Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data

Table 3 Summary of biases affecting the pre- and post-BS WGBS library preparation strategies

Bias	Main result figures	Pre-bisulfite		Post-bisulfite
Bias	Main result figures	GC-biasing	AT-biasing (KAPA)	Amplification-free	With amplification
Degradation bias (C depletion)	Figs. 1a and 2a, c	+++	+++	+	+
PCR (polymerase) bias	Figs. 2a–c and 5f	+++	+	+	+++
Modified C bias	Figs. 1d and 3d	++	++	+	++
Conversion artefacts	Fig. 4	++	+	+	+
Global 5mC overestimation	Fig. 2d	+++	++	+	++

For simplicity, pre-BS methods have been divided into GC-biasing (Heat, Alkaline and Am-BS) and AT-biasing (KAPA), and post-BS methods into amplification-free (PBAT) and with amplification (ampPBAT and EpiGnome). A plus sign indicates low bias, two plus signs medium bias and three high bias

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