Table 1.

Summary of all technologies discussed

	Technique	Principle	Advantages	Disadvantages	Citation(s)
N6-methyladenosine (m6A)	meRIP-seq/m6A-IP-seq	enrichment of m6A-containing fragments via IP with anti-m6A antibody	benchmark technique in this field	antibody cross-reactivity, relatively high input, can’t provide occupancy, low resolution	Meyer et al., 2012; Dominissini et al., 2012
	miCLIP-seq/PA-m6A-seq	enrichment of m6A-containing fragments via CLIP with anti-m6A antibody	cross-linking gives m6A a “unique chemical signature” that increases resolution	antibody cross-reactivity, can’t provide occupancy	Linder et al., 2015; Grozhik et al., 2017; Chen et al., 2015
	m6A-LAIC-seq	enrichment of m6A-containing full length RNAs via IP with anti-m6A antibody	isoform aware because of full-length RNA input, spike-ins allow measurement of percent modified RNA	antibody cross-reactivity, doesn’t give information about occupancy of specific sites	Molinie et al., 2016
	MAZTER-seq/m6A-REF-seq	utilizes the differential activity of the MazF nuclease toward m6A/A to distinguish the two	antibody-independent methods, can give a measure of occupancy	MazF only cuts at ACA sites, which are only ~25% of all m6A sites	Garcia-Campos et al., 2019; Zhang et al., 2019
	DART-seq	targets APOBEC1 to m6A sites with a fused YTH domain to leave a C-to-U scar nearby	antibody-independent method, low input	YTH domain of YTHDF2 may have sequence preferences for m6A recognition	Meyer, 2019
	m6A-SEAL-seq	converts m6A to hm6A with FTO, then further modifies with DTT to allow enrichment	antibody-independent method, adaptable to other modifications	low resolution	Wang et al., 2020
	m6A-label-seq	uses allyl-SeAM to produce an m6A derivative that induces RT misincorporations	antibody-independent method	allyl-SeAM may disrupt cellular metabolism, imperfect misincorporation rate	Shu et al., 2020
	SCARLET	measures the ratio of m6A/A at a specific site in RNA using RNase H cleavage and TLC	high specificity of RNaseH cleavage	requires radiation, complex protocol, low throughput	Liu et al., 2013
	SELECT	measures the ratio of m6A/A at a specific site in RNA using differential ligation	easier to implement that SCARLET	low throughput	Xiao et al., 2018
5-methylcytosine (5mC/m5C)	BS-seq	utilizes chemical conversion of unmodified Cs detectable by sequencing	benchmark technique in this field	cannot distinguish between 5mC and TET-mediated oxidation products, requires harsh reaction conditions that limit its usefulness for RNA	Frommer et al., 1992
	oxBS-seq	chemical oxidation with KRuO4 converts 5hmC to 5fC, which is susceptible to bisulfite deamination	allows differentiation of 5mC and 5hmC	cannot distinguish between 5mC and TET-mediated oxidation products, requires harsh reaction conditions that limit its usefulness for RNA	Booth et al., 2012
	TAB-seq	utilizes the enzymatic activity of TET to convert 5mC to 5caC (which is susceptible to bisulfite) while protecting 5hmC from deamination using βGT	allows differentiation of 5mC and 5hmC	cannot distinguish between 5mC and TET-mediated oxidation products, requires harsh reaction conditions that limit its usefulness for RNA	Yu et al., 2012
	meDIP-seq/m5C meRIP	enrichment of methylated DNA/RNA fragments via IP with anti-5mC/m5C antibody	bisulfite-free method, more amenable to RNA use	antibody cross-reactivity, can’t provide occupancy	Weber et al., 2005 Edelheit et al., 2013
	ACE-seq	utilizes differential deamination by APOBEC to differentiate C and 5mC from 5hmC	can distinguish between 5mC and 5hmC, does not require bisulfite treatment	complex protocol, not currently amenable for RNA	Schutsky et al., 2018
	TAPS	utilizes the enzymatic activity of TET to convert 5mC and 5hmC to 5caC followed by borane reduction, used in combination with βGT protection or KRuO4 oxidation	can distinguish between 5mC and 5hmC, does not require bisulfite treatment	complex protocol, not currently amenable for RNA	Liu et al., 2019c
	Aza-IP-seq	5-azaC analog covalently traps methyltransferases, allowing RNA targets to be identified by pull-down of transferase and sequencing	covalent trapping allows for accurate identification of RNA targets	better suited for some methyltransferases (single cysteine type) than others	Khoddami and Cairns, 2013
	miCLIP	mutant methyltransferases covalently trap RNA targets, allows capture	covalent trapping allows accurate identification of RNA targets	requires overexpression of mutant methyltransferase	Hussain et al., 2013b
Inosine (I)	Inosine mutational profiling (RDD)	Detecting A > G mutations in sequencing (I reads as G in sequencing)	benchmark technique in this field	computationally difficult to differentiate true positives from false positives	Levanon et al., 2004; Li et al., 2011; Bahn et al., 2012; Peng et al., 2012
	ICE-seq	modifying inosine with acetylnitrile to induce RT stops	more robust than standard mutational profiling	often unreliable for sites with low (>10%) or high (near 100%) editing frequency	Suzuki et al., 2015
	iSeq	differentiating I from G using glyoxal modification and RNase T1 cleavage	more robust than standard mutational profiling	unsuitable for clusters of inosines because of small fragment size	Cattenoz et al., 2013
	EndoVIPER-seq	using EndoV-MBP fusion protein in the presence of Ca2+ (to prevent catalysis) to selectively bind and IP inosine-containing transcripts	relatively easy to perform, does not require further chemical modification of RNA	cannot give occupancy at specific sites	Knutson et al., 2020
Pseudouridine (ψ)	Pseudo-seq/CeU-seq/PSI-seq/ψ-seq	chemically modifying ψ with CMCT to cause RT stops	allows high-throughput identification of ψ sites	removal of CMC from other bases may be incomplete	Carlile et al., 2014; Lovejoy et al., 2014; Schwartz et al., 2014; Li et al., 2015
	HydraPsiSeq	cleaves RNA at uridine sites (but not ψ) through hydrazine treatment	allows high-throughput identification and occupancy measurement of ψ sites	high read depth required, prone to false positives from highly structured uridines and modified uridines	Marchand et al., 2020
N4-acetylcytidine (ac4C)	acRIP-seq	enrichment of ac4C-containing fragments via IP	allows high-throughput identification of ac4C sites	antibody cross-reactivity, can’t provide occupancy, low resolution	Arango et al., 2018
	ac4C-seq	reduction of ac4C with NaCNB3, resulting in mutational profiling	antibody-independent method	unable to map ac4C in the presence of certain other modifications that also react with NaCNB3	Sas-Chen et al., 2020