A method for PCR-free library preparation for sequencing palaeogenomes

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.

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PLOS ONE | https://doi.org/10.1371/journal.pone.0319573 March 19, 2025 1 / 15
OPEN ACCESS
Citation: Henneberger K, Barlow A, Alberti F,
Preick M, Constantin S, Döppes D, et al. (2025)
A method for PCR-free library preparation for
sequencing palaeogenomes. PLoS ONE 20(3):
e0319573. https://doi.org/10.1371/journal.
pone.0319573
Editor: Mateusz Baca, University of Warsaw,
POLAND
Received: July 5, 2024
Accepted: February 4, 2025
Published: March 19, 2025
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.
Data availability statement: Raw data for
all samples are available on the European
Nucleotide Archive under Project accession
number PRJEB83706 and can be retrieved via
the following URL: https://www.ebi.ac.uk/ena/
browser/view/PRJEB83706.
Funding: JLAP is funded by the Marie
Skłodowska-Curie individual fellowship
“RESOURCEFUL” (101028348). This work
RESEARCH ARTICLE
A method for PCR-free library preparation
for sequencing palaeogenomes
Kirstin Henneberger1*, Axel Barlow2, Federica Alberti1¤, Michaela Preick1,
Silviu Constantin 3,4, Doris Döppes5, Wilfried Rosendahl5,6, Michael Hofreiter1,
Johanna L. A. Paijmans 2,7*
1 Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany, 2 School of
Environmental and Natural Sciences, Bangor University, Bangor, United Kingdom, 3 Department of
Geospeleology and Paleontology, Emil Racovita Institute of Speleology, Bucharest, Romania, 4 Centro
Nacional de Investigacion sobre la Evolucion Humana, CENIEH, Burgos, Spain, 5 Reiss-Engelhorn-
Museen, Mannheim, Germany, 6 Curt-Engelhorn-Centre for Archaeometry, Mannheim, Germany,
7 Department of Zoology, University of Cambridge, Cambridge, United Kingdom
¤ Current address: Institute of Pathology, Laboratory of Molecular Tumor Pathology, Charite
Universitätsmedizin Berlin, Berlin, Germany
* kihenneb@gmail.com (KH); paijmans.jla@gmail.com (JLAP)
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 amplication. Here we present an amplication-free library
preparation method based on a benchmark library preparation protocol in palaeogenom-
ics 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 rst
amplication-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 nd 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 pop-
ulation clustering analyses. Our amplication-free library preparation method could nd
application in experimental designs where the original template molecule needs to be
characterised more directly.
Introduction
A long-standing assumption in palaeogenetic research is that the genetic material present in
ancient samples occurs in very low quantities, and that the amount of exogenous DNA always
far outweighs the target or endogenous DNA. In order to overcome this limitation, genetic
analysis pipelines have always included a PCR amplification step in order to generate enough
genetic material for sequencing; either by species-specific primers, or library amplification
prior to sequencing. For this reason, PCR has often, and with justification, been hailed as the
most important development that enabled palaeogenetic research, leading to many unprece-
dented insights into evolution, ecology, human history and disease [reviewed in e.g. 1].

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PLOS ONE Amplication-free palaeogenome library preparation
In recent years, thanks to the development of new DNA extraction methods [e.g. 2], library
preparation methods [3–5] and improved tissue sampling strategies [6–8], the standard expec-
tations regarding the prevalence of DNA in an ancient sample have changed. Yields over 100
ng DNA per extract have been reported for high endogenous content Pleistocene samples (S1
Fig in S1 File), which for a typical mammalian species would correspond to over 16,000 dip-
loid copies of the nuclear genome, if all extracted DNA were endogenous. Such DNA yields
could make sequencing of even a high coverage genome without PCR amplification achiev-
able. Excluding PCR from the library construction protocol would further avoid duplications
as well as artefacts caused by amplification [9–11], with the potential to increase usable data
output from the sequencer [12,13]. Furthermore, amplification-free recovery of DNA would
allow for a more direct and unbiased characterisation of the genetic material preserved in the
ancient sample, potentially leading to a better understanding of e.g. copy number variations,
or the relative presence of alleles in a poolseq experimental design.
