Decoding cell lineage from acquired mutations using arbitrary deep sequencing

Department of Pathology, University of Washington School of Medicine, Seattle, Washington, USA.
Nature Methods (Impact Factor: 32.07). 11/2011; 9(1):78-80. DOI: 10.1038/nmeth.1781
Source: PubMed


Because mutations are inevitable, the genome of each cell in a multicellular organism becomes unique and therefore encodes a record of its ancestry. Here we coupled arbitrary single primer PCR with next-generation DNA sequencing to catalog mutations and deconvolve the phylogeny of cultured mouse cells. This study helps pave the way toward construction of retrospective cell-fate maps based on mutations accumulating in genomes of somatic cells.

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    • "Determining generation in vivo remains challenging as often it cannot be achieved by direct observation or cell stain methods. Estimating replicative history, cell depth and lineage trees has been proposed by measurement of average telomere length [21] [1] [68] [72] [53] [25] or by the number of somatic mutations, which are introduced during DNA duplication [57] [67] [58] [70] [50] [7]. While methods in this direction rely on inference rather than direct observation, they offer the possibility of tracing more than 10 generations in a wide range of species, including humans. "
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    ABSTRACT: For proliferating cells subject to both division and death, how can one estimate the average generation number of the living population without continuous observation or a division-diluting dye? In this paper we provide a method for cell systems such that at each division there is an unlikely, heritable one-way label change that has no impact other than to serve as a distinguishing marker. If the probability of label change per cell generation can be determined and the proportion of labeled cells at a given time point can be measured, we establish that the average generation number of living cells can be estimated. Crucially, the estimator does not depend on knowledge of the statistics of cell cycle, death rates or total cell numbers. We validate the estimator and illustrate its features through comparison with published data and physiologically parameterized stochastic simulations, using it to suggest new experimental designs.
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    • "Mouse studies were approved by the University of Washington Institutional Animal Care and Use Committee (Protocol 3015–04). Pold1+/eMlh1+/Δ mice were obtained from B. Preston (University of Washington) [16]. The DNA polymerase delta gene Pold1 retained an inactive exonuclease domain due to a single point mutation (D400A) [23,24], while the mismatch repair gene Mlh1 was dysfunctional due to the deletion of exon 2 [25]. "
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    ABSTRACT: Background The C. elegans cell fate map, in which the lineage of its approximately 1000 cells is visibly charted beginning from the zygote, represents a developmental biology milestone. Nematode development is invariant from one specimen to the next, whereas in mammals, aspects of development are probabilistic, and development exhibits variation between even genetically identical individuals. Consequently, a single defined cell fate map applicable to all individuals cannot exist. Results To determine the extent to which patterns of cell lineage are conserved between different mice, we have employed the recently developed method of “phylogenetic fate mapping” to compare cell fate maps in siblings. In this approach, somatic mutations arising in individual cells are used to retrospectively deduce lineage relationships through phylogenetic and—as newly investigated here—related analytical approaches based on genetic distance. We have cataloged genomic mutations at an average of 110 mutation-prone polyguanine (polyG) tracts for about 100 cells clonally isolated from various corresponding tissues of each of two littermates of a hypermutable mouse strain. Conclusions We find that during mouse development, muscle and fat arise from a mixed progenitor cell pool in the germ layer, but, contrastingly, vascular endothelium in brain derives from a smaller source of progenitor cells. Additionally, formation of tissue primordia is marked by establishment of left and right lateral compartments, with restricted cell migration between divisions. We quantitatively demonstrate that development represents a combination of stochastic and deterministic events, offering insight into how chance influences normal development and may give rise to birth defects.
    BMC Genomics 01/2013; 14(1):39. DOI:10.1186/1471-2164-14-39 · 3.99 Impact Factor
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    ABSTRACT: Next-generation DNA sequencing has revolutionized genomic studies and is driving the implementation of precision diagnostics. The ability of these technologies to disentangle sequence heterogeneity, however, is limited by their relatively high error rates. A Several single molecule barcoding strategies have been propose to reduce the overall error frequency. A Duplex Sequencing additionally exploits the fact that DNA is double-strand, with one strand reciprocally encoding the sequence information of its complement, and can eliminate nearly all sequencing errors by comparing the sequence of individually tagged amplicons derived from one strand of DNA with that of its complementary strand. This method reduces errors to fewer than one per ten million nucleotides sequenced.
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