Table 1.

Strengths and limitations of omics technologies in EDC research

Type	Biomarkers	Methods	Description	Strengths	Limitations
Genomics	DNA sequencing	Sanger Sequencing; Next-gen Sequencing	Determines the order of nucleotides within a DNA molecule and allows for full interrogation of the genome, both targeted and global	Not just limited to nuclear DNA - can be extrapolated to mtDNA‡	Can have high cost, especially for whole-genome sequencing
				Able to investigate all DNA variants and how they might be related to EDCs^	Information limited to the DNA sequence
	GWAS	Genome-wide Association Studies - microarray	Examines specific genetic variants across the genome in different individuals and can be used to establish associations between these variants and disease or quantative traits (4)	Not hypothesis driven - no prior gene information required thus allows for discovery analyses	Hard to use if certain EDC chemicals target variants other than SNPs* or CNV†
				Well established for investigating outcomes
				Allows for association to be examined, especially for environmental exposures
Transcriptomics	Gene-expression analyses	RNA-Seq; real-time-quantitative PCR, and microarray	Examines expression patterns of specific genes, an array of genes, or the entire transcriptome and reveals the presence and quantity of RNA in a biological sample (5)	Allows for associations to be examined between EDCs and specific expressed genes or an array of genes	Gene expression varies by tissue type making it more difficult to isolate the biological mechanism
				Primer design allows for study specific a priori genes to be examined and developed microarrays are readily available	Usually represents data at the point in time the sample was collected; limited in reflecting history of exposures over time
				Some toxicology or in vitro models in relation to EDCs^ (1)(2)	Relatively few RNA-seq EDC^ studies done in human population
Epigenomics	DNA Methylation	Pyrosequencing-Microarray; whole-genome sequencing	Examines the DNA methylome, ranging from gene specific areas to a microarray of about 850K sites to the entire DNA methylome (6) (7), which can impact gene expression	Can examine gene specific methylation and/or epigenome wide DNA methylation (up to 95%)	Tissue specific, so might not be the best representation if target tissue is not obtained
				Able to examine associations with both EDC^ exposures and outcomes	Only represents data at the point in time the sample was collected, might not reflect the windows of susceptibility
				Established methods within epidemiology studies that allow for replication of EDC^ findings
				Methods can also be used to measure methylation in mtDNA‡
	Histone Modifications	Chromatin immunoprecipitation - seq	Examines epigenetic marks on histones, including acetylation, phosphorylation, glycosylation, sumolation, methylation and ADP ribosylation, which can impact gene expression by altering chromatin structure (9)(10)	Previous studies have examined the relationship between histone modifications and environmental exposures (Nickel, Arsenic, and few EDCs) (11)(13)(14)	Still a relatively unstudied field in EDC^ (15)
				Some studies have linked histone modifications to outcomes, such as obesity(12)	Most studies examining histone modifications are in vitro or in toxicology models (11)(13)(14)
	Chromatin Remodeling	DNAse-seq; MNase-seq FAIRE-seq; ATAC-seq	Examines the dynamic modification of the chromatin architecture that allows for transcription machinery to adhere to the DNA which can impact gene expression (16)	Provides information on the actual chromatin conformation. Analyzes a cellular state intermediate between the epigenetics marks (e.g. DNA methylation, histone modifications) and gene expression	Most assays require high amounts of cells.
					Still not widely applied in EDC studies
Mitochondriomics	Mitochondrial Copy Number	Multi-plex real-time-PCR#; Digital-Droplet PCR#	Examines the number of copies of mtDNA‡ compared to nDNAº within a sample (17)	mtDNA‡ copy number can be altered by the presence of environmental chemicals (17)(19)	Measurements are relative to the controls used, so it can be hard to compare between studies
				This assay has been optimized for toxicologic, in Vitro, and human studies (18)	Still new to the EDC^ field and few studies have examined mtDNA‡ copy number in relation to EDCŝ (18)
	Mitochondrial Lesions	LongRange quantitative PCR# & Picogreen Fluoroescence	Examines the number of DNA lesions within a fragment of mtDNA‡	This assay can be used in human studies, allowing for reliable and senstive measures	Measurements are relative to controls used, so it can be hard to compare between studies
				Low cost, small amount of DNA needed to start, and PCR# based allows for easy set up and running of the assay	Cannot distinguish the nature or location of DNA damage
					Not all types of lesions are captured by this method
					Emerging technology, has not been studied with EDC^
	Mitochondrial Sequencing	Next-gen sequencing; MiSeq, MitoExome; sanger sequencing	Like genomic sequencing, allows for gaining the order of nucleotides and allows for full interrogation of the mtDNA‡ genome	Able to investigate all DNA variants and how they might be related to EDCs^	Information limited to the mtDNA sequence
				Measure of mtDNA heteroplasmy vary over time and are in principle influenced by environmental exposures	Emerging technology, has not been studied with EDCs^
				mtDNA‡ hyper variable region could be used as a tool for exposure fingerprinting (20)	mtDNA sequence variation is tissue specific, so mechanisms can be missed if measuring in a different tissue type
					Potential coamplification of nuclear homologs of mtDNA which can lead to inaccurate measures(21)

^*SNP Single Polymorphic Nucleotide

^†CNV Copy Number Variant

^‡mtDNA Mitochondrial DNA

^ºnDNA Nuclear DNA

^{^}EDC Endocrine Disrupting Chemical

^#PCR Polymerase Chain Reaction