title: Exemplary multiplex bisulfite amplicon data used to demonstrate the utility of Methpat.
abstract: BACKGROUND
  DNA methylation is a complex epigenetic marker that can be analyzed using a wide variety of methods. Interpretation and visualization of DNA methylation data can mask complexity in terms of methylation status at each CpG site, cellular heterogeneity of samples and allelic DNA methylation patterns within a given DNA strand. Bisulfite sequencing is considered the gold standard, but visualization of massively parallel sequencing results remains a significant challenge.
  
  
  RESULTS
  We created a program called Methpat that facilitates visualization and interpretation of bisulfite sequencing data generated by massively parallel sequencing. To demonstrate this, we performed multiplex PCR that targeted 48 regions of interest across 86 human samples. The regions selected included known gene promoters associated with cancer, repetitive elements, known imprinted regions and mitochondrial genomic sequences. We interrogated a range of samples including human cell lines, primary tumours and primary tissue samples. Methpat generates two forms of output: a tab-delimited text file for each sample that summarizes DNA methylation patterns and their read counts for each amplicon, and a HTML file that summarizes this data visually. Methpat can be used with publicly available whole genome bisulfite sequencing and reduced representation bisulfite sequencing datasets with sufficient read depths.
  
  
  CONCLUSIONS
  Using Methpat, complex DNA methylation data derived from massively parallel sequencing can be summarized and visualized for biological interpretation. By accounting for allelic DNA methylation states and their abundance in a sample, Methpat can unmask the complexity of DNA methylation and yield further biological insight in existing datasets.
authors.affiliation: Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, VIC 3084 Australia ; Murdoch Childrens Research Institute, The Royal Children's Hospital, Parkville, VIC 3052 Australia ; Department of Paediatrics, The University of Melbourne, Parkville, VIC 3052 Australia ; Present Addresses: Pacific Edge Biotechnology Ltd, Dunedin, Otago 9016 New Zealand.
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Wong
authors.affiliation: Victorian Life Sciences Computation Initiative (VLSCI), The University of Melbourne, Parkville, VIC 3052 Australia ; Department of Computing and Information Systems, The University of Melbourne, Parkville, VIC 3052 Australia.
authors.forename: Bernard J
authors.initials: BJ
authors.lastname: Pope
authors.affiliation: Department of Microbiology and Immunology, The University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, VIC 3010 Australia ; Department of Pathology, The University of Melbourne, Parkville, VIC 3052 Australia.
authors.forename: Ida
authors.initials: I
authors.lastname: Candiloro
authors.affiliation: Centre for Personalised NanoMedicine, Australian Institute of Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072 Australia.
authors.forename: Darren
authors.initials: D
authors.lastname: Korbie
authors.affiliation: Centre for Personalised NanoMedicine, Australian Institute of Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072 Australia ; School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD 4072 Australia.
authors.forename: Matt
authors.initials: M
authors.lastname: Trau
authors.affiliation: Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002 Australia ; Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002 Australia ; Translational Research Laboratory, Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002 Australia.
authors.forename: Stephen Q
authors.initials: SQ
authors.lastname: Wong
authors.affiliation: Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, VIC 3084 Australia ; Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002 Australia ; School of Cancer Medicine, La Trobe University, Bundoora, VIC 3084 Australia ; Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, VIC 3002 Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Mikeska
authors.affiliation: St Vincent's Institute of Medical Research, 9 Princes Street, Fitzroy, 3065 Australia.
authors.forename: Bryce J W
authors.initials: BJ
authors.lastname: van Denderen
authors.affiliation: Institute of Health and Biomedical Innovation and School of Biomedical Sciences, Queensland University of Technology, Brisbane, QLD 4059 Australia.
authors.forename: Erik W
authors.initials: EW
authors.lastname: Thompson
authors.affiliation: Murdoch Childrens Research Institute, The Royal Children's Hospital, Parkville, VIC 3052 Australia.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Eggers
authors.affiliation: Department of Animal, Plant and Soil Sciences, La Trobe University, Bundoora, VIC 3086 Australia.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Doyle
authors.affiliation: Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, VIC 3084 Australia ; Department of Pathology, The University of Melbourne, Parkville, VIC 3052 Australia ; Division of Cancer Medicine, La Trobe University, Bundoora, VIC 3086 Australia ; School of Cancer Medicine, La Trobe University, Bundoora, VIC 3084 Australia.
authors.forename: Alexander
authors.initials: A
authors.lastname: Dobrovic
mesh_terms: D002460:Cell Line; D019175:DNA Methylation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009369:Neoplasms / Q000235:genetics; D009928:Organ Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software
keywords: Bisulfite sequencing; Cancer; DNA methylation; Epialleles; Epigenetics; PCR; Visualization