Table 1:

Methods for Investigating DNA methylation

Approach	Utility	Caveats	Examples from Inner Ear or Related Tissues
Bisulfite PCR with high resolution melt analysis	Rapid, cost-efficient method to determine if a specific region or multiple regions, of interest are methylated.	1. Not useful for querying many regions of interest or for coverage of the whole genome. 2. Binary output (Y/N) may fail to reveal multiple methylation sites in a given region and does allow for comparison of methylation levels across experimental groups.	N/A
Bisulfite sequencing PCR, Nested-MSP	Rapid, cost-efficient methods to determine if a specific region or multiple regions of interest are methylated or may have multiple sites of methylation.	1. Not useful for querying many regions of interest or for coverage of the whole genome.	Mutai et al., 2009 Wu et al., 2014 Lin et al., 2017
EpiTyper (bisulfite PCR, transcription, MALDI-TOF MS)	Useful for determining the ratio of methylated vs unmethylated status of a particular region or several regions of the genome from a pool of cells or tissue of interest	1. Not ideal for whole genome interrogation 2. Can be costly 3. Requires expertise and equipment for MALDI-TOF mass spectrometry	Waldhaus et al., 2012 Xia et al., 2015
BS-seq, WGBS	Useful for mapping methylation sites across the whole genome. If coupled with single cell isolation (scBS-seq) can detect monoallelic methylation. Allows for quantification and therefore comparison across experimental groups.	1. Can be costly. 2. May require additional bioinformatic tools or other expertise with next generation sequencing and subsequent data analysis	Yizhar-Barnea et al., 2018
MeDIP-qPCR	Rapid, cost-effective method for determining whether there is methylated DNA in a specific genomic region or in a handful of regions of interest and how those levels may compare across experimental groups	1. Not ideal for many regions of interest or whole genome interrogation 2. Does not provide absolute quantification, only relative comparison across groups	Zhou and Hu, 2016
MeDIP-seq	Useful for mapping methylation sites across the whole genome and providing quantitation and comparison across experimental groups.	1. Can be costly. 2. May require additional bioinformatics and next generation sequencing expertise. 3. Single cell MeDIP is not yet well-established and cell sorting to obtain homogenous populations is therefore critical.	N/A
AIMS	A versatile approach that offers similar benefits to BS-PCR, but can be used to provide information about global rather than local levels of DNA methylation. PCR amplification instead of sequencing results in cost savings. Gel extraction and sequencing of bands of interest can provide spatial localization of methylated sites of interest within the genome. DNA methylome footprints can be compared across samples.	1. Use of sequence-specific endonucleases limits detection and biases the results toward sequences that are recognized by the endonuclease used. 2. Amplification rather than sequencing prevents localization of methylation sites across the genome. 3. Comparisons across groups are limited to quantitation of the number of sites that are overwhelmingly methylated within a sample. Frequencies of methylation at specific regions within a cell population cannot be determined or compared using this method.	Mutai et al., 2009
CMH	Provides site-specific methylation information across the genome. Use of microarrays instead of next-gen sequencing provides cost savings. Unlike AIMS, frequencies of methylation in a cell population can be determined and compared across samples.	1. Genome coverage by microarray is limited compared to direct sequencing. 2. Sequence-specific endonucleases limit detection of all methylated sites similar to AIMS.	N/A
LC-MS	Highly sensitive for detecting global levels of DNA methylation and does not require special enzymes or pretreatment.	1. Can be costly. 2. Requires specialized equipment and expertise in mass spectrometry. 3. Does not provide location-specific information, only global levels of DNA methylation in a given sample.	N/A
Genetic or pharmacological manipulation of DNMTs or TETs	Can uncover important processes and genes that are regulated by DNA methylation. Genetic manipulations can be designed to be cell-type specific obviating the need for cell sorting.	1. Disruption of an enzyme may be fatal or, if not, may lead to complex and overlapping phenotypes due to targeting multiple loci. 2. Partiality of enzymes means coverage is not likely to be global. 3. Redundancy of enzymes means that genes that are regulated by methylation may be missed. 4. Context dependent divalence of enzymes to methylate or demethylate substrates may lead to further complexity of phenotypic results. 5. Potential off-target effects of pharmacological compounds may confound results.	Chen et al., 2013 Zhou & Hu, 2015 Zhou & Hu, 2016 Roellig & Bronner, 2016

NOTE for Table 1: All of the techniques listed above with the exception of “genetic or pharmacological manipulation of DNMTs or TETs” are ideally performed using homogeneous or sorted cell populations or isolated single cells. Cellular heterogeneity can introduce a large degree of noise into the data and result in incorrect estimations of the frequencies and locations of DNA methylation in the genomes of cell types of interest.