To date, no specific protocol for recovering ancient DNA without the need for PCR
amplification exists. Although amplification-free protocols have been developed for modern
DNA [14], the recovery of DNA from ancient and other degraded samples require specialised
protocols that target very short and damaged template molecules [3]. The requirements for
standard and/or commercial amplification-free protocols (25-200 ng of several hundred bp
long double-stranded DNA fragments; see e.g. https://www.illumina.com/products/by-type/
sequencing-kits/library-prep-kits/dna-pcr-free-prep.html) are very difficult to achieve for
ancient samples. To our knowledge, only one amplification-free protocol has been developed
for low input single-stranded DNA, for recovering cell-free fetal DNA [15]. Although this
protocol is based on the single-stranded library protocol for ancient DNA, it is unclear how its
adaptations would impact the recovery of ancient DNA, and the downstream compatibility of
the data with the single-stranded protocol [16–18].
Here, we present an amplification-free adaptation of a well-established protocol for ancient
DNA, which was the first single-stranded library preparation developed for Illumina sequenc-
ing [3,19]. We show the effectiveness of our amplification-free protocol through application to
a range of samples of various ages (from museum samples to Pleistocene sub-fossil samples)
and tissue types (bone, preserved skin, formalin fixed tissue), and compare its performance to
the standard single-stranded library preparation protocol. Finally, we utilise the technique to
sequence a Pleistocene nuclear genome without any PCR amplification prior to sequencing,
and compare the data with those recovered using the standard single-stranded protocol to
gauge for downstream biases. Our amplification-free protocol could have application in fields
where low-quantity input DNA is common, such as palaeogenomics, museomics or forensics,
and where a reduction of potential PCR bias is desired.
Materials and methods
Library protocol
The amplification-free protocol is based on the first published single-stranded library
method for ancient DNA (Fig 1) [3,4,19]. After removal of terminal phosphate groups
and denaturation of the template DNA, a biotinylated adapter is ligated to the resulting
single-stranded template molecules. Then, instead of annealing a truncated adapter to the
biotinylated adapter, our new amplification-free protocol anneals a specifically modified
oligonucleotide consisting of the full adapter sequence including the inline index, which is
then extended along the template. The second adapter is added using blunt-end ligation,
again using a modified oligonucleotide mix that contains the full-length double-stranded
adapter including the inline barcode. After another heat denaturation to release the new
was also supported by the European Research
Council (consolidator grant GeneFlow #310763
to M.H.) and the Klaus Tschira Stiftung
Heidelberg within the project “Eiszeitfenster
Oberrheingraben” (FA, MH, DD and WR). SC
acknowledges the financial support through the
EEA Grant 216/2019 – “KARSTHIVES2”.
Competing interests: The authors have
declared that no competing interests exist.

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PLOS ONE Amplication-free palaeogenome library preparation
template molecule, it is then converted into a double-stranded molecule using a fill-in reac-
tion in which the original template DNA molecule is displaced. This fill-in reaction replaces
the indexing PCR in the standard single-stranded protocol (Fig 1). A detailed protocol is
presented as S1 Protocol in S1 File.
Investigation of library conversion
We investigated the conversion of template molecules into the final sequencing library using
two experiments:
Experiment 1. To calculate the conversion rate – defined here as the total number of
molecules in the final product versus the number of molecules with library adapters – of
the amplification-free protocol compared to the standard single-stranded protocol, both
types of libraries were prepared from the test oligo CL104 (Fig 1C; [also see 3]), and the
Fig 1. Overview of both the amplification-free protocol (A) and the standard single-stranded protocol (B) [3,19]. Denaturation and immo-
bilisation steps are identical between the protocols. The priming sites for the quantification assays are indicated in panel C. For more details, see
S1 Protocol and S5 Table in S1 File.
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conversion rates calculated using qPCR as described in Gansauge and Meyer [3]. The
procedure in brief: 1 µl of a 0.1 µ M dilution of the CL104 oligo was used as input for library
preparation, using a total of five replicates each (one replicate set of the original six failed).
