Efficiency of whole genome amplification of single circulating tumor cells enriched by CellSearch and sorted by FACS

Joost Franciscus Swennenhuis, J. Reumers, K. Thys, J. Aerssens, Leonardus Wendelinus Mathias Marie Terstappen

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Abstract

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.
Original languageEnglish
Article number106
Pages (from-to)1-11
JournalGenome medicine
Volume5
Issue number11
DOIs
Publication statusPublished - 2013

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Circulating Neoplastic Cells
Flow Cytometry
Genome
DNA
Exome
Lung Neoplasms
Blood Cells

Keywords

  • IR-90098
  • METIS-300984

Cite this

@article{0312ef00f05349279100a0a9426203f3,
title = "Efficiency of whole genome amplification of single circulating tumor cells enriched by CellSearch and sorted by FACS",
abstract = "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.",
keywords = "IR-90098, METIS-300984",
author = "Swennenhuis, {Joost Franciscus} and J. Reumers and K. Thys and J. Aerssens and Terstappen, {Leonardus Wendelinus Mathias Marie}",
note = "Open access",
year = "2013",
doi = "10.1186/gm510",
language = "English",
volume = "5",
pages = "1--11",
journal = "Genome medicine",
issn = "1756-994X",
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Efficiency of whole genome amplification of single circulating tumor cells enriched by CellSearch and sorted by FACS. / Swennenhuis, Joost Franciscus; Reumers, J.; Thys, K.; Aerssens, J.; Terstappen, Leonardus Wendelinus Mathias Marie.

In: Genome medicine, Vol. 5, No. 11, 106, 2013, p. 1-11.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Efficiency of whole genome amplification of single circulating tumor cells enriched by CellSearch and sorted by FACS

AU - Swennenhuis, Joost Franciscus

AU - Reumers, J.

AU - Thys, K.

AU - Aerssens, J.

AU - Terstappen, Leonardus Wendelinus Mathias Marie

N1 - Open access

PY - 2013

Y1 - 2013

N2 - 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.

AB - 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.

KW - IR-90098

KW - METIS-300984

U2 - 10.1186/gm510

DO - 10.1186/gm510

M3 - Article

VL - 5

SP - 1

EP - 11

JO - Genome medicine

JF - Genome medicine

SN - 1756-994X

IS - 11

M1 - 106

ER -