Ancient DNA (aDNA) techniques have rapidly evolved in recent years, especially the application of single-stranded DNA library construction protocol and the automation of lab work using liquid handling robots that has greatly improved the efficiency of ancient DNA research.

However, laboratory background contaminants introduced when using different protocols is still unclear, which has been one of the great challenges in the field of ancient DNA.

A research team led by Prof. FU Qiaomei and Prof. LIU Yichen from the Institute of Vertebrate Paleontology and Paleoanthropology (IVPP) of the Chinese Academy of Sciences has evaluated the microbial DNA introduced in different ancient DNA experimental protocols to better understand the background contaminant DNA in ultra-clean laboratories and to minimize contamination. 

The study was published in Science Bulletin on Dec. 28.

The researchers profiled and analyzed the metagenomes of 40 negative controls processed with different protocols in the ultra-clean laboratory. These samples contain extraction negative controls samples generated manually using double-stranded DNA library protocol, library negative controls samples generated manually using double-stranded DNA library protocol, extraction negative controls samples generated manually using single-stranded DNA library protocol, library negative controls samples generated manually using single-stranded DNA library protocol, extraction negative controls samples generated by robots using single-stranded DNA library protocol, and library negative controls samples generated by robots using single-stranded DNA library protocol.

They found that the single-stranded DNA library preparation protocol performed better in retrieving highly damaged DNA, whereas the protocol was lengthy and thus robot served as a preferable choice.

"The environmental microbial DNA like those from water, soil, air, and human skin are all potential sources of laboratory background contaminants and no obvious ancient DNA damage patterns were observed," said Prof. FU.

Manually processed samples showed higher richness (237 species) and diversity (Shannon Index) compared with samples processed by robots (37 species).

"The combination of single-stranded DNA library construction protocol and the automation of lab work using liquid handling robots can significantly reduce the diversity of background microorganisms and reduce the possible background contaminants in the experiments," said Prof. LIU.