Following library preparation, two qPCR assays and a standard dilution series were used
to determine the number of molecules successfully converted into library molecules, as
described in Gansauge and Meyer [3], using triplicate qPCR reactions for each library.
qPCR assay A involved amplification using the IS7/IS8 primer pair, which amplifies only
complete library molecules, indicating successful ligation of a P5 and P7 adapter on each
end of the test oligo CL104 (Fig 1C). Assay B involved amplification using primers CL107/
CL108, which detect oligo CL104 regardless of whether it has been successfully converted
into a library molecule or not (Fig 1C). A standard dilution series of a known quantity of
CL104 (amplified from pUC19 DNA) was used for absolute quantification. qPCR analysis
was carried out on a Roche LightCycler 480 and intensity values converted into Ct values
using R v4.1.0 [20] with the package ‘qpcR’ [21].
Experiment 2. To further compare library conversion of the amplification-free and
standard protocol when applied to real samples, an assay of six ancient bones samples (three
replicates each; Table 1) was used. 50 mg of bone powder was taken from each sample and
DNA extracted following Dabney et al. [2]. These extracts were then used to prepare both
amplification-free and standard (i.e. PCR amplified) single-stranded libraries. By using qPCR
assay A (described above), we performed absolute quantification of the successfully converted
library molecules.
Data consistency and performance on different sample types
In order to test the efficacy of the amplification-free protocol for different tissue types and
preservation contexts, a range of samples were selected for sequencing using both the
amplification-free and the standard single-stranded library preparation protocols.
Table 1. Details of samples used in this study.
Sample name Organism Sample type Approximate age Experiment Sample provider
NK64 steppe bison petrosa dex 39.0 ka BP uncal 2 Reiss-Engelhorn-Museen
NK65 steppe bison petrosa sin 40.1 ka BP uncal 2 Reiss-Engelhorn-Museen
NK67 steppe bison petrosa sin 41.4 ka BP uncal 2 Reiss-Engelhorn-Museen
NK69 steppe bison petrosa 39.4 ka BP uncal 2 Reiss-Engelhorn-Museen
NK71 aurochs petrosa sin 42.6 ka BP uncal 2 Reiss-Engelhorn-Museen
MENG21 aurochs petrosa sin 43.6 ka BP uncal 2 Reiss-Engelhorn-Museen
33961 king cobra formalin-fixed, ethanol preserved 1970s AD 3 Natural History Museum Berlin
PLI-19 linsang soft tissue 1896 AD 3 Naturalis Museum Leiden
50746NB leopard nasal bone 1962 AD 3 Natural History Museum Berlin
70423NB leopard nasal bone unknown (historical) 3 Natural History Museum Berlin
56097ST leopard soft tissue 1904 AD 3 Natural History Museum Berlin
56407ST leopard soft tissue 1907 AD 3 Natural History Museum Berlin
SP347 cave bear bone - not compact Pleistocene 3 Institute of Palaeontology, University of Vienna
PM-V6-IV-II cave bear petrous Pleistocene 3 Emil Racovita Institute of Speleology, Romania
PM-T6-I-II-102 cave bear petrous Pleistocene 3 Emil Racovita Institute of Speleology, Romania
PU-D2-VIII-101 cave bear petrous Pleistocene 3 Emil Racovita Institute of Speleology, Romania
PM-T6-I-II-101 cave bear bone - compact Pleistocene 3 Emil Racovita Institute of Speleology, Romania
O34-16 bear petrous Pleistocene 3, 4 Emil Racovita Institute of Speleology, Romania
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Experiment 3. DNA was extracted from Pleistocene bear bone material (compact and
non-compact), archival leopard bone, formalin-fixed snake tissue in ethanol, and preserved
skin samples from leopards and an Asiatic linsang (Table 1). Bone samples were extracted
following Dabney et al. [2]. For the tissue samples, digestion was performed using a non-
destructive buffer containing guanidine-thiocyanate [22], followed by the washing and
binding steps from Dabney et al. (2013) [2,23,24]. Between 7 and 13 ng of DNA extract were
used for the preparation of both the single-stranded libraries and the amplification-free
libraries, as input amounts over 13 ng negatively impacts the efficiency of the single-stranded
protocol [3] (S1 Table in S1 File). Libraries were then sequenced on the NextSeq 500 platform
using custom primers CL72 and Gesaffelstein [3,25] generating 75 bp single-end reads (S2
Table in S1 File).
