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Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing

BACKGROUND: The current literature on single cell genomic analyses on the DNA level is conflicting regarding requirements for cell quality, amplification success rates, allelic dropouts and resolution, lacking a systematic comparison of multiple cell input down to the single cell. We hypothesized th...

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Autores principales: Simonsen, Anita T., Hansen, Marcus C., Kjeldsen, Eigil, Møller, Peter L., Hindkjær, Johnny J., Hokland, Peter, Aggerholm, Anni
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142419/
https://www.ncbi.nlm.nih.gov/pubmed/30223769
http://dx.doi.org/10.1186/s12864-018-5063-5
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author Simonsen, Anita T.
Hansen, Marcus C.
Kjeldsen, Eigil
Møller, Peter L.
Hindkjær, Johnny J.
Hokland, Peter
Aggerholm, Anni
author_facet Simonsen, Anita T.
Hansen, Marcus C.
Kjeldsen, Eigil
Møller, Peter L.
Hindkjær, Johnny J.
Hokland, Peter
Aggerholm, Anni
author_sort Simonsen, Anita T.
collection PubMed
description BACKGROUND: The current literature on single cell genomic analyses on the DNA level is conflicting regarding requirements for cell quality, amplification success rates, allelic dropouts and resolution, lacking a systematic comparison of multiple cell input down to the single cell. We hypothesized that such a correlation assay would provide an approach to address the latter issues, utilizing the leukemic cell line OCI-AML3 with a known set of genetic aberrations. RESULTS: By analyzing single and multiple cell replicates (2 to 50 cells) purified by micromanipulation and serial dilution we stringently assessed the signal-to-noise ratio (SNR) from single as well as a discrete number of cells based on a multiple displacement amplification method, with whole exome sequencing as signal readout. In this setting, known OCI-AML3 mutations as well as large copy number alterations could be identified, adding to the current knowledge of cytogenetic status. The presence of DNMT3A R882C, NPM1 W288 fs and NRAS Q61L was consistent, in spite of uneven allelic read depths. In contrast, at the level of single cells, we observed that one-third to half of all variants were not reproduced in the replicate sample, and this allelic mismatch displayed an exponential function of cell input. Large signature duplications were discernible from 5 cells, whereas deletions were visible down to the single cell. Thus, even under highly optimized conditions, single cell whole genome amplification and interpretation must be taken with considerable caution, given that allelic change is frequent and displays low SNR. Allelic noise is rapidly alleviated with increased cell input, and the SNR is doubled from 2 to 50 cells. CONCLUSIONS: In conclusion, we demonstrate noisy allele distributions, when analyzing genetic aberrations within single cells relative to multiple cells. Based on the presented data we recommend that single cell analyses should include replicate cell dilution assays for a given setup for relative assessment of procedure-specific SNR to ensure that the resolution supports the specific hypotheses. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12864-018-5063-5) contains supplementary material, which is available to authorized users.
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spelling pubmed-61424192018-09-20 Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing Simonsen, Anita T. Hansen, Marcus C. Kjeldsen, Eigil Møller, Peter L. Hindkjær, Johnny J. Hokland, Peter Aggerholm, Anni BMC Genomics Research Article BACKGROUND: The current literature on single cell genomic analyses on the DNA level is conflicting regarding requirements for cell quality, amplification success rates, allelic dropouts and resolution, lacking a systematic comparison of multiple cell input down to the single cell. We hypothesized that such a correlation assay would provide an approach to address the latter issues, utilizing the leukemic cell line OCI-AML3 with a known set of genetic aberrations. RESULTS: By analyzing single and multiple cell replicates (2 to 50 cells) purified by micromanipulation and serial dilution we stringently assessed the signal-to-noise ratio (SNR) from single as well as a discrete number of cells based on a multiple displacement amplification method, with whole exome sequencing as signal readout. In this setting, known OCI-AML3 mutations as well as large copy number alterations could be identified, adding to the current knowledge of cytogenetic status. The presence of DNMT3A R882C, NPM1 W288 fs and NRAS Q61L was consistent, in spite of uneven allelic read depths. In contrast, at the level of single cells, we observed that one-third to half of all variants were not reproduced in the replicate sample, and this allelic mismatch displayed an exponential function of cell input. Large signature duplications were discernible from 5 cells, whereas deletions were visible down to the single cell. Thus, even under highly optimized conditions, single cell whole genome amplification and interpretation must be taken with considerable caution, given that allelic change is frequent and displays low SNR. Allelic noise is rapidly alleviated with increased cell input, and the SNR is doubled from 2 to 50 cells. CONCLUSIONS: In conclusion, we demonstrate noisy allele distributions, when analyzing genetic aberrations within single cells relative to multiple cells. Based on the presented data we recommend that single cell analyses should include replicate cell dilution assays for a given setup for relative assessment of procedure-specific SNR to ensure that the resolution supports the specific hypotheses. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s12864-018-5063-5) contains supplementary material, which is available to authorized users. BioMed Central 2018-09-17 /pmc/articles/PMC6142419/ /pubmed/30223769 http://dx.doi.org/10.1186/s12864-018-5063-5 Text en © The Author(s). 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Simonsen, Anita T.
Hansen, Marcus C.
Kjeldsen, Eigil
Møller, Peter L.
Hindkjær, Johnny J.
Hokland, Peter
Aggerholm, Anni
Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing
title Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing
title_full Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing
title_fullStr Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing
title_full_unstemmed Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing
title_short Systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing
title_sort systematic evaluation of signal-to-noise ratio in variant detection from single cell genome multiple displacement amplification and exome sequencing
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142419/
https://www.ncbi.nlm.nih.gov/pubmed/30223769
http://dx.doi.org/10.1186/s12864-018-5063-5
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