Assessing the fidelity of ancient DNA sequences amplified from nuclear genes.
ABSTRACT To date, the field of ancient DNA has relied almost exclusively on mitochondrial DNA (mtDNA) sequences. However, a number of recent studies have reported the successful recovery of ancient nuclear DNA (nuDNA) sequences, thereby allowing the characterization of genetic loci directly involved in phenotypic traits of extinct taxa. It is well documented that postmortem damage in ancient mtDNA can lead to the generation of artifactual sequences. However, as yet no one has thoroughly investigated the damage spectrum in ancient nuDNA. By comparing clone sequences from 23 fossil specimens, recovered from environments ranging from permafrost to desert, we demonstrate the presence of miscoding lesion damage in both the mtDNA and nuDNA, resulting in insertion of erroneous bases during amplification. Interestingly, no significant differences in the frequency of miscoding lesion damage are recorded between mtDNA and nuDNA despite great differences in cellular copy numbers. For both mtDNA and nuDNA, we find significant positive correlations between total sequence heterogeneity and the rates of type 1 transitions (adenine --> guanine and thymine --> cytosine) and type 2 transitions (cytosine --> thymine and guanine --> adenine), respectively. Type 2 transitions are by far the most dominant and increase relative to those of type 1 with damage load. The results suggest that the deamination of cytosine (and 5-methyl cytosine) to uracil (and thymine) is the main cause of miscoding lesions in both ancient mtDNA and nuDNA sequences. We argue that the problems presented by postmortem damage, as well as problems with contamination from exogenous sources of conserved nuclear genes, allelic variation, and the reliance on single nucleotide polymorphisms, call for great caution in studies relying on ancient nuDNA sequences.
- SourceAvailable from: Viviane Despres[show abstract] [hide abstract]
ABSTRACT: Maize was domesticated from teosinte, a wild grass, by approximately 6300 years ago in Mexico. After initial domestication, early farmers continued to select for advantageous morphological and biochemical traits in this important crop. However, the timing and sequence of character selection are, thus far, known only for morphological features discernible in corn cobs. We have analyzed three genes involved in the control of plant architecture, storage protein synthesis, and starch production from archaeological maize samples from Mexico and the southwestern United States. The results reveal that the alleles typical of contemporary maize were present in Mexican maize by 4400 years ago. However, as recently as 2000 years ago, allelic selection at one of the genes may not yet have been complete.Science 12/2003; 302(5648):1206-8. · 31.20 Impact Factor
- Current Biology 02/2004; 14(1):R9-10. · 9.49 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The complete 17,009-bp mitochondrial genome of the domestic cat, Felis catus, has been sequenced and conforms largely to the typical organization of previously characterized mammalian mtDNAs. Codon usage and base composition also followed canonical vertebrate patterns, except for an unusual ATC (non-AUG) codon initiating the NADH dehydrogenase subunit 2 (ND2) gene. Two distinct repetitive motifs at opposite ends of the control region contribute to the relatively large size (1559 bp) of this carnivore mtDNA. Alignment of the feline mtDNA genome to a homologous 7946-bp nuclear mtDNA tandem repeat DNA sequence in the cat, Numt, indicates simple repeat motifs associated with insertion/deletion mutations. Overall DNA sequence divergence between Numt and cytoplasmic mtDNA sequence was only 5.1%. Substitutions predominate at the third codon position of homologous feline protein genes. Phylogenetic analysis of mitochondrial gene sequences confirms the recent transfer of the cytoplasmic mtDNA sequences to the domestic cat nucleus and recapitulates evolutionary relationships between mammal species.Genomics 05/1996; 33(2):229-46. · 3.01 Impact Factor
Copyright ? 2006 by the Genetics Society of America
Assessing the Fidelity of Ancient DNA Sequences Amplified From
Jonas Binladen,* Carsten Wiuf,†M. Thomas P. Gilbert,‡Michael Bunce,§Ross Barnett,**
Greger Larson,** Alex D. Greenwood,††,‡‡James Haile,** Simon Y. W. Ho,**
Anders J. Hansen* and Eske Willerslev*,1
