title: MethPat: a tool for the analysis and visualisation of complex methylation patterns obtained by massively parallel sequencing.
abstract: BACKGROUND
  DNA methylation at a gene promoter region has the potential to regulate gene transcription. Patterns of methylation over multiple CpG sites in a region are often complex and cell type specific, with the region showing multiple allelic patterns in a sample. This complexity is commonly obscured when DNA methylation data is summarised as an average percentage value for each CpG site (or aggregated across CpG sites). True representation of methylation patterns can only be fully characterised by clonal analysis. Deep sequencing provides the ability to investigate clonal DNA methylation patterns in unprecedented detail and scale, enabling the proper characterisation of the heterogeneity of methylation patterns. However, the sheer amount and complexity of sequencing data requires new synoptic approaches to visualise the distribution of allelic patterns.
  
  
  RESULTS
  We have developed a new analysis and visualisation software tool "Methpat", that extracts and displays clonal DNA methylation patterns from massively parallel sequencing data aligned using Bismark. Methpat was used to analyse multiplex bisulfite amplicon sequencing on a range of CpG island targets across a panel of human cell lines and primary tissues. Methpat was able to represent the clonal diversity of epialleles analysed at specific gene promoter regions. We also used Methpat to describe epiallelic DNA methylation within the mitochondrial genome.
  
  
  CONCLUSIONS
  Methpat can summarise and visualise epiallelic DNA methylation results from targeted amplicon, massively parallel sequencing of bisulfite converted DNA in a compact and interpretable format. Unlike currently available tools, Methpat can visualise the diversity of epiallelic DNA methylation patterns in a sample.
authors.affiliation: Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, 3084, Australia. nwon@unimelb.edu.au.|Murdoch Childrens Research Institute, The Royal Children's Hospital, Parkville, Victoria, 3052, Australia. nwon@unimelb.edu.au.|Department of Paediatrics, The University of Melbourne, Parkville, Victoria, 3052, Australia. nwon@unimelb.edu.au.|Present Address: Pacific Edge Biotechnology Ltd, Dunedin, Otago, 9016, New Zealand. nwon@unimelb.edu.au.
authors.forename: Nicholas C
authors.initials: NC
authors.lastname: Wong
authors.affiliation: Victorian Life Sciences Computation Initiative (VLSCI), The University of Melbourne, Parkville, Victoria, 3052, Australia. bjpope@unimelb.edu.au.|Department of Computing and Information Systems, The University of Melbourne, Parkville, Victoria, 3052, Australia. bjpope@unimelb.edu.au.
authors.forename: Bernard J
authors.initials: BJ
authors.lastname: Pope
authors.affiliation: Department of Pathology, The University of Melbourne, Parkville, Victoria, 3010, Australia. ic85@hotmail.com.
authors.forename: Ida L
authors.initials: IL
authors.lastname: Candiloro
authors.affiliation: Centre for Personalised NanoMedicine, Australian Institute of Nanotechnology and Bioengineering, The University of Queensland, Brisbane, Queensland, 4072, Australia. d.korbie@uq.edu.au.
authors.forename: Darren
authors.initials: D
authors.lastname: Korbie
authors.affiliation: Centre for Personalised NanoMedicine, Australian Institute of Nanotechnology and Bioengineering, The University of Queensland, Brisbane, Queensland, 4072, Australia. m.trau@uq.edu.au.|School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland, 4072, Australia. m.trau@uq.edu.au.
authors.forename: Matt
authors.initials: M
authors.lastname: Trau
authors.affiliation: Molecular Pathology Research and Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, Victoria, 3002, Australia. Stephen.Wong@petermac.org.|Present Address: Translational Research Laboratory, Division of Cancer Research, Peter MacCallum Cancer Centre, East Melbourne, Victoria, 3002, Australia. Stephen.Wong@petermac.org.
authors.forename: Stephen Q
authors.initials: SQ
authors.lastname: Wong
authors.affiliation: Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, 3084, Australia. Thomas.Mikeska@onjcri.org.au.|School of Cancer Medicine, La Trobe University, Bundoora, Victoria, 3084, Australia. Thomas.Mikeska@onjcri.org.au.
authors.forename: Thomas
authors.initials: T
authors.lastname: Mikeska
authors.affiliation: BioInfoRx Inc., Madison, WI, USA. xinmin@bioinforx.com.
authors.forename: Xinmin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: BioResearch Software Consultants, Battle Ground, WA, USA. mark@bioresearchsoftware.com.
authors.forename: Mark
authors.initials: M
authors.lastname: Pitman
authors.affiliation: Murdoch Childrens Research Institute, The Royal Children's Hospital, Parkville, Victoria, 3052, Australia. steffi.eggers@gmail.com.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Eggers
authors.affiliation: Department of Animal, Plant and Soil Sciences, La Trobe University, Bundoora, Victoria, 3086, Australia. s.doyle@latrobe.edu.au.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Doyle
authors.affiliation: Translational Genomics and Epigenomics Laboratory, Olivia Newton-John Cancer Research Institute, Heidelberg, Victoria, 3084, Australia. alex.dobrovic@onjcri.org.au.|Department of Pathology, The University of Melbourne, Parkville, Victoria, 3010, Australia. alex.dobrovic@onjcri.org.au.|School of Cancer Medicine, La Trobe University, Bundoora, Victoria, 3084, Australia. alex.dobrovic@onjcri.org.au.
authors.forename: Alexander
authors.initials: A
authors.lastname: Dobrovic
mesh_terms: D019175:DNA Methylation / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D017422:Sequence Analysis, DNA / Q000379:methods*