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Efficiency of whole genome amplification of Single Circulating Tumor Cells enriched by CellSearch and sorted by FACS

ABSTRACT: Efficiency of whole genome amplification of Single Circulating Tumor Cells enriched by CellSearch and sorted by FACS

PROVIDER: PRJEB4979 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR372251_1.fastq.gz	Fastqsanger.gz
	ERR372251_2.fastq.gz	Fastqsanger.gz
	ERR372252_1.fastq.gz	Fastqsanger.gz
	ERR372252_2.fastq.gz	Fastqsanger.gz
	ERR372253_1.fastq.gz	Fastqsanger.gz

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Project description:BACKGROUND: Tumor cells in the blood of patients with metastatic carcinomas are associated with poor survival. Knowledge of the cells' genetic make-up can help to guide targeted therapy. We evaluated the efficiency and quality of isolation and amplification of DNA from single circulating tumor cells (CTC). METHODS: The efficiency of the procedure was determined by spiking blood with SKBR-3 cells, enrichment with the CellSearch system, followed by single cell sorting by fluorescence-activated cell sorting (FACS) and whole genome amplification. A selection of single cell DNA from fixed and unfixed SKBR-3 cells was exome sequenced and the DNA quality analyzed. Single CTC from patients with lung cancer were used to demonstrate the potential of single CTC molecular characterization. RESULTS: The overall efficiency of the procedure from spiked cell to amplified DNA was approximately 20%. Losses attributed to the CellSearch system were around 20%, transfer to FACS around 25%, sorting around 5% and DNA amplification around 25%. Exome sequencing revealed that the quality of the DNA was affected by the fixation of the cells, amplification, and the low starting quantity of DNA. A single fixed cell had an average coverage at 20× depth of 30% when sequencing to an average of 40× depth, whereas a single unfixed cell had 45% coverage. GenomiPhi-amplified genomic DNA had a coverage of 72% versus a coverage of 87% of genomic DNA. Twenty-one percent of the CTC from patients with lung cancer identified by the CellSearch system could be isolated individually and amplified. CONCLUSIONS: CTC enriched by the CellSearch system were sorted by FACS, and DNA retrieved and amplified with an overall efficiency of 20%. Analysis of the sequencing data showed that this DNA could be used for variant calling, but not for quantitative measurements such as copy number detection. Close to 55% of the exome of single SKBR-3 cells were successfully sequenced to 20× depth making it possible to call 72% of the variants. The overall coverage was reduced to 30% at 20× depth, making it possible to call 56% of the variants in CellSave-fixed cells.

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Efficiency of whole genome amplification of Single Circulating Tumor Cells enriched by CellSearch and sorted by FACS

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