Single-molecule, quantitative detection of low-abundance somatic mutations by high-throughput sequencing

Maslov, Alexander Y.; Makhortov, S. D.; Sun, Shixiang; Heid, Johanna; Dong, Xiao; Lee, Moonsook; Vijg, Jan

doi:10.1126/sciadv.abm3259

Cited by 20 publications

(9 citation statements)

References 19 publications

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“…5A). This is in keeping with previously published ENU-induced mutational spectra by us and others (Barbaric, Wells et al 2007, Maslov, Makhortov et al 2022). We then inferred SNV mutational signatures de novo using non-negative matrix factorization (Supplementary Fig.…”

Section: Resultssupporting

confidence: 93%

“…2A). These results were orthogonally validated with our recently developed single-molecule assay (Maslov, Makhortov et al 2022) (Supplementary Note 1, Supplementary Figs. 4B-E).…”

Section: Repeated Mutagen Treatment Only Slightly Affects Cell Growthmentioning

confidence: 57%

“…200ng gDNA was used as input for library construction for SNV analysis. Library preparations were performed as described elsewhere (Maslov, Makhortov et al 2022). mRNA libraries were prepared and sequenced by Novogene.…”

Section: Methodsmentioning

confidence: 99%

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Negative selection allows human primary fibroblasts to tolerate high somatic mutation loads induced by N-ethyl-N-nitrosourea

Heid,

Cutler,

Sun

et al. 2024

Preprint

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High-throughput sequencing at the single-cell and single-molecule level has shown that mutation rate is much higher in somatic cells than in the germline, with thousands of mutations accumulating with age in most human tissues. While there is now ample evidence that some of these mutations can clonally amplify and lead to disease, most notably cancer, the total burden of mutations a cell can tolerate without functional decline remains unknown. Here we addressed this question by exposing human primary fibroblasts multiple times to low doses of N-ethyl-N-nitrosourea (ENU) and quantitatively analyzing somatic mutation burden using single-cell whole genome sequencing. The results indicate that individual cells can sustain ~60,000 single-nucleotide variants (SNVs) with only a slight adverse effect on growth rate. We found evidence for selection against potentially deleterious variants in gene coding regions as well as depletion of mutations in sequences associated with genetic pathways expressed in these human fibroblasts, most notably those relevant for maintaining basic cellular function and growth. However, no evidence of negative selection was found for variants in non-coding regions. We conclude that actively proliferating fibroblasts can tolerate very high levels of somatic mutations without major adverse effects on growth rate via negative selection against damaging coding mutations. Since most tissues in adult organisms have very limited capacity to select against mutations based on a growth disadvantage, these results suggest that a causal effect of somatic mutations in aging and disease cannot be ruled out.

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Section: Resultssupporting

confidence: 93%

“…2A). These results were orthogonally validated with our recently developed single-molecule assay (Maslov, Makhortov et al 2022) (Supplementary Note 1, Supplementary Figs. 4B-E).…”

Section: Repeated Mutagen Treatment Only Slightly Affects Cell Growthmentioning

confidence: 57%

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Negative selection allows human primary fibroblasts to tolerate high somatic mutation loads induced by N-ethyl-N-nitrosourea

Heid,

Cutler,

Sun

et al. 2024

Preprint

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“…These DNA strands can be amplified by PCR, enabling detection with single-molecule sensitivity. ( 51, 52 ) Thus, the sensitivity of our method ultimately depends on the efficiency of PEA. Using the best-performing antibodies, we have demonstrated that PEA can quantitatively convert 10 amol of PTC amino acids to DNA reporters in a 20 µL reaction containing approximately 1,000 beads ( Fig.…”

Section: Discussionmentioning

confidence: 99%

Peptide sequencing via reverse translation of peptides into DNA

Zheng,

Sun,

Eisenstein

et al. 2024

Preprint

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Scalable methods that can accurately sequence peptides at single-amino acid resolution could significantly advance proteomic studies. We present a protein sequencing method based on the “reverse translation” of peptide sequence information into DNA barcodes that document the identity, position, and the originating peptide of each amino acid. We employ a modified Edman degradation process that converts peptides into DNA-barcoded amino acids, which are subsequently detected by proximity extension assay, yielding multi-barcoded DNA outputs that can be PCR amplified and sequenced. Using our method, we sequenced multiple consecutive amino acids within a model peptide. This method also enables the differentiation of single amino acid substitutions, and the identification of post-translational modifications and their positions within multiple peptides simultaneously. With further development, we anticipate that this method will enable highly parallelde novoprotein sequencing with single-molecule sensitivity.

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“…However, because they tend to be rare, and are distributed randomly across in the genome, it can be difficult to detect them among the overwhelming background of WT DNA. To solve this problem, a number of highly sophisticated genome-wide detection techniques have been developed that greatly improve the detection of mutations, including Cypher-seq (13), circle-sequencing(30), o2nsequencing (31), SMM-seq (32) and duplex sequencing (19).…”

Section: Discussionmentioning

confidence: 99%

MADDD-seq, a novel massively parallel sequencing tool for simultaneous detection of DNA damage and mutations

Vermulst,

Paskvan,

Chung

et al. 2023

Preprint

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Our genome is exposed to a wide variety of DNA-damaging agents. If left unrepaired, this damage can be fixed into mutations that promote carcinogenesis and the development of genetically inherited diseases. As a result, it is crucial that we can detect DNA damage and mutations with exquisite sensitivity. Here, we describe a modified version of double barcoding sequencing technology termed Mutation And DNA Damage Detection-seq (MADDD-seq) that can detect DNA damage and mutations simultaneously, with a single assay. To demonstrate the utility of MADDD-seq as a multifunctional detection tool, we treated yeast cells with a DNA-damaging agent and tracked the presence of DNA damage and mutations over a 24-hour timespan. These experiments allowed us to identify thousands of adducts and mutations in a single sequencing run and expose the kinetics of DNA repair and mutagenesis in remarkable detail.

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Single-molecule, quantitative detection of low-abundance somatic mutations by high-throughput sequencing

Cited by 20 publications

References 19 publications

Negative selection allows human primary fibroblasts to tolerate high somatic mutation loads induced by N-ethyl-N-nitrosourea

Negative selection allows human primary fibroblasts to tolerate high somatic mutation loads induced by N-ethyl-N-nitrosourea

Peptide sequencing via reverse translation of peptides into DNA

MADDD-seq, a novel massively parallel sequencing tool for simultaneous detection of DNA damage and mutations

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