RESUMO
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.
Assuntos
Mapeamento Cromossômico/métodos , DNA/genética , Genoma/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Linhagem Celular Tumoral , Cromossomos Humanos Par 19/genética , Cromossomos Humanos Par 4/genética , Evolução Clonal/genética , Neoplasias Colorretais/genética , Genômica/métodos , Humanos , Deleção de Sequência/genéticaRESUMO
We present a single mode, single polarization, distributed feedback polymer dye laser, based on a short high order Bragg grating defined in a dye doped polymer layer between two secondary polymer layers. The Bragg grating is defined solely with standard I-line UV lithography. In this device we obtain single mode operation in a multimode structure by means of mode loss differentiation without using sub-wavelength structures. The laser is fabricated using micro-fabrication technology, is pumped by a pulsed frequency doubled Nd:YAG laser, and emits light in the chip plane at 551.39 nm, with a FWHM linewidth below 150 pm.