Adapter sequences were trimmed from the raw reads using cutadapt v4 [26], using a
minimum length filter of 30 bp and a minimal match between read and adapter sequence of 1
bp (-O 1), and then mapped to an appropriate reference genome available at the time of data
processing (Tables 1 and S2 in S1 File) using the ‘aln’ algorithm of bwa [27] with the program’s
default parameters (-n 0.04). SAMtools v1.12 [28] was used to discard reads with low mapping
quality (MapQ < 30). MarkDupsByStartEnd v0.2.1 (https://github.com/dariober/Java-cafe)
was used to remove duplicates that share the same 5’-end and 3’-end positions. AMBER
(https://github.com/tvandervalk/AMBER; commit b8fa6fe) [29] was used for generating ter-
minal C > T deamination, as well as nucleotide misincorporation and read length plots (S2 and
S3 Figs in S1 File).
The following additional variables were recorded from the data: endogenous content
(calculated as a percentage of filtered reads that map to the reference genome), duplication
rate (calculated as the percentage of reads flagged as duplicates from the total mapped reads),
recovered fragment lengths, and GC content. All these statistics were extracted directly from
the bam files using samtools and standard BASH code. Statistical analyses and plotting were
performed in R v4.1.0 including utilities from the tidyverse library [30].
Amplification free Pleistocene genome
A Late Pleistocene cave bear from Peștera cu Oase cave in Romania (“O34-16”) was selected
for whole genome sequencing to 1x coverage, to validate the consistency of data generated
from the amplification-free library for population genetic analyses.
Experiment 4. 75 bp single-end sequencing was carried out on the amplification-free
library of sample O34-16, aiming for 1x coverage. Further sequencing (10 million reads) was
additionally carried out for the standard single-stranded library from the same specimen to
assess library complexity, and this sequencing run employed 75 bp paired-end sequencing
(S2 Table in S1 File). Reads were trimmed as described above (using cutadapt and discarding
reads less than 30bp), and overlapping paired-end reads were merged using Flash v1.2.11
[31] with a minimum overlap (-m) of 10bp and a maximum (-M) of 76bp. All data were then
mapped as described above and analysed alongside previously published cave, brown and
polar bear genomic data [32,33] (S4 Table in S1 File).
Principal component analysis was carried out by calculating an allele covariance matrix in
ANGSD v0.933 [34] by sampling a random base for each position (doIBS 1), ignoring reads
with a base or mapping quality below 30. Singleton sites, transition sites, and sites where any
individual had missing data were also excluded. Principal components were then calculated
from the covariance matrix using the eigen() function in R v4.3.1. A neighbour-joining tree
was computed by calculating a distance matrix in ANGSD, using the same filters as described
above, from which a neighbour-joining tree was generated using the ‘ape’ [35] and ‘phytools’

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[36] packages in R. The tree was rooted on a known root node in cave bear phylogenetic rela-
tionships, separating the Caucasus and European/Uralian cave bear clades [33].
Results and discussion
We successfully developed a modified version of the well-established single-stranded library
preparation method that avoids the need for a PCR amplification step (S1 Protocol). Using
this modified protocol, we were able to successfully generate libraries for all samples investi-
gated in this study (Tables 1 and S1 in S1 File). Using low level sequencing of these libraries
(<1 million sequence reads each; S2 Table in S1 File), we were able to assess the performance
of the amplification-free library preparation protocol in terms of basic data characteristics
such as endogenous content, GC content, duplication and read length. Furthermore, we gen-
erated a palaeogenome dataset of a Pleistocene sample without any pre-sequencing amplifica-
tion (S2 Table in S1 File).
Conversion
Comparisons of absolute conversion using the test oligo CL104 indicated that conversion rates
for the amplification-free protocol are lower (22%; Fig 2A and S1 Data in S1 File) than for the
standard single-stranded protocol (47%). Comparisons using Pleistocene steppe bison bone
samples showed the same pattern overall, but revealed that absolute library yield – defined as
the absolute quantity of library molecules – for both protocols are in general highly variable
between individual samples, and in some cases between experimental replicates (Fig 2B). As
expected, we find that library yield is higher for samples where a higher amount of input DNA
was used, both for the standard single-stranded protocol and amplification-free method (S1
Table in S1 File and Fig 2B). On average, we achieved 1.8x (range 1.4-2.1x) better yield for the
Pleistocene samples with the standard single-stranded library protocol than the amplification-
free method, which is in line with the absolute conversion rates measured using the
CL104 oligo.
