Table 1.

Ancient DNA damage

Damage	Type of process	Effects on DNA molecule	Possible solutions in aDNA classical sequencing methodologies
Oxidative damage	Formation of strand breaks (single-stranded nicks)	Cleavage of the phosphodiester backbone	PCR of overlapping fragments of short length
		Depurination resulting in a baseless site	Multiple independent PCR Cloning and sequencing of several clones
		Breakage of the sugar backbone through b-elimination	Uracil-N-glycolase treatment
		Results in lesions blocking the polymerase enzyme, and promoting chimeric sequences through ‘jumping’ PCR	Blocking primers Single primer extension or Spex
Degradation by microorganisms’ nucleases in the post mortem cell	Strand breaks	Short fragment length	PCR of overlapping fragments of short length
DNA crosslinks	Inter-strand crosslinks by alkylation	May prevent the amplification of endogenous template molecules	PTB (N-phenylacyl thiazolium bromide)
	Intermolecular crosslinks by Maillard reaction	Increases the risk of contamination
Hydrolysis damage	Results in miscoding lesions, for example, deamination of cytosine and adenine to uracil and hypoxathine, respectively	Results in the incorporation of erroneous bases during amplification and change of coding	Multiple independent PCR Cloning and sequencing of several clones UNG treatment

UNG : Uracil-N-glycosylase; Spex: Single Primer Extension; PTB: N-phenylacyl thiazolium bromide.