Table 4. Cross-contaminations and their possible prevention in two-step PCR settings for NGS library generation.

	Possible contamination site
Source of contamination	First amplification PCR mix	Second amplification PCR mix
Amplicon from the first PCR	(A)	(C)
	Prevention: UTP/UNG system	Prevention and detection:
	Detection: PCR with first amplification tail-specific primers	K-box protection (this manuscript)
Amplicon from the second PCR	(B)	(D)
	Prevention: UTP/UNG system	Prevention and detection:
	Detection: PCR with first or second amplification tail- or adaptor-specific primers	K-box protection (this manuscript)

A & B: Contaminations of the first amplification by PCR products derived from another first or second amplification can be prevented by the UTP/UNG system and detected by tail- or adaptor-specific primers (19–21). C & D: Contaminations of the second amplification by PCR products derived from another first or second amplification can be prevented and detected by the K-box protection method described in this manuscript.