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Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells

BACKGROUND: Whole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material preservation is still elusive. RESULTS: In respect to the accuracy of SNP/mutation, indel, and...

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Autores principales: Babayan, Anna, Alawi, Malik, Gormley, Michael, Müller, Volkmar, Wikman, Harriet, McMullin, Ryan P., Smirnov, Denis A., Li, Weimin, Geffken, Maria, Pantel, Klaus, Joosse, Simon A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Impact Journals LLC 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593545/
https://www.ncbi.nlm.nih.gov/pubmed/28915574
http://dx.doi.org/10.18632/oncotarget.10701
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author Babayan, Anna
Alawi, Malik
Gormley, Michael
Müller, Volkmar
Wikman, Harriet
McMullin, Ryan P.
Smirnov, Denis A.
Li, Weimin
Geffken, Maria
Pantel, Klaus
Joosse, Simon A.
author_facet Babayan, Anna
Alawi, Malik
Gormley, Michael
Müller, Volkmar
Wikman, Harriet
McMullin, Ryan P.
Smirnov, Denis A.
Li, Weimin
Geffken, Maria
Pantel, Klaus
Joosse, Simon A.
author_sort Babayan, Anna
collection PubMed
description BACKGROUND: Whole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material preservation is still elusive. RESULTS: In respect to the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling, the HiSeq2000 platform outperformed IonProton in all aspects. Furthermore, more accurate SNP/mutation and indel calling was demonstrated using single tumor cells obtained from EDTA-collected blood in respect to CellSave-preserved blood, whereas CNA analysis in our study was not detectably affected by fixation. Although MDA-based WGA yielded the highest DNA amount, DNA quality was not adequate for downstream analysis. PCR-based WGA demonstrates superiority over MDA-PCR combining technique for SNP and indel analysis in single cells. However, SNP calling performance of MDA-PCR WGA improves with increasing amount of input DNA, whereas CNA analysis does not. The performance of PCR-based WGA did not significantly improve with increase of input material. CNA profiles of single cells, amplified with MDA-PCR technique and sequenced on both HiSeq2000 and IonProton platforms, resembled unamplified DNA the most. MATERIALS AND METHODS: We analyzed the performance of PCR-based, multiple-displacement amplification (MDA)-based, and MDA-PCR combining WGA techniques (WGA kits Ampli1, REPLI-g, and PicoPlex, respectively) on single and pooled tumor cells obtained from EDTA- and CellSave-preserved blood and archival material. Amplified DNA underwent exome-Seq with the Illumina HiSeq2000 and ThermoFisher IonProton platforms. CONCLUSION: We demonstrate the feasibility of single cell genotyping of differently preserved material, nevertheless, WGA and NGS approaches have to be chosen carefully depending on the study aims.
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spelling pubmed-55935452017-09-14 Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells Babayan, Anna Alawi, Malik Gormley, Michael Müller, Volkmar Wikman, Harriet McMullin, Ryan P. Smirnov, Denis A. Li, Weimin Geffken, Maria Pantel, Klaus Joosse, Simon A. Oncotarget Research Paper BACKGROUND: Whole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material preservation is still elusive. RESULTS: In respect to the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling, the HiSeq2000 platform outperformed IonProton in all aspects. Furthermore, more accurate SNP/mutation and indel calling was demonstrated using single tumor cells obtained from EDTA-collected blood in respect to CellSave-preserved blood, whereas CNA analysis in our study was not detectably affected by fixation. Although MDA-based WGA yielded the highest DNA amount, DNA quality was not adequate for downstream analysis. PCR-based WGA demonstrates superiority over MDA-PCR combining technique for SNP and indel analysis in single cells. However, SNP calling performance of MDA-PCR WGA improves with increasing amount of input DNA, whereas CNA analysis does not. The performance of PCR-based WGA did not significantly improve with increase of input material. CNA profiles of single cells, amplified with MDA-PCR technique and sequenced on both HiSeq2000 and IonProton platforms, resembled unamplified DNA the most. MATERIALS AND METHODS: We analyzed the performance of PCR-based, multiple-displacement amplification (MDA)-based, and MDA-PCR combining WGA techniques (WGA kits Ampli1, REPLI-g, and PicoPlex, respectively) on single and pooled tumor cells obtained from EDTA- and CellSave-preserved blood and archival material. Amplified DNA underwent exome-Seq with the Illumina HiSeq2000 and ThermoFisher IonProton platforms. CONCLUSION: We demonstrate the feasibility of single cell genotyping of differently preserved material, nevertheless, WGA and NGS approaches have to be chosen carefully depending on the study aims. Impact Journals LLC 2016-07-19 /pmc/articles/PMC5593545/ /pubmed/28915574 http://dx.doi.org/10.18632/oncotarget.10701 Text en Copyright: © 2017 Babayan et al. http://creativecommons.org/licenses/by/3.0/ This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/) (CC-BY), which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Research Paper
Babayan, Anna
Alawi, Malik
Gormley, Michael
Müller, Volkmar
Wikman, Harriet
McMullin, Ryan P.
Smirnov, Denis A.
Li, Weimin
Geffken, Maria
Pantel, Klaus
Joosse, Simon A.
Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
title Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
title_full Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
title_fullStr Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
title_full_unstemmed Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
title_short Comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
title_sort comparative study of whole genome amplification and next generation sequencing performance of single cancer cells
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593545/
https://www.ncbi.nlm.nih.gov/pubmed/28915574
http://dx.doi.org/10.18632/oncotarget.10701
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