A comparison of genomic methods to assess DNA replication timing
Abstract Replication timing (RT), the temporal order in which genomic regions replicate, is considered a functional feature of multiple cellular processes and chromatin organization. Two approaches to measure RT are the Repli-seq and DNA copy number (also called S/G1) methods. We previously adapted...
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Nature Portfolio
2025-05-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-025-02699-0 |
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| author | Emily Wheeler Leigh Mickelson-Young Emily E. Wear Mason Burroughs Hank W. Bass Lorenzo Concia William F. Thompson Linda Hanley-Bowdoin |
| author_facet | Emily Wheeler Leigh Mickelson-Young Emily E. Wear Mason Burroughs Hank W. Bass Lorenzo Concia William F. Thompson Linda Hanley-Bowdoin |
| author_sort | Emily Wheeler |
| collection | DOAJ |
| description | Abstract Replication timing (RT), the temporal order in which genomic regions replicate, is considered a functional feature of multiple cellular processes and chromatin organization. Two approaches to measure RT are the Repli-seq and DNA copy number (also called S/G1) methods. We previously adapted Repli-seq using 5-ethynyl-2’- deoxyuridine (EdU) pulse-labeling and bivariate flow sorting, and while the approach offers high resolution and exposes heterogeneity in timing, the S/G1 method is a simpler, faster and less resource-intensive assessment. Here we modified the S/G1 technique by using EdU labeling (EdU-S/G1) to facilitate better separation of replicating from non-replicating nuclei during flow sorting, which enables the collection of a more pure sample of G1-phase nuclei. When comparing the three methods we found that profiles from the S/G1 and EdU-S/G1 methods are highly correlated with each other and with Repli-seq profiles for early replication. We also found that the EdU-S/G1 approach offers a better representation of replication in early and late S phase than the conventional S/G1 method. However, the high reproducibility of RT profiles among all three methods indicates that considerations of cost and sample availability can drive the decision of which method to choose. |
| format | Article |
| id | doaj-art-61b6fa8ad6df43d9a049b8f4fe6c29ab |
| institution | DOAJ |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Nature Portfolio |
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| spelling | doaj-art-61b6fa8ad6df43d9a049b8f4fe6c29ab2025-08-20T03:08:40ZengNature PortfolioScientific Reports2045-23222025-05-0115111310.1038/s41598-025-02699-0A comparison of genomic methods to assess DNA replication timingEmily Wheeler0Leigh Mickelson-Young1Emily E. Wear2Mason Burroughs3Hank W. Bass4Lorenzo Concia5William F. Thompson6Linda Hanley-Bowdoin7Department of Plant and Microbial Biology, North Carolina State UniversityDepartment of Plant and Microbial Biology, North Carolina State UniversityDepartment of Plant and Microbial Biology, North Carolina State UniversityDepartment of Plant and Microbial Biology, North Carolina State UniversityDepartment of Biological Science, Florida State UniversityTexas Advanced Computing Center, University of Texas at AustinDepartment of Plant and Microbial Biology, North Carolina State UniversityDepartment of Plant and Microbial Biology, North Carolina State UniversityAbstract Replication timing (RT), the temporal order in which genomic regions replicate, is considered a functional feature of multiple cellular processes and chromatin organization. Two approaches to measure RT are the Repli-seq and DNA copy number (also called S/G1) methods. We previously adapted Repli-seq using 5-ethynyl-2’- deoxyuridine (EdU) pulse-labeling and bivariate flow sorting, and while the approach offers high resolution and exposes heterogeneity in timing, the S/G1 method is a simpler, faster and less resource-intensive assessment. Here we modified the S/G1 technique by using EdU labeling (EdU-S/G1) to facilitate better separation of replicating from non-replicating nuclei during flow sorting, which enables the collection of a more pure sample of G1-phase nuclei. When comparing the three methods we found that profiles from the S/G1 and EdU-S/G1 methods are highly correlated with each other and with Repli-seq profiles for early replication. We also found that the EdU-S/G1 approach offers a better representation of replication in early and late S phase than the conventional S/G1 method. However, the high reproducibility of RT profiles among all three methods indicates that considerations of cost and sample availability can drive the decision of which method to choose.https://doi.org/10.1038/s41598-025-02699-0 |
| spellingShingle | Emily Wheeler Leigh Mickelson-Young Emily E. Wear Mason Burroughs Hank W. Bass Lorenzo Concia William F. Thompson Linda Hanley-Bowdoin A comparison of genomic methods to assess DNA replication timing Scientific Reports |
| title | A comparison of genomic methods to assess DNA replication timing |
| title_full | A comparison of genomic methods to assess DNA replication timing |
| title_fullStr | A comparison of genomic methods to assess DNA replication timing |
| title_full_unstemmed | A comparison of genomic methods to assess DNA replication timing |
| title_short | A comparison of genomic methods to assess DNA replication timing |
| title_sort | comparison of genomic methods to assess dna replication timing |
| url | https://doi.org/10.1038/s41598-025-02699-0 |
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