Fig 2. Absolute quantification of libraries. (A) Results of experiment 1: Boxplots showing absolute conversion – defined as the total number of molecules in the qPCR
input product versus the number of molecules with library adapters – for the amplification-free (AF) and single-stranded (SS) libraries, using the test oligo CL104. The
number of CL104 molecules amplifiable with the primers CL107/CL108 (internal; displayed in orange) correspond to the total number of input CL104 molecules (i.e.
all those present after library preparation), while the primer pair IS7/IS8 (external; light blue) only amplifies correctly formed library molecules. Points show values for
individual experimental replicates. (B) Results of experiment 2: Absolute quantification of successfully converted library molecules for six Pleistocene bovid sample
extracts, for amplification-free (AF; red circles) and standard single-stranded (SS; blue triangles) libraries.
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Data consistency and performance on different sample types
Analysis of sequencing data from a wide range of sample types revealed that all investigated
variables -- endogenous content, GC content, duplication rates, fragment lengths and terminal
deamination -- are highly similar between the amplification-free and standard single-stranded
protocols (Figs 3 and S2 in S1 File), which are based on equal amounts of input DNA for each
pair of library protocols. Only minor differences between the protocols are observed for one
Pleistocene bone sample (SP347) with a very low number of reads mapping to the reference
genome (0.02%), suggesting that this is likely a stochastic effect (S2 Table in S1 File). We fur-
thermore calculated all variables also for equal subsamples between the pairs of amplification-
free and standard single-stranded libraries (S3 Table in S1 File). The results are consistent
between the total and the subsampled data, except for the duplication levels which is lower as
expected for subsampled data. Overall, our results indicate that, at the level of coverage tested
here, the amplification-free protocol is consistent with the well-established protocol, and can
likely be broadly applied across many different sample types. Previous studies have shown that
data produced by different library protocols can be highly heterogeneous, with the potential
to mislead certain types of analysis [18]. Therefore, the observed consistency in data recovery
between amplification-free and standard single-stranded protocol is an important indicator
that combining data from these protocols will be less prone to such confounding effects.
Sequence duplication
A surprising aspect of our sequence data analysis are the similar levels of apparent PCR dupli-
cation between amplification-free and standard single-stranded libraries (both approximately
2%; Fig 3 and S2 Table in S1 File). As no PCR amplification was performed in the library
preparation, the presence of apparent PCR duplicates in the amplification-free datasets is arte-
factual. This result does indicate that for the DNA extracts and sequencing depths investigated
in this study, the standard single-stranded protocol produces libraries of sufficient complexity
that the duplication generated by PCR does not impact the sequencing yield. However, PCR
duplication has led to a substantial loss in usable sequence data in other ancient DNA studies;
for example a recent ancient giant panda genome dataset [37] contained up to 60% dupli-
cates. In such cases, the amplification-free library preparation could be a potential avenue
to improve sequence yield by reducing the amount of data lost to PCR duplication, and thus
leverage the budgeted sequencing funds.
The absence of PCR duplicates in the amplification free data also provides an opportunity
to investigate the potential origin of the artefactual duplication, particularly duplication gener-
ated during the sequencing process. We find a spatial pattern of increased duplication at the
edges of the flow cell (Fig 4), where duplicates are generated during the sequencing process
itself, when the same cluster is falsely identified as different clusters during the imaging stage
of sequencing (discussed on https://www.biostars.org/p/229842/). At the levels of sequencing
generated in this study, these false optical edge duplications account for approximately half of
all duplicates – however it is unknown how this effect scales with increased sequencing levels.
In cases where this type of duplications impact data recovery, a coordinate-aware deduplica-
tion strategy -- where duplicates are removed for reads near the edge of the flow cell, but not
for those in the centre -- could be a method to avoid losing reads to false duplicates.
