Feasibility of a workflow for the molecular characterization of single cells by next generation sequencing

Publication date: Available online 12 September 2015 Source:Biomolecular Detection and Quantification Author(s): Francesca Salvianti, Giada Rotunno, Francesca Galardi, Francesca De Luca, Marta Pestrin, Alessandro Maria Vannucchi, Angelo Di Leo, Mario Pazzagli, Pamela Pinzani The purpose of the study was to explore the feasibility of a protocol for the isolation and molecular characterization of single circulating tumor cells (CTCs) from cancer patients using a single-cell next generation sequencing (NGS) approach. To reach this goal we used as a model an artificial sample obtained by spiking a breast cancer cell line (MDA-MB-231) into the blood of a healthy donor. Tumor cells were enriched and enumerated by CellSearch® and subsequently isolated by DEPArray™ to obtain single or pooled pure samples to be submitted to the analysis of the mutational status of multiple genes involved in cancer. Upon whole genome amplification, samples were analysed by NGS on the Ion Torrent PGM™ system (Life Technologies) using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies), designed to investigate genomic “hot spot” regions of 50 oncogenes and tumor suppressor genes. We successfully sequenced five single cells, a pool of 5 cells and DNA from a cellular pellet of the same cell line with a mean depth of the sequencing reaction ranging from 1581 to 3479 reads. We found 27 sequence variants in 18 genes, 15 of which already reported in the COSMIC or dbSN...
Source: Biomolecular Detection and Quantification - Category: Molecular Biology Source Type: research