*Ancient DNA and Evolution Group, Centre for Ancient Genetics, Niels Bohr Institute and Biological Institute, University of Copenhagen,
Copenhagen DK-2100, Denmark,†Bioinformatics Research Center, University of Aarhus, Aarhus DK-8000, Denmark,‡Ecology and
Evolutionary Biology, University of Arizona, Tucson, Arizona 85721,§Department of Anthropology, McMaster University, Hamilton,
Ontario L8S 4L9, Canada, **Henry Wellcome Ancient Biomolecules Centre, Department of Zoology, University of Oxford,
Oxford OX1 3PS, United Kingdom,††Department of Vertebrate Zoology, American Museum of Natural History,
New York, New York 10024 and‡‡Institute of Molecular Virology, GSF-National Research Center for
Environment and Health, 85764 Neuherberg, Germany
Manuscript received August 17, 2005
Accepted for publication October 25, 2005
To date, the field of ancient DNA has relied almost exclusively on mitochondrial DNA (mtDNA) se-
(nuDNA) sequences, thereby allowing the characterization of genetic loci directly involved in phenotypic
traits of extinct taxa. It is well documented that postmortem damage in ancient mtDNA can lead to the
generation of artifactual sequences. However, as yet no one has thoroughly investigated the damage spec-
trum in ancient nuDNA. By comparing clone sequences from 23 fossil specimens, recovered from environ-
the mtDNA and nuDNA, resulting in insertion of erroneous bases during amplification. Interestingly, no
significant differences in the frequency of miscoding lesion damage are recorded between mtDNA and
nuDNA despite great differences in cellular copy numbers. For both mtDNA and nuDNA, we find significant
positive correlations between total sequence heterogeneity and the rates of type 1 transitions (adenine /
guanine and thymine / cytosine) and type 2 transitions (cytosine / thymine and guanine / adenine),
respectively. Type 2 transitions are by far the most dominant and increase relative to those of type 1 with
damage load. The results suggest that the deamination of cytosine (and 5-methyl cytosine) to uracil (and
thymine) is the main cause of miscoding lesions in both ancient mtDNA and nuDNA sequences. We argue
that the problems presented by postmortem damage, as well as problems with contamination from exoge-
nous sources of conserved nuclear genes, allelic variation, and the reliance on single nucleotide poly-
morphisms, call for great caution in studies relying on ancient nuDNA sequences.
on the environment and subsequent conditions of stor-
age (Pa ¨a ¨bo 1989; Lindahl 1993; Ho ¨ss et al. 1996;
Poinar et al. 1996; Hofreiter et al. 2001a; Smith et al.
2001; Pa ¨a ¨bo et al. 2004; Willerslev et al. 2004a;
Willerslev and Cooper 2005). Postmortem DNA is
subject to degradation by microorganisms, soil inver-
tebrates, and cellular nucleases, in addition to mod-
ifications by spontaneous chemical reactions such as
hydrolysis and oxidation (Lindahl 1993; Hofreiter
et al. 2001a; Gilbert and Hansen 2006). While some
HEN an organism dies, its DNA starts degrading
at a rate that is believed to be highly dependent
sion of polymerase enzymes, thus rendering the mole-
cules unsuitable as template for PCR, others, termed
miscoding lesions, allow for amplification, but result in
the incorporation of erroneous bases during PCR
(Pa ¨a ¨bo 1989; Lindahl 1993; Ho ¨ss et al. 1996; Poinar
et al. 1998; Hansen et al. 2001; Hofreiter et al. 2001b;
Gilbert et al. 2003a,b; Banerjee and Brown 2004;
Willerslev et al. 2004b; Mitchell et al. 2005; Gilbert
and Hansen 2006). The most commonly observed
miscoding lesions in ancient DNA (aDNA) studies are
the transitions adenine/ guanine (A / G), cytosine /
thymine (C / T), G / A, and T / C (Hansen et al.
2001; Hofreiter et al. 2001b; Gilbert et al. 2003b).
Although four different transitions have been observed,
it has been argued that it is possible to differentiate
the transitions into only two complementary groups,
termed type 1 (TS1: A / G/T / C) and type 2 (TS2:
C / T/G / A) transitions, caused putatively by the
Sequence data from this article have been deposited with the EMBL/
GenBank Data Libraries under accession nos. DQ318533–DQ318562.
1Corresponding author: Ancient DNA and Evolution Group, Centre for
of Copenhagen, Juliane Maries vej 30, DK-2100, Denmark.
Genetics 172: 733–741 (February 2006)
deamination of adenine to hypoxanthine (A / H) and
the deamination of cytosine (and its homolog 5-methyl
cytosine) to uracil (and thymine), respectively (Hansen
et al. 2001; Hofreiter et al. 2001b; Gilbert et al.
