Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox

Sjors Middelkamp; Freek Manders; Flavia Peci; Markus J van Roosmalen; Diego Montiel González; Eline J M Bertrums; Inge van der Werf; Lucca L M Derks; Niels M Groenen; Mark Verheul; Laurianne Trabut; Cayetano Pleguezuelos-Manzano; Arianne M Brandsma; Evangelia Antoniou; Dirk Reinhardt; Marc Bierings; Mirjam E Belderbos; Ruben van Boxtel

doi:10.1016/j.xgen.2023.100389

Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox

Sjors Middelkamp, Freek Manders, Flavia Peci, Markus J van Roosmalen, Diego Montiel González, Eline J M Bertrums, Inge van der Werf, Lucca L M Derks, Niels M Groenen, Mark Verheul, Laurianne Trabut, Cayetano Pleguezuelos-Manzano, Arianne M Brandsma, Evangelia Antoniou, Dirk Reinhardt, Marc Bierings, Mirjam E Belderbos, Ruben van Boxtel

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Detection of somatic mutations in single cells has been severely hampered by technical limitations of whole-genome amplification. Novel technologies including primary template-directed amplification (PTA) significantly improved the accuracy of single-cell whole-genome sequencing (WGS) but still generate hundreds of artifacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolbox (PTATO), to accurately detect single base substitutions, insertions-deletions (indels), and structural variants in PTA-based WGS data. PTATO includes a machine learning approach and filtering based on recurrence to distinguish PTA artifacts from true mutations with high sensitivity (up to 90%), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that hematopoietic stem cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS, have normal somatic single base substitution burdens but increased numbers of deletions. Our results show that PTATO enables studying somatic mutagenesis in the genomes of single cells with unprecedented sensitivity and accuracy.

Originele taal-2	Engels
Pagina's (van-tot)	100389
Tijdschrift	Cell genomics
Volume	3
Nummer van het tijdschrift	9
DOI's	https://doi.org/10.1016/j.xgen.2023.100389
Status	Gepubliceerd - 13 sep. 2023

Trefwoorden

Fanconi anemia
cancer
hematopoietic stem cells
mutational signatures
primary template-directed amplification
single-cell sequencing
somatic mutations
structural variants
whole-genome amplification
whole-genome sequencing

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10.1016/j.xgen.2023.100389

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Middelkamp, S., Manders, F., Peci, F., van Roosmalen, M. J., González, D. M., Bertrums, E. J. M., van der Werf, I., Derks, L. L. M., Groenen, N. M., Verheul, M., Trabut, L., Pleguezuelos-Manzano, C., Brandsma, A. M., Antoniou, E., Reinhardt, D., Bierings, M., Belderbos, M. E., & van Boxtel, R. (2023). Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox. Cell genomics, 3(9), 100389. https://doi.org/10.1016/j.xgen.2023.100389

@article{32d76d67fb864743acf1fae92b397960,

title = "Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox",

abstract = "Detection of somatic mutations in single cells has been severely hampered by technical limitations of whole-genome amplification. Novel technologies including primary template-directed amplification (PTA) significantly improved the accuracy of single-cell whole-genome sequencing (WGS) but still generate hundreds of artifacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolbox (PTATO), to accurately detect single base substitutions, insertions-deletions (indels), and structural variants in PTA-based WGS data. PTATO includes a machine learning approach and filtering based on recurrence to distinguish PTA artifacts from true mutations with high sensitivity (up to 90%), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that hematopoietic stem cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS, have normal somatic single base substitution burdens but increased numbers of deletions. Our results show that PTATO enables studying somatic mutagenesis in the genomes of single cells with unprecedented sensitivity and accuracy.",

keywords = "Fanconi anemia, cancer, hematopoietic stem cells, mutational signatures, primary template-directed amplification, single-cell sequencing, somatic mutations, structural variants, whole-genome amplification, whole-genome sequencing",

author = "Sjors Middelkamp and Freek Manders and Flavia Peci and {van Roosmalen}, {Markus J} and Gonz{\'a}lez, {Diego Montiel} and Bertrums, {Eline J M} and {van der Werf}, Inge and Derks, {Lucca L M} and Groenen, {Niels M} and Mark Verheul and Laurianne Trabut and Cayetano Pleguezuelos-Manzano and Brandsma, {Arianne M} and Evangelia Antoniou and Dirk Reinhardt and Marc Bierings and Belderbos, {Mirjam E} and {van Boxtel}, Ruben",