Amplification-free Pleistocene genome
The consistency of data generated using the amplification-free protocol with that generated
with the standard single-stranded library protocol is further confirmed by analysis of the
approximately 1x Pleistocene cave bear palaeogenome from Romania (sample “O34-16”). For

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this sample, we generated 1.5Gb (0.7x) and 0.17Gb (0.07x) of the nuclear genome using the
amplification-free and standard single-stranded protocols, respectively. Previous studies have
shown that different library preparation protocols can significantly alter the properties of the
resulting sequencing data, including fragment length recovery, GC content and damage patterns
[17,38], leading to a potential bias in downstream analysis when mixing different library prepa-
ration methods in a single study. Furthermore, it has been shown that such biases can impact
downstream population clustering analysis, for example resulting in a pronounced branch
Fig 3. Results of experiment 3: (A) Endogenous content, measured by the percentage of trimmed, unique reads that map to the reference
genome. (B) GC content of mapped reads. (C) Percentage of duplications. (D) Boxplots showing fragment length distributions of mapped reads
in the 30–75 bp range.
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lengthening effect in phylogenetic trees [29]. In order to test if the amplification-free protocol
would result in a bias in downstream analyses, we performed basic population clustering analy-
ses with the datasets generated with the amplification-free protocol and standard single-stranded
protocol from the Romanian cave bear. The genetic clustering analyses, both the principal
component analysis and the distance-based neighbour-joining tree, generally show no difference
when the amplification-free data or the standard single-stranded data is used (Fig 5; based on
938,121 and 157,672 variable sites for the amplification-free and single-stranded data, respec-
tively). For the principal component analysis, the individual clusters as expected with other cave
bears from the same evolutionary lineage when considering the first two principal components
that together account for more than 50% of the total variation (European cave bears, see [33];
Fig 5). The phylogenetic analyses performed on published data and data generated using the
amplification-free protocol shows the same topology for the amplification-free and standard
single-stranded libraries, and no apparent differences in branch lengths. We do note that in
neither analysis the individual O34-16, identified as an ingressus cave bear based on morphology,
clusters with the published ingressus genome, but rather with the spelaeus genome which can be
considered a separate evolutionary lineage [39,40]. Further investigations of cave bear popula-
tion structure is needed to resolve this apparent discrepancy. However, as this result is identical
between the two library preparation protocols, this cannot be the result of inadvertent bias of
the amplification-free library preparation protocol. Thus, we confirm that the amplification-free
protocol enables the sequencing of palaeogenomes without PCR amplification, and that the
resulting data can be readily combined and integrated with existing single-stranded datasets for
evolutionary and population genetics analyses.
Fig 4. Tile-edge duplications. (A) Visual representation of the distribution of unique vs non-unique (duplicate) clusters on the flowcell for sample “O34-16”,
amplification-free library (test sequencing). (B-C) Density plots for unique and duplicate clusters along the X axis (B) and Y axis (C) for the same library further show-
ing the increased density of duplicates around the edges of the flow cell (deep sequencing).
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Limitations of the amplification-free protocol
During the development and testing of the amplification free protocol we noted some
limitations. The first is that, unlike the standard single-stranded libraries, the total quan-
tity of library available for sequencing is directly related to the quantity of input DNA.
Repeated quantification and sequencing runs will rapidly diminish the available library
stock, unlike PCR amplified libraries where a large stock can be amplified for use in many
experiments. This means that the level of coverage that can be achieved for an individ-
ual library is limited by its volume, rather than complexity. To achieve the 1x coverage
(2.9 mapped Gb) for sample O34-16, 3.17µl of the final library product (~25µl) was used
for sequencing, meaning that this particular library would be limited to an estimated 4x
sequence coverage. Although subsequent amplification of the amplification-free library
is possible using standard primers, this would remove the unique aspect of the approach
used to generate it.
During preparation of amplification-free libraries, we observed an increased presence of
adapter dimers in the purified product compared to the standard single-stranded protocol
(Fig 6), likely due to the increased length of the oligonucleotides used (Fig 1). At time of this
Fig 5. Results of experiment 4: Population clustering of the cave bear data generated with the amplification-free
method as well as the standard single-stranded method within existing bear data. (A/B). Principal component analysis
of cave bears using the amplification-free (A) and standard (B) methods. (C/D): neighbour-joining phylogenetic analyses
using the amplification-free (C) and standard (D) methods.
