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Table 2 Current next-generation sequencing technologies that are suitable for pharmacogene regulatory element discovery

From: Pharmacogene regulatory elements: from discovery to applications

Technology Description References
DNA methylation
MethylC-Seq Unmethylated cytosines are converted into uracil by bisulfite treatment, and the converted DNA is sequenced. Methylation is detected with single-base resolution by comparing the sequence of the converted DNA with that of the unconverted DNA, assuming efficient cytosine conversion [7, 8]
RRBS Restriction digested DNA is size-selected, treated with bisulfite for cytosine conversion, and sequenced. Methylation is detected as described for MethylC-Seq. The reduced representational approach lessens the complexity of analysis and covers a reproducible fraction of the genomic space, but regions with a lack of restriction sites might be missed [80, 81]
MeDIP Methylated DNA elements are enriched with an antibody that binds to 5-methylcytosine, and then sequenced [82]
MBD-Seq Methylated DNA elements are enriched with recombinant methyl CpG binding domain of MBD2, and then sequenced [83]
CAP-Seq The chromatography-based method uses the CXXC domain, which has a high affinity for unmethylated CpG sites, to enrich for DNA elements with methylated CpG sites [84]
MRE-Seq A methylation-sensitive restriction enzyme is used, leaving unmethylated CpG sites available for sequencing [85]
Identification of regulatory elements
ChIP-Seq DNA is crosslinked and isolated via immunoprecipitation typically using an antibody for a transcription factor, regulatory co-factor, or chromatin mark. Subsequent sequencing allows identification of the binding sites of the DNA-associated protein or histone mark [9, 10, 8690]
DNase-Seq DNase I hypersensitive sites are known to harbor regulatory elements. In this method, they are identified by sequencing DNase-digested DNA fragments [121, 144, 145]
FAIRE-Seq Nucleosome-depleted DNA elements associated with regulatory activities are isolated via phenol-chloroform phase separation and sequenced [122, 123]
ChIP-exo Similar to ChIP-Seq, it is used for identification of DNA elements interacting with a protein of interest. It provides better resolution over ChIP-Seq by using an exonuclease to trim unbound DNA around the transcription factor binding site [133]
Expression profiling
RNA-Seq RNA fragments are converted to cDNA and sequenced for gene expression, isoform, and splicing analysis. Novel transcripts and isoforms can be recovered, but low-expression level transcripts may be missed unless sequencing coverage is sufficient [1113, 99102, 109]
Chromosomal interactions
Hi-C DNA elements in close spatial proximity are crosslinked, ligated and sequenced. It allows for a genome-wide, high-resolution analysis of interacting DNA elements [127]
ChIA-PET DNA elements interacting with a protein of interest are enriched via chromatin immunoprecipitation, crosslinked and ligated so that long-range interactions can be identified by sequencing [128]
  1. CAP, CXXC affinity purification; ChIA-PET, chromatin interaction analysis by paired-end tag sequencing; ChIP, chromatin immunoprecipitation; DNase, deoxyribonuclease I; FAIRE, formaldehyde-assisted isolation of regulatory elements; Hi-C, high-throughput chromosome capture; MBD, methylated DNA binding domain sequencing; MeDIP, methylated DNA immunoprecipitation; MRE, methylation-sensitive restriction enzyme; RRBS, reduced representation bisulfite sequencing.