note = "{\textcopyright} 2023 The Author(s).",

year = "2023",

month = sep,

day = "13",

doi = "10.1016/j.xgen.2023.100389",

language = "English",

volume = "3",

pages = "100389",

journal = "Cell genomics",

issn = "2666-979X",

publisher = "Cell Press",

number = "9",

}

Middelkamp, S, Manders, F, Peci, F, van Roosmalen, MJ, González, DM, Bertrums, EJM, van der Werf, I, Derks, LLM, Groenen, NM, Verheul, M, Trabut, L, Pleguezuelos-Manzano, C, Brandsma, AM, Antoniou, E, Reinhardt, D, Bierings, M, Belderbos, ME & van Boxtel, R 2023, 'Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox', Cell genomics, vol. 3, nr. 9, blz. 100389. https://doi.org/10.1016/j.xgen.2023.100389

TY - JOUR

T1 - Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox

AU - Middelkamp, Sjors

AU - Manders, Freek

AU - Peci, Flavia

AU - van Roosmalen, Markus J

AU - González, Diego Montiel

AU - Bertrums, Eline J M

AU - van der Werf, Inge

AU - Derks, Lucca L M

AU - Groenen, Niels M

AU - Verheul, Mark

AU - Trabut, Laurianne

AU - Pleguezuelos-Manzano, Cayetano

AU - Brandsma, Arianne M

AU - Antoniou, Evangelia

AU - Reinhardt, Dirk

AU - Bierings, Marc

AU - Belderbos, Mirjam E

AU - van Boxtel, Ruben

PY - 2023/9/13

Y1 - 2023/9/13

N2 - Detection of somatic mutations in single cells has been severely hampered by technical limitations of whole-genome amplification. Novel technologies including primary template-directed amplification (PTA) significantly improved the accuracy of single-cell whole-genome sequencing (WGS) but still generate hundreds of artifacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolbox (PTATO), to accurately detect single base substitutions, insertions-deletions (indels), and structural variants in PTA-based WGS data. PTATO includes a machine learning approach and filtering based on recurrence to distinguish PTA artifacts from true mutations with high sensitivity (up to 90%), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that hematopoietic stem cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS, have normal somatic single base substitution burdens but increased numbers of deletions. Our results show that PTATO enables studying somatic mutagenesis in the genomes of single cells with unprecedented sensitivity and accuracy.

AB - Detection of somatic mutations in single cells has been severely hampered by technical limitations of whole-genome amplification. Novel technologies including primary template-directed amplification (PTA) significantly improved the accuracy of single-cell whole-genome sequencing (WGS) but still generate hundreds of artifacts per amplification reaction. We developed a comprehensive bioinformatic workflow, called the PTA Analysis Toolbox (PTATO), to accurately detect single base substitutions, insertions-deletions (indels), and structural variants in PTA-based WGS data. PTATO includes a machine learning approach and filtering based on recurrence to distinguish PTA artifacts from true mutations with high sensitivity (up to 90%), outperforming existing bioinformatic approaches. Using PTATO, we demonstrate that hematopoietic stem cells of patients with Fanconi anemia, which cannot be analyzed using regular WGS, have normal somatic single base substitution burdens but increased numbers of deletions. Our results show that PTATO enables studying somatic mutagenesis in the genomes of single cells with unprecedented sensitivity and accuracy.

KW - Fanconi anemia

KW - cancer

KW - hematopoietic stem cells

KW - mutational signatures

KW - primary template-directed amplification

KW - single-cell sequencing

KW - somatic mutations

KW - structural variants

KW - whole-genome amplification

KW - whole-genome sequencing

UR - https://www.mendeley.com/catalogue/e8c2bbd0-b54d-3500-a87a-94b8279b472e/

U2 - 10.1016/j.xgen.2023.100389

DO - 10.1016/j.xgen.2023.100389

M3 - Article

C2 - 37719152

SN - 2666-979X

VL - 3

SP - 100389

JO - Cell genomics

JF - Cell genomics

IS - 9

ER -

Comprehensive single-cell genome analysis at nucleotide resolution using the PTA Analysis Toolbox

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