Properties of the set of 893 patterns. (a) Number of patterns with a given number of probe sets associated with a score of 90%. (b) Number of patterns with a given number of samples segregated in the high expression group.

Precision/Recall curves for genes selected by the gap method and k-means. Precision/recall curves in red are associated with the gap method, and the point marked with a circle denotes the precision/recall associated with patterns associated with 63 probe sets or less. Gray curves show precision/recall for all replicates of k-means clustering, and the average expected precision/recall curve is shown in black. Recall values are based on the 71 stem cell markers defined in Table 2, whereas precision is the fraction of marker genes identified in the total number of predicted marker genes.

Sample segregation for selected markers. Samples segregated in the high expression group for probe set markers from the nuclear receptor (nr2f1 and nr2f2), cytochrome P450 family (cytp1a1 and cytp1b1), serpin family (Serpins a1b, a3g, a3n, b9, g1, and h1), and Rab GTPase (rab3d) superfamilies. The nr2f1/nr2f2 and cytp1a1/cytp1b1 gene pairs are highly similar in sequence but their expression patterns are notably different. Undifferentiated and differentiated sample pairs are shaded in pink and blue, respectively.

Heatmap indicating the distribution of patterns for markers. The horizontal axis shows 241 mouse samples used in this study. The vertical axis shows patterns for 1,301 markers either predicted to have high reliability (scoring 100%, n = 1,252; blue) or probe sets belonging to genes ascribed marker roles based on evidence in the literature (n = 49; yellow). Rows were clustered and diagonalized. Vertical separators were used to distinguish major sample cell types. Sample identities are grouped as follows: embryonic, blue; P19 embryonal carcinoma, orange; fibroblasts, purple; spheres, yellow; hematopoietic, red. Gene names are indicated only for the 49 stem cell markers.

Phylogenetic distribution of stem cell markers and their close paralogs in four protein families. Major taxa along the Coelomata lineage are depicted in bold black text with deduced numbers of genes from these families in parentheses. Phylogenetic relations between species were taken from the National Center for Biotechnology Information's Taxonomy Browser, except for the relation between Cephalocordata and Urochordata, which was taken from Vienne and Pontarotti [72]. Species names are given in italic black text. Colored text indicates paralogs for four families: red for cyp1, orange for ebf, blue for nr2f1, and green for rab3. Numbers in parentheses indicate multiple copies of gene (for example, in Actinopterygii genes). Many genes are duplicated in Actinopterygii because of a whole-genome duplication event postulated to have occurred along the ray-finned fish lineage [73]. Most expansion of these four families occurs after divergence of Deuterostomia and before divergence of Teleostei. Underlined genes for mouse indicate genes identified in the selected marker set. The database identifiers of the sequences are given in Additional data file 4. The inset on the lower right corner shows schematic phylogenetic trees for the murine members of the four families. All branches shown had bootstrap values above 0.5. Outlier sequences used for each family (not displayed): D. melanogaster rab3 for rab3, S. purpuratus cytp1 for cytp1, D. melanogaster svp for nrf2f1, and D. melanogaster knot for ebf.

Distributions of hybridization values for probe sets. Each histogram depicts the number of replicates (from a total of 241) with a given hybridization value for a given probe set. For illustrative purposes, we display the distribution of hybridization values of three probe sets selected using the gap method as markers corresponding to (a) a known neural stem cell marker (Nestin; probe set 1449022_at), (b) a novel stem cell marker encoding a protein of known function (phospholipase Pla2g7; probe set 1430700_a_at) that we observed to be upregulated in bone marrow mast cell precursors and in undifferentiated mammospheres, and (c) a novel stem cell marker corresponding to an uncharacterized transcript upregulated in undifferentiated V6.5 and J1 murine embryonic stem cells (2410146L05Rik; probe set 1460471_at). Details on these three cases can be obtained through our online webserver [21] by viewing group numbers 73, 497, and 265, respectively. (d) For comparison, we show the distribution for a housekeeping gene (Eef1a1; probe set 1424635_at), a ribosomal translation elongation factor protein, which is always expressed and was not classified as a marker in our analysis. For robustness, the segregation of the samples (indicated by red and blue bars in the three marker distributions for the down and upregulated groups, respectively) is derived by analysis of the global set of patterns (see Materials and methods) and might not correspond perfectly to the distribution observed here. See, for example, a single replicate in the distribution of Nestin, which is left of the gap in the distribution but was (correctly) associated to the upregulated (blue) group.