Simultaneous genomic identification and profiling of a single cell using semiconductor-based next generation sequencing

Publication date: 1 September 2014 Source:Applied & Translational Genomics, Volume 3, Issue 3 Author(s): Manabu Watanabe , Junko Kusano , Shinsaku Ohtaki , Takashi Ishikura , Jin Katayama , Akira Koguchi , Michael Paumen , Yoshiharu Hayashi Combining single-cell methods and next-generation sequencing should provide a powerful means to understand single-cell biology and obviate the effects of sample heterogeneity. Here we report a single-cell identification method and seamless cancer gene profiling using semiconductor-based massively parallel sequencing. A549 cells (adenocarcinomic human alveolar basal epithelial cell line) were used as a model. Single-cell capture was performed using laser capture microdissection (LCM) with an Arcturus® XT system, and a captured single cell and a bulk population of A549 cells (≈106 cells) were subjected to whole genome amplification (WGA). For cell identification, a multiplex PCR method (AmpliSeq™ SNP HID panel) was used to enrich 136 highly discriminatory SNPs with a genotype concordance probability of 1031–35. For cancer gene profiling, we used mutation profiling that was performed in parallel using a hotspot panel for 50 cancer-related genes. Sequencing was performed using a semiconductor-based bench top sequencer. The distribution of sequence reads for both HID and Cancer panel amplicons was consistent across these samples. For the bulk population of cells, the percentages of sequence covered at coverage of more...

Source: Applied and Translational Genomics - October 12, 2014 Category: Genetics & Stem Cells Source Type: research