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

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.

Sculpturing wafer-scale nanofluidic devices for DNA single molecule analysis

“…Integrated micro-and nanofluidic devices are valuable tools for handling, manipulating and detecting ultra-low quantities of biomolecules. [3][4][5] In particular, on-chip analysis of DNA 6,7 can have an impact on early disease diagnosis, on personalized medicine and on effective cancer treatment. 8 The DNA molecules can be stretched inside nanochannels and analyzed, for instance, by optical mapping.…”

Microfluidic long DNA sample preparation from cells

“…Being a microfluidic approach, our simple design has the potential to be integrated with genomics chips, serving as a powerful tool to deliver ultra-long DNA directly from cells to genomic analysis technologies without any human intervention. Although single-cell DNA isolation for either linearization in nanochannels 49 or optical mapping in nanoslits 50 have been demonstrated on a single device, there is yet no generic sample preparation platform that can be used upstream of any genomic analysis technology.…”

Detailed molecular cytogenetic characterisation of the myeloid cell line U937 reveals the fate of homologous chromosomes and shows that centromere capture is a feature of genome instability

“…Metaphase FISH is a single cell analysis tool which can help ll in some of these gaps. More recently, optical mapping [32] and nanopore sequencing [33][34][35][36][37] are making the description of highly complex karyotypes more comprehensive, and these will allow the exploration of chromosome rearrangements with greater resolution, including long read sequencing across centromeres. One advantage of our approach is that the distribution of the homologues can readily be interpreted.…”