Table 1.

Comparison of the targeted bisulfite sequencing methods

Method	BSPP			SHBS-seq		ACBS-seq	Bisulfite-Patch PCR	Microdroplet PCR
Reference	[47]	[48]	[49]	[51]	[52]	[58]	[60]	[62]
Capture probes/primer	ssDNA	ssDNA	ssDNA	Biotinylated RNA	Biotinylated RNA	ssDNA	Partial dsDNA	ssDNA
Probe/primer size	150 bp	150 bp	150 bp	160 bp	120 bp	60 bp	40–50 bp	20–30 bp
Number of probes/primers	30,000	9,552	330,000	51,551	n/a	240,000	94 pairs	3,500 pairs
Probe/primer manufacturer	Agilent	Agilent	Agilent	Agilent	Agilent	Agilent	Sigma	RainDance
Capture method	Annealing, extension and circularization	Annealing, extension and circularization	Annealing, extension and circularization	Hybridization in solution	Hybridization in solution	Hybridization on array	Annealing, ligation, and PCR	Microdroplet PCR
Amount of input DNA	200 ng bisulfite-treated	1 µg bisulfite-treated	200 ng bisulfite-treated	20–30 µg native	2–3 µg native	20 µg bisulfite-treated and amplified	250 ng bisulfite-treated	2 µg bisulfite-treated
Bisulfite treatment	Pre-capture	Pre-capture	Pre-capture	Post-capture	Pre-capture	Pre-capture	Pre-capture	Pre-capture
Sequencing platform	Illumina	Illumina	Illumina	Illumina	Illumina	Illumina	454	Illumina
Target size	2.1Mb	n/a	34Mb	8.2Mb	38Mb	258.9kb	25.5kb	1.35Mb
Number of targets	10,582	9552	140,749	51,551	n/a	324	94	3,500
Target features	CGI, ENCODE, (TSS±1000bp)	ENCODE regions	DMRs, CTCF, DNase I sites	CGI, TSS	Exon	CGI	Promoter	Promoter (TSS±1000bp)
Targets covered	n/a	68% (>10 reads)	n/a	85–88% (>10 reads)	90% (>10 reads)	~92%	100%	99%
On-target rate (%)	n/a	n/a	96%	77–84%	71–75%	6–12%	90%	90%
Number of CpG	66,000	6,400	500,0000	0.9–1 million	n/a	25,044	n/a	77,674
Coverage per CpG	n/a	n/a	n/a	86–146 (mean)	58–73	95–105 (median)	444 (median)	>100 (97% of CpGs)
Reproducibility (r)	0.97	0.965	0.97–98	0.94	n/a	n/a	0.91	n/a
Advantages	Highly specific and reproducible, require lower amount of input DNA			Flexible probe design; easy to scale up to target large regions		Use bisulfite-treated and amplified DNA	Require lower amount of DNA, use regular PCR primers	Highly multiplex PCR, low PCR bias
Disadvantages	Expensive probes, complex probe design			Capture small amount of DNA; challenges in bisulfite treatment		Low capture specificity, require specific instruments	Uncertainty about scale-up in throughput	Require specific instruments.