Validation array coverage.

<p>Validation array coverage.</p

Sequencing of iPSC clones.

<p><b>A.</b> Number of variants identified per iPSC clone. <b>B.</b> Variant allele frequencies of all validated mutations for each clone. Samples are ranked by the number of variants in decreasing order. No mutations were identified in Ax2-11, thus it is not listed in panel B.</p

Comparison of hematopoietic potential of iPSCs to mouse ESCs.

<p>Fractions of Lin⁻ cells (A), KLS cells (B), Kit+Lin-Sca-1- Progenitors (C), GMPs (D), CMPs (E), and MEPs (F) from iPSCs relative to mouse ESCs after 7 days of OP9 coculture (unsorted).</p

Generation of iPSC clones from a single mouse C57BL/6 male mouse.

<p><b>A.</b> Schematic of experimental approach (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120585#sec002" target="_blank">Material and Methods</a> for full protocol). <b>B.</b> Representative bright-field images (left) and alkaline-phosphastase stains (right) of B6/BLU ESCs (top) and a representative iPSC (bottom, Ax1-10). All images at 100x magnification. <b>C.</b> Images from the teratoma derived from Ax1-10 demonstrating ectoderm (neural tissue), mesoderm (cartilage) and endoderm (ciliated respiratory epithelium).</p

Hematopoietic differentiation potential of the 24 iPSC clones.

<p>100,000 cells from OP9 cocultured mESCs (B6/BLU) or iPSCs were plated in methylcellulose media containing hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). <b>A.</b> CFUs were counted after 7 additional days of culture. The relative number of CFUs per 100,000 cells plated from Day7 iPSC-derived progenitors vs. Day7 ESC (B6/BLU)-derived progenitors are shown. iPSC clones are ranked from the highest to the lowest average of two independent experiments. Error bars represent the means +/− one standard deviation. <b>B.</b> Morphology of day 7 OP9 cocultured ESC-derived cells after 7–8 days of additional culture in MethoCult media containing hematopoietic cytokines (SCF, IL-3, IL-6, and Epo). A scale bar of 20 μm is shown. <b>(C-E)</b>. Fractions of CD11b⁺ (<b>C</b>), CD34⁺Kit⁺ (<b>D</b>), and Ter119⁺ (<b>E</b>) cells obtained after 7 days of methylcellulose culture containing hematopoietic cytokines (SCF, IL-3, IL-6, and Epo), comparing iPSC-derived progenitors relative to ESC-derived progenitors, in the same order as panel A. <b>F.</b> Lack of correlation between the number of mutations and the hematopoietic differentiation potential of the iPSC clones (r² = 0.0006065).</p

Hematopoietic differentiation from murine ESCs.

<p><b>A.</b> Morphology of wild type ESC-derived cells after 7 days of OP9 coculture (unsorted) by Wright-Giemsa staining. A scale bar of 20 μm is shown. (B-D). Immunophenotyping of hematopoietic progenitor cells from wild-type mouse bone marrow cells (panel B), murine ESCs after 7 days of OP9 coculture (panel C), and iPSC clone Ax1-14 after 7 days of OP9 coculture (panel D). Lineage⁻ (Lin⁻), KLS (Lin⁻Kit⁺Sca-1⁺), progenitors (Lin⁻Kit⁺Sca-1⁻), CMPs (Lin⁻Kit⁺Sca-1⁻CD34⁺FCγ⁻), GMPs (Lin⁻Kit⁺Sca-1⁻CD34⁺FCγ⁺), and MEPs (Lin⁻Kit⁺Sca-1⁻CD34⁻FCγ-).</p

Whole exome sequencing coverage.

<p>Whole exome sequencing coverage.</p

Single-cell mutation profiles.

<p>Variant profiles across targeted somatic mutations in single-cell samples (sAML bone marrow) in (<b>A</b>) UPN461282, (<b>B</b>) UPN182896, and (<b>C</b>) UPN288033. Rows display positive and negative variant calls color-coded by mutation cluster for each single-cell sample, and columns indicate specific SNVs somatic at sAML diagnosis. Variants are grouped and color-coded by cluster as predicted from sequencing unfractionated material (uppermost track in each panel). Each cell is grouped by the clone it is inferred to represent. Outlier SNVs (purple) were those which could not be confidently clustered based on bulk sequencing. Here, many of these are merged into predicted clusters based upon their presence/absence in single-cell libraries (i.e., harboring the same pattern as well-defined clones). Positions where reference calls were made are colored grey; positions where no call was made (<25× coverage) are colored white. Pairs of variants that always travel in the same state (reference or variant) likely arose in the same clonal expansion. Pairs of variants that are called together in some cells but not others are likely related by linear evolution. Pairs of variants that are mutually exclusive suggest evolutionary branch points, and were rare. This suggested that variants in subclone 5 in UPN461282 (<b>A</b>), and subclone 1 in UPN288033 (<b>C</b>) were divided among additional subclones (now 5A/5B, 2A/2B). See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004462#pgen.1004462.s007" target="_blank">Figure S7</a></b> for data presentation with unmodified clone and cluster definitions (derived from bulk sequencing).</p

Depth and distribution of coverage for each sequencing library (n = 56).

<p>(<b>A</b>) Cumulative coverage represented as the proportion of the capture target (y-axis) with read depth greater than or equal to specific coverage thresholds (x-axis). Coverage values are derived from quality-filtered data (de-duplicated, phred-scaled alignment quality ≥10, phred- scaled base quality ≥13). The intersection of each curve with y = 0.5 identifies the median coverage. Higher coverage was obtained for the unsorted samples (median 228×), compared to the single- or two-cell samples (median 28×). (<b>B</b>) Lorenz curve detailing uniformity of coverage as proportion of targeted bases versus proportion of sequenced bases. Dashed line (y = x) represents a perfectly uniform distribution of read depth across the capture target. Libraries prepared from WGA samples (single- and two-cells) exhibit significantly less uniform representation, compared to libraries derived from unfractionated material. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004462#pgen-1004462-t001" target="_blank"><b>Table 1</b></a> and <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004462#pgen.1004462.s011" target="_blank">Table S3</a></b> for additional details.</p

Performance of variant calling at germline SNPs.

<p>True Positive (TP): ≥1 non-reference allele called by Affymetrix array, ≥1 non-reference allele called by sequencing.</p><p>True Negative (TN): 0 non-reference alleles called by Affymetrix array, 0 non-reference alleles called by sequencing.</p><p>False Positive (FP): 0 non-reference alleles called by Affymetrix array, ≥1 non-reference allele called by sequencing.</p><p>False Negative (FN): ≥1 non-reference allele called by Affymetrix array, ≥0 non-reference alleles called by sequencing.</p><p>True Positive Rate (TPR): TP/(TP+FN) = sensitivity = power.</p><p>False Positive Rate (FPR): FP/(FP+TN) = 1-specificity.</p><p>False Negative Rate (FNR): FN/(TP+FN) = 1 - sensitivity = type II error.</p