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

Abstract

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.

Keywords: Heterogeneity; Laser capture microdissection; Semiconductor-based sequencing; Single cell identification.

Fig. 1

Workflow used for this study. A) Procedures used and the time required for an experiment. The total time required for a single experiment was approximately 21 h. B) Summary of single-cell identification and simultaneous functional sequence analysis with a semiconductor-based sequencer. Amplifications using a population of cells and a whole-genome amplified single cell from the same bulk population were compared.

Fig. 2

Results for single-cell capture and WGA. A) Image of a single cell. This cell was captured by LCM using an Arcturus® XT system (Life Technologies). B) Captured single cells and a blank sample included as a negative control were lysed and WGA was performed using single cell WGA kits (New England Bio Laboratories). Successful amplification of the samples was checked by agarose gel electrophoresis.

Fig. 3

Correlations for read depths between two A549 single-cell replicates and between an A549 single cell and an A549 population of cells. Comparative analyses were conducted for two A549 single-cell replicates and for an A549 single cell and an A549 population of cells. A) Correlation for read depths between single cell Library prep replicates #1 and #2 (#1 was from 50 ng of DNA templates and # 2 was from 10 ng of DNA templates). These results indicated a high correlation between these replicates. B) Correlation for read depths between A549 single cell Library prep replicate #1 and an A549 population of cells.

Fig. 4

Allelic description plots as replication study using TaqMan® SNP genotyping assays. To validate SNP HID sequencing results, allele-specific real-time PCR was performed. Four representative plots showing performance of four assays in analysis of A549 samples and reference samples. VIC signal (x-axis) is associated with the probe for allele A (graph (1), (3)) and allele C (graph (2), (4)), while FAM (y-axis) labels the allele G (graph (1), (3)) and allele T (graph (2), (4)) probes. Aqua blue × symbols indicate A549 bulk cells and a single cell with NGS reads data. Circles symbols and black × symbols indicate 20 Coriell gDNA samples as reference.