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Direct, genome-wide assessment of DNA mutations in single cells.

Gundry M, Li W, Maqbool SB, Vijg J - Nucleic Acids Res. (2011)

Bottom Line: One way to circumvent this problem and simultaneously account for the mutational heterogeneity within tissues is whole genome sequencing of a representative number of single cells.Here, we show elevated mutation levels in single cells from Drosophila melanogaster S2 and mouse embryonic fibroblast populations after treatment with the powerful mutagen N-ethyl-N-nitrosourea.This method can be applied as a direct measure of exposure to mutagenic agents and for assessing genotypic heterogeneity within tissues or cell populations.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Albert Einstein College of Medicine, New York, NY 10461, USA.

ABSTRACT
DNA mutations are the inevitable consequences of errors that arise during replication and repair of DNA damage. Because of their random and infrequent occurrence, quantification and characterization of DNA mutations in the genome of somatic cells has been difficult. Random, low-abundance mutations are currently inaccessible by standard high-throughput sequencing approaches because they cannot be distinguished from sequencing errors. One way to circumvent this problem and simultaneously account for the mutational heterogeneity within tissues is whole genome sequencing of a representative number of single cells. Here, we show elevated mutation levels in single cells from Drosophila melanogaster S2 and mouse embryonic fibroblast populations after treatment with the powerful mutagen N-ethyl-N-nitrosourea. This method can be applied as a direct measure of exposure to mutagenic agents and for assessing genotypic heterogeneity within tissues or cell populations.

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Somatic point mutation spectra and localization. (A) Mutation spectra for the control and ENU-treated S2 and MEF single cells. Due to a limited number of point mutations, the control cells were combined for the analysis. (B) Strand of origin for ENU-induced mutations found in genic regions of MEFs. (C) Strand of origin for ENU-induced mutations found in genic regions of S2 cells.
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gkr949-F3: Somatic point mutation spectra and localization. (A) Mutation spectra for the control and ENU-treated S2 and MEF single cells. Due to a limited number of point mutations, the control cells were combined for the analysis. (B) Strand of origin for ENU-induced mutations found in genic regions of MEFs. (C) Strand of origin for ENU-induced mutations found in genic regions of S2 cells.

Mentions: A major advantage of direct sequencing is that the mutation spectra can immediately be visualized across the genome. The ENU-induced spectrum was highly consistent across individual cells from the same population, but a larger fraction of C:G →T:A mutations was found in the S2 cells (Figure 3A). This may be due to a more efficient repair of O6-ethyl-guanine adducts by the mouse O6-alkylguanine-DNA alkyltransferase gene (Mgmt) compared with its Drosophila homologue. In spite of this difference, the similarity between the two species also at this level is striking. The spontaneous mutation spectra observed in the untreated cells showed considerably lower levels of A:T →T:A mutations, which are known to be highly enriched following treatment with alkylating agents (12,22).Figure 3.


Direct, genome-wide assessment of DNA mutations in single cells.

Gundry M, Li W, Maqbool SB, Vijg J - Nucleic Acids Res. (2011)

Somatic point mutation spectra and localization. (A) Mutation spectra for the control and ENU-treated S2 and MEF single cells. Due to a limited number of point mutations, the control cells were combined for the analysis. (B) Strand of origin for ENU-induced mutations found in genic regions of MEFs. (C) Strand of origin for ENU-induced mutations found in genic regions of S2 cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3300019&req=5

gkr949-F3: Somatic point mutation spectra and localization. (A) Mutation spectra for the control and ENU-treated S2 and MEF single cells. Due to a limited number of point mutations, the control cells were combined for the analysis. (B) Strand of origin for ENU-induced mutations found in genic regions of MEFs. (C) Strand of origin for ENU-induced mutations found in genic regions of S2 cells.
Mentions: A major advantage of direct sequencing is that the mutation spectra can immediately be visualized across the genome. The ENU-induced spectrum was highly consistent across individual cells from the same population, but a larger fraction of C:G →T:A mutations was found in the S2 cells (Figure 3A). This may be due to a more efficient repair of O6-ethyl-guanine adducts by the mouse O6-alkylguanine-DNA alkyltransferase gene (Mgmt) compared with its Drosophila homologue. In spite of this difference, the similarity between the two species also at this level is striking. The spontaneous mutation spectra observed in the untreated cells showed considerably lower levels of A:T →T:A mutations, which are known to be highly enriched following treatment with alkylating agents (12,22).Figure 3.

Bottom Line: One way to circumvent this problem and simultaneously account for the mutational heterogeneity within tissues is whole genome sequencing of a representative number of single cells.Here, we show elevated mutation levels in single cells from Drosophila melanogaster S2 and mouse embryonic fibroblast populations after treatment with the powerful mutagen N-ethyl-N-nitrosourea.This method can be applied as a direct measure of exposure to mutagenic agents and for assessing genotypic heterogeneity within tissues or cell populations.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Albert Einstein College of Medicine, New York, NY 10461, USA.

ABSTRACT
DNA mutations are the inevitable consequences of errors that arise during replication and repair of DNA damage. Because of their random and infrequent occurrence, quantification and characterization of DNA mutations in the genome of somatic cells has been difficult. Random, low-abundance mutations are currently inaccessible by standard high-throughput sequencing approaches because they cannot be distinguished from sequencing errors. One way to circumvent this problem and simultaneously account for the mutational heterogeneity within tissues is whole genome sequencing of a representative number of single cells. Here, we show elevated mutation levels in single cells from Drosophila melanogaster S2 and mouse embryonic fibroblast populations after treatment with the powerful mutagen N-ethyl-N-nitrosourea. This method can be applied as a direct measure of exposure to mutagenic agents and for assessing genotypic heterogeneity within tissues or cell populations.

Show MeSH
Related in: MedlinePlus