Comparison of methylation status in ctDMRs in HMF (Fb), HMEC and cancer cell lines (CCL)

<p>Methylation status of CpG sites 87 ctDMRs was analyzed by MassARRAY. Individual CpG and sample types were ordered by hierarchical clustering. Within heatmap, red and green correspond to hypermethylation and hypomethylation respectively.</p

Comparison of ctDMRs with breast cancer specific DMRs.

<p>Number of unique and common DMRs is shown in Venn diagrams. Data for breast cancer specific DMR were reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052299#pone.0052299-Novak2" target="_blank">[23]</a>. Numbers in Venn diagrams show the number of regions; number of probes on the microarrays which are covering these region are show as numbers in parenthesis. Histograms below Venn diagrams show expected number of overlapping regions if positions of DMRs are random. Histogram is based on 5000 iterations of simulation. Red line shows the observed number of overlapping DMR.</p

Number of DMR.

<p>Number of DMR.</p

Distribution of ctDMRs.

<p>A) Position of ctDMR relative to TSS. Negative and positive distances correspond to sequences upstream and downstream to TSS, respectively. <b>B)</b> Position of ctDMR relative to the nearest CpG island. For both charts, pink lines show the expected distribution if DMRs have random localization and uneven microarray coverage is taken into consideration. 99% confidence interval based on the simulation is shown.</p

Validation of microarray results using MALDI-TOF mass spectrometry (MassARRAY).

<p>CtDMRs are labeled by the gene symbol of the closest gene. Each ctDMR is shown as a heatmap; methylation level for each informative CpG fragment is shown in columns and analyzed samples are organized in rows. Each colored rectangle corresponds to 1 CpG unit. All three genotypes were analyzed. White spaces are shown when the quality of data was not sufficient to assess methylation. <b>A</b>) ctDMRs which are methylated in HMF according to microarray. <b>B)</b> ctDMRs which are methylated in HMEC according to microarray. A more detailed view for each analyzed region is included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052299#pone.0052299.s003" target="_blank">Information S1</a>.</p

Summary of sample types utilized for each of the studies performed.

<p>Summary of sample types utilized for each of the studies performed.</p

Summary of analysis results for each of the studies performed. Sens = sensitivity.

<p>Spec = specificity; NA = Not applicable.</p

Paired comparison of z-scores.

<p>Z-scores were calculated for paired samples with previously described GC normalized, repeat masked z-scores on the x-axis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057381#pone.0057381-Palomaki1" target="_blank">[7]</a> and z-scores from the same libraries sequenced in 12-plex on the y-axis. Samples classified by karyotype analysis as trisomies for A) Chromosome 21, B) Chromosome 13, or C) Chromosome 18 are shown in blue; unaffected samples for each aneuploidy condition are shown in gray. Red horizontal and vertical lines in each plot represent the respective classification cutoff for that chromosome (z = 3 for chromosome 21, z = 3.95 for chromosomes 13 and 18).</p

Paired comparison of z-scores.

<p>Z-scores were calculated for 1269 paired samples with previously described GC normalized, repeat masked z-scores on the x-axis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057381#pone.0057381-Palomaki1" target="_blank">[7]</a> and z-scores from the high-throughput assay on the y-axis. Samples classified by karyotype analysis as trisomies for A) Chromsome 21, B) Chromosome 13, or C) Chromosome 18 are shown in blue; unaffected samples for each aneuploidy condition are shown in gray. Red horizontal and vertical lines in each plot represent the respective classification cutoff for that chromosome (z = 3 for chromosome 21, z = 3.95 for chromosomes 13 and 18). Black line in plot represents y = x.</p