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

## TL;DR

This paper introduces a PCR-free method for preparing ancient DNA samples for sequencing, successfully applied to a Pleistocene cave bear genome.

## Contribution

The novel PCR-free library preparation method enables direct sequencing of ancient DNA without amplification bias.

## Key findings

- The PCR-free method produced consistent DNA metrics comparable to standard protocols.
- The method generated the first amplification-free nuclear genome of a Pleistocene cave bear.
- Downstream population analyses showed no bias from the PCR-free approach.

## Abstract

In recent years, methodological advances have substantially improved our ability to recover DNA molecules from ancient samples, raising the possibility to sequence palaeogenomes without PCR amplification. Here we present an amplification-free library preparation method based on a benchmark library preparation protocol in palaeogenomics based on single-stranded DNA, and demonstrate suitability of the new method for a range of sample types. Furthermore, we use the method to generate the first amplification-free nuclear genome of a Pleistocene cave bear, and analyse the resulting data in the context of cave bear population genetics and phylogenetics using standard genomic clustering analyses. We find that the PCR-free adaptation provides endogenous DNA contents, GC contents and fragment lengths consistent with the standard protocol, although with reduced conversion efficiency, and shows no biases in downstream population clustering analyses. Our amplification-free library preparation method could find application in experimental designs where the original template molecule needs to be characterised more directly.