TY - JOUR

T1 - A method for PCR-free library preparation for sequencing palaeogenomes

AU - Henneberger, Kirstin

AU - Barlow, Axel

AU - Alberti, Federica

AU - Preick, Michaela

AU - Constantin, Silviu

AU - Döppes, Doris

AU - Rosendahl, Wilfried

AU - Hofreiter, Michael

AU - Paijmans, Johanna L A

N1 - Copyright: © 2025 Henneberger et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

PY - 2025/3/19

Y1 - 2025/3/19

N2 - 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.

AB - 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.

KW - Gene Library

KW - Animals

KW - Ursidae/genetics

KW - Sequence Analysis, DNA/methods

KW - Polymerase Chain Reaction/methods

KW - Phylogeny

KW - DNA, Ancient/analysis

KW - DNA, Single-Stranded/genetics

U2 - 10.1371/journal.pone.0319573

DO - 10.1371/journal.pone.0319573

M3 - Article

C2 - 40106766

VL - 20

SP - e0319573

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 3

ER -