIs
    Publication statusPublished - 2018

    Keywords

    • DNA
    • Nanofluidics
    • Optical mapping
    • Sequencing
    • Single cell

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