. Author manuscript; available in PMC: 2013 Oct 1.

Published in final edited form as: Nat Protoc. 2012 Sep 27;7(10):1897–1908. doi: 10.1038/nprot.2012.103

TABLE 2.

Troubleshooting table.

Step	Problem	Possible reason	Solutions
5	Inaccurate genomic concentration	Genomic DNA is not completely dissolved and is very viscous;	Shear the DNA with needle or sharp tips; alternatively, after AFA shearing, measure the DNA concentration again
		RNA contamination	Treat DNA with an increased amount of RNase and for a longer time
20	Incomplete bisulfite conversion (< 99%)	Genomic DNA amount is > 500 ng during reaction	Dilute DNA and perform the reaction
		Bisulfite solution is too old	Freshly prepare the bisulfite solution
		Reaction conditions are not optimized	Prolong the reaction time and change the reaction temperature
			Treat samples twice with bisulfite; overtreatment with bisulfite could impair library integrity and can result in less-useful reads from high-throughput sequencing
48	Adapter and primer dimer contamination	Incomplete removal of the adapter and primer dimer	Remove the adapter and primer dimer once or several times more with AmpurXP beads