Simultaneous genomic identification and profiling of a single cell using semiconductor-based next generation sequencing.
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 (≈ 10(6) 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 10(31-35). 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 than 100 × were 99.04% for the HID panel and 98.83% for the Cancer panel, while for the single cell percentages of sequence covered at coverage of more than 100 × were 55.93% for the HID panel and 65.96% for the Cancer panel. Partial amplification failure or randomly distributed non-amplified regions across samples from single cells during the WGA procedures or random allele drop out probably caused these differences. However, comparative analyses showed that this method successfully discriminated a single A549 cancer cell from a bulk population of A549 cells. Thus, our approach provides a powerful means to overcome tumor sample heterogeneity when searching for somatic mutations.
Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan.
Manabu
M
Watanabe
Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan.
Junko
J
Kusano
Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan.
Shinsaku
S
Ohtaki
Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan.
Takashi
T
Ishikura
Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan.
Jin
J
Katayama
Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan.
Akira
A
Koguchi
Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan.
Michael
M
Paumen
Life Technologies Japan Ltd., a part of Thermo Fisher Scientific. 4-2-8 Shibaura, Minato-ku Tokyo 108-0023, Japan.
Yoshiharu
Y
Hayashi
Heterogeneity; Laser capture microdissection; Semiconductor-based sequencing; Single cell identification