Method for identifying cells based on DNA replication domain timing profiles
Methods for identifying and/or distinguishing a homogeneous population of cells based on their replication domain timing profile using high resolution genomic arrays or sequencing procedures are provided. These methods may be used to compare the replication timing profile for a population of cells to another replication timing profile(s), a replication timing fingerprint, and/or one or more informative segments of a replication timing fingerprint, which may be simultaneously or previously determined and/or contained in a database, to determine whether there is a match between them. Based on such information, the identity of the population of cells may be determined, or the identity of the population of cells may be distinguished from other populations of cells or cell types. Methods for determining a replication timing fingerprint for particular cell types are also provided.
1. A method for identifying cells comprising the following steps:
(a) separating a population of sample cells having a particular cell type into subpopulations of sample cells based on cell cycle stages, wherein each subpopulation of sample cells consists essentially of sample cells from a specific cell cycle phase;
(b) isolating genomic DNA from each subpopulation of sample cells;
(c) separately sequencing genomic DNA isolated from each subpopulation of sample cells, thereby generating a replication timing test profile for the population of sample cells; and
(d) identifying the particular cell type of the population of sample cells by comparing the replication timing test profile to a set of replication timing reference profiles for a set of known cell types.
2. The method of claim 1 , wherein the population of sample cells comprises a cell line.
3. The method of claim 1 , wherein the population of sample cells comprises primary cells derived from an individual.
4. The method of claim 1 , wherein the population of sample cells comprises embryonic stem cells, precursor cells, iPS cells, or differentiated cells.
5. The method of claim 1 , wherein the population of sample cells comprises diseased, transformed, or tumorigenic cells.
6. The method claim 1 , wherein the population of sample cells comprises a population of mammalian cells.
7. The method of claim 1 , wherein each replication timing reference profile of the set of replication timing reference profiles comprises a replication timing fingerprint for a particular known cell type.
8. The method of claim 7 , wherein the replication timing fingerprint is defined as at least one region of a chromosome from cells of the particular known cell type that differs in replication timing ratio values by at least about 0.5 across a distance of at least about 50 kilobases (kb) compared to different cell types.
9. The method of claim 1 ,
wherein the population of sample cells are separated into a subpopulation of G1-phase sample cells and a subpopulation of S-phase sample cells,
wherein the replication timing test profile comprises loess-smoothed replication timing ratio values for the population of sample cells based on sequencing the genomic DNA of the subpopulation of G1-phase sample cells and the genomic DNA of the subpopulation of S-phase sample cells, and
wherein each loess-smoothed replication timing ratio value of the loess-smoothed replication timing ratio values is equal to log 2 (G1-phase/S-phase replication).
10. The method of claim 9 , wherein the separating step comprises separating cells by fluorescent-activated cell sorting (FACS).
11. The method of claim 1 ,
wherein the population of sample cells are separated into a subpopulation of early S-phase sample cells and a subpopulation of late S-phase sample cells,
wherein the replication timing test profile comprises loess-smoothed replication timing ratio values for the population of sample cells based on sequencing the genomic DNA of the subpopulation of early S-phase sample cells and the genomic DNA of the subpopulation of late S-phase sample cells, and
wherein each loess-smoothed replication timing ratio value of the loess-smoothed replication timing ratio values is equal to log 2 (early/late S-phase replication).
12. The method of claim 1 comprising,
generating the set of replication timing reference profiles for a set of known cell types, wherein each replication timing reference profile is generated by:
separating a population of reference cells having a particular known cell type into a subpopulation of G1-phase reference cells and a subpopulation of S-phase reference cells;
isolating genomic DNA separately from the subpopulation of G1-phase reference cells and the subpopulation of S-phase reference cells; and
separately sequencing genomic DNA isolated from the subpopulation of G1-phase reference cells and genomic DNA isolated from the subpopulation of S-phase reference cells, thereby generating a replication timing reference profile for the particular known cell type of the population of reference cells.
13. The method of claim 1 comprising generating the set of replication timing reference profiles for a set of known cell types, wherein each replication timing reference profile is generated by:
separating a population of reference cells having a particular known cell type into a subpopulation of early S-phase reference cells and a subpopulation of late S-phase reference cells;
isolating genomic DNA separately from the subpopulation of early S-phase reference cells and the subpopulation of late S-phase reference cells; and
separately sequencing genomic DNA isolated from the subpopulation of early S-phase reference cells and the subpopulation of late S-phase reference cells, thereby generating a replication timing profile for the particular known cell type of the population of reference cells.
14. The method of claim 1 , wherein the replication timing test profile comprises loess-smoothed replication timing ratio values for the population of sample cells,
wherein each replication timing reference profile of the set of replication timing reference profiles comprises loess-smoothed replication timing ratio values for a particular known cell type,
wherein the particular cell type of the population of sample cells is identified as one of the set of known cell types if the loess-smoothed replication timing ratio values for the population of sample cells are substantially the same as loess-smoothed replication timing ratio values for the one of the set of known cell types.
15. The method of claim 14 , wherein the loess-smoothed replication timing ratio values for the population of sample cells are substantially the same as loess-smoothed replication timing ratio values for the one of the set of known cell types if at least about 95% of the loess-smoothed replication timing ratio values for the population of sample cells and the loess-smoothed replication timing ratio values for the one of the set of known cell types differ by a differential value of less than about 0.5.
16. The method of claim 14 , wherein the loess-smoothed replication timing ratio values for the population of sample cells are substantially the same as loess-smoothed replication timing ratio values for the one of the set of known cell types if less than about 5% of the loess-smoothed replication timing ratio values for the population of sample cells and the loess-smoothed replication timing ratio values for the one of the set of known cell types differ by a differential value of more than about 0.5.
17. The method of claim 1 , wherein step (d) is performed by a computer.