The resulting genome sequences therefore contain intermixed seque

The resulting genome sequences therefore contain intermixed sequences from different tumour clones, as well as from admixed normal cells. Computational methods can determine

which mutations are clonal (present in all tumour cells) and which are subclonal [15]. In addition, by analyzing point mutation and copy number data further with bioinformatics algorithms, phylogenetic trees of different tumour subclones can be inferred [12]. Although these methods GSK458 ic50 provide important information on the genomes of distinct cell populations within the tumour, the number of tumour cell populations they can disentangle is limited, and inferring rare subclonal populations remains difficult. Recent advances have made it possible to profile the genomes of single cells. The isolation

of single cancer cells, followed by amplification of the DNA and array profiling or next-generation sequencing (Figure 1), opens avenues to study tumour subclonal architecture and tumour evolution in unprecedented depth. Here, we provide an overview of current methods to profile genomes of single cells. We discuss their strengths and limitations and the perspectives they offer for cancer research and therapy monitoring. To isolate single cells from solid tumours, two main approaches have been developed. The first method exploits the precision of modern flow cytometry to sort nuclei from single cells [16 and 17••]. Tissue-cubes of ∼1 mm3, cut off a (frozen) solid tumour, are teased apart in cell lysis buffer, containing DAPI, a fluorescent DNA-intercalator, and the resulting single Epacadostat solubility dmso nuclei are flow-sorted based on DNA content. This technique provides the advantage of allowing identification and isolation of tumour subpopulations on the basis of ploidy [16 and 17••]. Although the cytoplasm is lost, extensions to analyses of the transcriptome per se are possible [ 18]. However, this approach also entails limitations. Beta adrenergic receptor kinase In particular, micronuclei may be lost. Micronuclei are not merely by-products from genomic instability but are likely prone to DNA-replication stress and further

DNA-mutational processes [ 19] and therefore may be important players in tumour evolution. A second method disperses the tissue from fresh solid tumour biopsies in a single-cell suspension, using enzymatic treatments, including, for example, collagenases [20•]. Intact individual cells can subsequently be isolated using (mouth-controlled) pipetting, modern cell-sorting or microfluidics systems with or without applying immunocytochemistry. Microfluidics devices provide the advantage that in addition to capturing individual cells, they also provide nanoliter reaction chambers to further process the nucleic acids of multiple individual cells in parallel under highly standardized conditions at significantly reduced reagent costs.

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