Assessment of contaminants associated with gold-standard ancient DNA protocols.

Ancient DNA(aDNA)techniques have rapidly evolved in the past decade,including the introduction of next-generation sequencing(NGS)techniques,the modification and optimization of various protocols to extract highly degraded DNA,and the incor-poration of these molecules into sequencing libraries[1].Two major milestones are the application of single-stranded DNA library construction protocol and the automation of lab work using liquid handling robots[1].However,despite the great potential of these methodologies in effectively retrieving aDNA,it is unclear how the combination of different protocols can affect the profiles of contaminants in aDNA libraries,which is still one of the greatest challenges in the aDNA field.In a low-biomass environment like aDNA laboratories,the biological signal within samples can be easily surpassed and dominated by contaminant DNA from the lab-oratory environments[2,3].Including negative controls is an effec-tive way to monitor the background contaminant introduced during experiments,and the metagenomes identified from the negative controls can be excluded from downstream analyses to mitigate the biases introduced by contaminant DNA[4-6].