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work, a combination of Tapestation and Qubit quantification of libraries was performed prior
to sequencing on the in-house sequencer (NextSeq 500), which has been shown to be highly
successful for achieving reliable cluster densities for standard single-stranded libraries [25].
We found that the cluster density achieved for the amplification-free library was substantially
lower than the standard cluster densities (84 k/mm2, compared to optimal cluster densities
of 180-220 k/mm2 following Illumina recommendations). The method of pre-sequencing
library quantification we used does not distinguish between sequenceable (i.e. with the cor-
rect adapter on either end of the molecule) and non-sequenceable dimers or artefacts. The
majority of adapter dimers observed in the amplification-free libraries are highly likely to be
of non-sequenceable type, as the amount of adapter dimers in the sequencing data (defined
here as reads reduced to 0 bp after adapter trimming) is low (S2 Table in S1 File). Therefore,
the issue of low cluster density can likely be mitigated by using qPCR for quantification of the
library prior to sequencing, as described in the original single-stranded protocol [3]. Alter-
natively, adjusting the adapter amounts or an approach as followed in Karlsson et al. [15]
with a random overhang added to oligo CL9_Phos_index (Fig 1) could potentially reduce the
formation of adapter dimers.
Conclusions
The amplification-free adaptation of the standard single-stranded library preparation protocol
we present here successfully generated sequence data from Pleistocene, Holocene and histori-
cal samples. We find that the data is highly consistent between the amplification-free and
single-stranded protocols, suggesting that PCR does not introduce major biases in the
standard single-stranded library construction. Our amplification-free adaptation of the
single-stranded library preparation method represents a method that, through the removal
of the indexing PCR, excludes a step that is often associated with the introduction of biases.
Thus, the amplification-free approach provides a more accurate representation of the origi-
nal template molecules in an ancient DNA extract. We use our amplification-free protocol to
Fig 6. TapeStation results from the libraries for the standard single-stranded protocols (A) (Standard D1000 tape)
and amplification-free (B) (High Sensitivity D1000 Tape). Peaks at 25 bp (basepair) and 1500 bp are standard Tapestation
markers. For the amplification-free libraries, adapter dimers have been detected in addition to the expected library peak:
an extra peak can be seen around ~ 71 bp, and a disruption of the distribution of DNA fragment lengths around 130 bp,
suggesting the presence of multiple adapter dimers.
https://doi.org/10.1371/journal.pone.0319573.g006

PLOS ONE | https://doi.org/10.1371/journal.pone.0319573 March 19, 2025 12 / 15
PLOS ONE Amplication-free palaeogenome library preparation
generate a nuclear genome of a Pleistocene cave bear, and perform basic population clustering
methods to show that this method is not expected to cause any biases in downstream analysis
compared to the standard single-stranded protocol.
Our study represents a proof-of-concept study, showcasing a method for sequencing
ancient and other degraded DNA without any amplification prior to sequencing.
Amplification-free library preparation from degraded samples could find a range of appli-
cations both within and outside of palaeogenomics. The ability to more directly characterise
DNA content of such samples has the potential to provide new insights into the properties
of damaged DNA and its degradation processes. Furthermore, amplification-free library
preparation can be of interest for e.g. (ancient) poolseq and metagenomics, where PCR could
introduce a bias in the relative presences of alleles, individuals or species present in the input
material [41]. Similarly, the study of individual genome copy number variation and structural
variants could benefit from the amplification-free method.
One further exciting future research avenue would be to implement the protocol presented
here for the recovery and sequencing of ancient RNA. The field of ancient RNA is still emerg-
ing, and our protocol could allow for a direct characterisation of ancient RNA molecules and
RNA specific damage occurring over time. As amplification could be problematic for compar-
ing expression levels across different genes, the use of an amplification-free protocol specifi-
cally designed for ancient samples could mitigate some of these issues.
