A method for PCR-free library preparation for sequencing palaeogenomes.

Area of Science:

• Ancient DNA research
• Palaeogenomics
• Molecular evolution

Background:

• Methodological advances have improved ancient DNA recovery from samples.
• Sequencing palaeogenomes without Polymerase Chain Reaction (PCR) amplification is now a possibility.
• Existing protocols often rely on PCR, which can introduce biases.

Purpose of the Study:

• To present a novel, amplification-free library preparation method for ancient DNA.
• To demonstrate the method's suitability across diverse ancient sample types.
• To generate the first amplification-free nuclear genome from a Pleistocene cave bear.

Main Methods:

• Developed an amplification-free library preparation protocol based on single-stranded DNA.
• Applied the method to ancient samples, including a Pleistocene cave bear.
• Analyzed resulting palaeogenomic data using population genetics and phylogenetic clustering.

Main Results:

• The PCR-free method yielded comparable endogenous DNA content, GC content, and fragment lengths to standard protocols.
• A reduced conversion efficiency was observed compared to the standard protocol.
• Downstream population clustering analyses showed no biases introduced by the amplification-free method.

Conclusions:

• The developed amplification-free library preparation method is suitable for palaeogenomics.
• This technique allows for more direct characterization of original ancient DNA template molecules.
• It offers a valuable alternative for studies sensitive to PCR-induced biases.