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. 2014 Apr;24(4):545–553. doi: 10.1101/gr.166439.113

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© 2014 Lay et al.; Published by Cold Spring Harbor Laboratory Press

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported), as described at http://creativecommons.org/licenses/by-nc/3.0/.

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Figure 1. — Inflammatory cell-specific DNA methylation markers as surrogate markers of neobladder sample purity. (A) Urine sediment collected from a neobladder patient is highly cellular and often thick with mucus due to the presence of mucus-producing goblet cells. Inflammatory cells may also contaminate the urine samples due to increased inflammation in patients immediately after surgery. Mucus production and white blood cell count, however, decrease over time. (B) Methylation level of KLHL6, a blood-specific gene, is measured in all matched samples using pyrosequencing. KLHL6 is hypomethylated in all blood samples (7.57 ± 2.45%) and highly methylated in the normal small intestine (83.09 ± 8.01%). Hypermethylation of this marker in the neobladder indicates enrichment for intestinal epithelial cells, while low or intermediate methylation indicates a significant presence of an inflammatory cell population. (*) Neobladder samples that passed the quality-control threshold, which is a methylation level within 10% of the matched small intestine. For downstream analysis, HM450 data were generated for neobladder samples that are enriched for intestinal cells as indicated by the high methylation level in at least two of the three inflammatory cell markers (see Supplemental Fig. 1 and Supplemental Table 2).