Supporting information
S1 File. S1 Fig. DNA yield (total and endogenous fraction in grey and black, respectively)
from 14 cave bear petrous bones, showcasing the potential genome recovery from Pleis-
tocene samples. For each sample, 50 mg bone powder was sampled from the otic capsule of
the petrous bone as described in Alberti et al. [7]. DNA was extracted using the methods of
Dabney et al. [2] and quantified using a Qubit fluorometer high sensitivity assay of 1µl of the
DNA extracted. The estimated concentration was multiplied by the 25 µl elution volume to
obtain an estimate of the total DNA yield. Single stranded libraries [3] were prepared from
the extracts and low level sequencing carried out on an Illumina NextSeq platform [25]. The
resulting data were mapped to the reference genome of the polar bear using the procedure
described in Barlow et al. [32,33] to provide an estimate of endogenous content. S2 Fig.
Nucleotide misincorporation and read length plots for deeper sequencing of sample O34-16,
for the standard single-stranded library protocol (SS; red) and the amplification-free protocol
(AF; grey). Plots were generated using AMBER [29] with default parameters. (A) mismatch
frequency and (B) nucleotide misincorporation at each position in the read, (C) read length
and (D) depth in 1kb windows. Note that the amplification-free library was sequenced using
a 75bp single-end sequencing method, and the single-stranded library using 75bp paired-end
method. This leads to an accumulation of all fragments that were > 75bp at a read length of
74-75bp for the amplification-free library which can be seen in panel C (AF; grey). S3 Fig.
Nucleotide misincorporation plots for all samples, for the standard single-stranded library
protocol (SS) and the amplification-free protocol (AF). Plots were generated using AMBER
[29] with default parameters. Panels for each figure are the same as defined in S2 Fig leg-
end. S1 Table. Wetlab information. S2 Table. Sequence data statistics. S3 Table. Subsampled
sequence data statistics. S4 Table. Published cave bear datasets included in population clus-
tering analysis. S5 Table. Oligonucleotides used in this study. Lowercase bases indicate index
sequence. For more information, please refer to the single-stranded protocol (Gansauge &
Meyer 2013). S1 Protocol. S1 Data. Raw qPCR results (csv file).
(ZIP)

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PLOS ONE Amplication-free palaeogenome library preparation
Acknowledgements
We thank Joerns Fickel (IZW, Berlin) for contributing sample PLI-19 and feedback on this
manuscript. Sample collection for PLI-19 was supported by the “Sunda shelf project”, and the
sample was provided by Naturalis Museum Leiden. We also thank Frieder Mayer, Mark-
Oliver Roedel and Johannes Penner (Natural History Museum Berlin) for supplying sam-
ples from the museum collections. We also thank Gernot Rabeder (University of Vienna) for
providing a cave bear sample and Frank Menger (Groß-Rohrheim, Germany) for providing
an aurochs sample.
Author contributions
Conceptualization: Axel Barlow, Johanna L. A. Paijmans.
Data curation: Johanna L. A. Paijmans.
Formal analysis: Kirstin Henneberger, Axel Barlow, Federica Alberti, Johanna L. A. Paijmans.
Funding acquisition: Silviu Constantin, Doris Döppes, Wilfried Rosendahl, Michael
Hofreiter, Johanna L. A. Paijmans.
Investigation: Kirstin Henneberger, Federica Alberti, Michaela Preick.
Methodology: Axel Barlow, Michael Hofreiter, Johanna L. A. Paijmans.
Project administration: Johanna L. A. Paijmans.
Resources: Silviu Constantin, Doris Döppes, Wilfried Rosendahl.
Software: Johanna L. A. Paijmans.
Supervision: Axel Barlow, Michael Hofreiter, Johanna L. A. Paijmans.
Validation: Kirstin Henneberger, Johanna L. A. Paijmans.
Visualization: Kirstin Henneberger, Axel Barlow, Federica Alberti, Johanna L. A. Paijmans.
Writing – original draft: Johanna L. A. Paijmans.
Writing – review & editing: Kirstin Henneberger, Axel Barlow, Federica Alberti, Michaela
Preick, Silviu Constantin, Doris Döppes, Wilfried Rosendahl, Michael Hofreiter, Johanna
L. A. Paijmans.
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