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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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Observed somatic point mutations. (A) Genome-wide sequence coverage and mutation localization in an untreated cell and an ENU-treated cell. The outer track represents binned coverage with an upper cutoff of 50×. The inner track shows the location of detected point mutations (represented as dark points). (B) Histograms of mutant read frequencies for point mutations on chr2L and chrX. The dotted lines indicate a normal distribution with mean of 25 for chr2L and 50 for chrX with standard deviations of 22.
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gkr949-F2: Observed somatic point mutations. (A) Genome-wide sequence coverage and mutation localization in an untreated cell and an ENU-treated cell. The outer track represents binned coverage with an upper cutoff of 50×. The inner track shows the location of detected point mutations (represented as dark points). (B) Histograms of mutant read frequencies for point mutations on chr2L and chrX. The dotted lines indicate a normal distribution with mean of 25 for chr2L and 50 for chrX with standard deviations of 22.

Mentions: The results indicated sufficient coverage (20×) for genotyping at between 40% and 80% of target bases (Table 1). The incomplete coverage is due to amplification bias, which can be pronounced especially with small template amounts (19). While we optimized the MDA protocol, locus dropout was still observed, as was a significant level of allele dropout. The latter was measured using both heterozygous SNPs present in our S2 cell line population and the mutant read frequency distribution, which produced similar results. A comparison of the mutation load in the control and ENU-treated cells showed a 7.5-fold induction of point mutations by ENU on average in the exposed cells (Figure 2A, Table 1). We chose multiple somatic mutations for validation with Sanger sequencing using the remaining amplified material from our single cells and found no evidence of false positives (for an example, see Supplementary Figure S2).Figure 2.


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

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

Observed somatic point mutations. (A) Genome-wide sequence coverage and mutation localization in an untreated cell and an ENU-treated cell. The outer track represents binned coverage with an upper cutoff of 50×. The inner track shows the location of detected point mutations (represented as dark points). (B) Histograms of mutant read frequencies for point mutations on chr2L and chrX. The dotted lines indicate a normal distribution with mean of 25 for chr2L and 50 for chrX with standard deviations of 22.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkr949-F2: Observed somatic point mutations. (A) Genome-wide sequence coverage and mutation localization in an untreated cell and an ENU-treated cell. The outer track represents binned coverage with an upper cutoff of 50×. The inner track shows the location of detected point mutations (represented as dark points). (B) Histograms of mutant read frequencies for point mutations on chr2L and chrX. The dotted lines indicate a normal distribution with mean of 25 for chr2L and 50 for chrX with standard deviations of 22.
Mentions: The results indicated sufficient coverage (20×) for genotyping at between 40% and 80% of target bases (Table 1). The incomplete coverage is due to amplification bias, which can be pronounced especially with small template amounts (19). While we optimized the MDA protocol, locus dropout was still observed, as was a significant level of allele dropout. The latter was measured using both heterozygous SNPs present in our S2 cell line population and the mutant read frequency distribution, which produced similar results. A comparison of the mutation load in the control and ENU-treated cells showed a 7.5-fold induction of point mutations by ENU on average in the exposed cells (Figure 2A, Table 1). We chose multiple somatic mutations for validation with Sanger sequencing using the remaining amplified material from our single cells and found no evidence of false positives (for an example, see Supplementary Figure S2).Figure 2.

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