2003b). For example, consider a single cytosine to ura-
cil (C / U) deamination event on the light strand of
mitochondrial DNA (mtDNA). The C / U event will
be observed as a C / T transition on any light strand
sequences amplified directly from the original dam-
aged template, but any of the derived complementary
heavy strand sequences will exhibit a G / A transition.
Similarly, any sequences derived directly from the
amplification of an A / H event will be exhibited as
either an A / G or T / C transition. Intriguingly, even
though miscoding lesions generating both type 1 and
type 2 transitions are recorded for DNA in solution
(deamination of cytosine with a rate ?30–50 times
higher than that for adenine; Lindahl 1993), it is still
debated whether the deamination of adenine actually
plays a role in the generation of type 1 events in aDNA
or whether they are simply due to regular DNA poly-
merase errors (Hofreiter et al. 2001b; Gilbert et al.
2003b; Pa ¨a ¨bo et al. 2004).
Although mtDNA is most frequently used in aDNA
research, a number of recent studies have reported the
successful retrieval of low- and single-copy nuclear DNA
(nuDNA) sequences (Greenwood et al. 1999, 2001;
et al. 2003; Orlando et al. 2003; Poinar et al. 2003;
Noonan et al. 2005). Such studies represent important
breakthroughs, as nuDNA can be used to answer pre-
viously intractable questions, such as the establishment
Despres et al. 2003) and the resolution of deep phylo-
genetic splits (Poinar et al. 2003). However, nuclear
genes are typically 5000–10,000 times less abundant per
cell than those of mitochondrial origin, which is
probably the reason for ancient nuDNA being much
more difficult to amplify than mtDNA from the same
aDNA extracts (Poinar et al. 2003).
To date, studies that characterize DNA damage in
fossil remains have been restricted to mtDNA (Ho ¨ss
et al. 1996; Hansen et al. 2001; Hofreiter et al. 2001a;
Gilbert et al. 2003a,b; Threadgoldand Brown 2003;
Banerjee and Brown 2004; Gilbert et al. 2005).
Although one study has reported substitutions in
PCR-amplified ancient nuDNA sequences, the authors
attribute the observed changes to mutagenic effects in-
troduced under PCR reactions (Pusch et al. 2004),
Despite the lack of publications, the characterization
of damage in ancient nuDNA remains important to
the field. In particular, the fact that miscoding lesions
can result in modification of the consensus sequence
amplified from ancient mtDNA (Handt et al. 1996;
Gilbert et al. 2003a; Banerjee and Brown 2004;
Hebsgaard et al. 2005), along with the overall low
plates, carries the implication that sequences amplified
from ancient nuDNA are at increased risk of contain-
ing sequence errors. Furthermore, nuDNA sequences
generated from fossil remains often are single nucleo-
tide polymorphisms (SNPs), and the determination of
SNPs as real or as the result of postmortem damage is
In this study we investigate the frequency and types of
miscoding lesions in various mtDNA and nuDNA mark-
ers across a variety of fossil specimens of different ages
addition, we discuss some of the factors that need to be
considered when amplifying, sequencing, and inter-
preting nuclear data from archival and fossil remains.
MATERIALS AND METHODS
We analyzed previously unreported DNA sequences ex-
tracted from permafrost-preserved bones of the woolly rhi-
noceros (Coelodonta antiquitatis, n ¼ 2) and the lion (Panthera
leo spelaea, n ¼ 6), as well as temperate-preserved bones of the
pig (Sus scrofa, n ¼ 5) and the female moa (Dinornis robustus,
n ¼ 4). Not included in the comparative study were amplifi-
cation results from one woolly rhinoceros, two pigs, and 28
lion specimens yielding mtDNA sequences but no nuDNA
sequences and from three woolly rhinoceroses and 88 lion
specimens yielding neither mtDNA nor nuDNA sequences.
Additionally, published mtDNA and nuDNA clone sequences
from desert-preserved ground sloth coprolites and permafrost-
preserved woolly mammoth and woolly rhinoceros bones and
2003; Greenwood et al. 1999, 2001; Orlando et al. 2003).
Published sequences from ancient human remains were not
included due to the high risk of contamination in such studies
(Cooper and Poinar 2001; Hofreiter et al. 2001b; Pa ¨a ¨bo
comprised mtDNA and nuDNA sequences from 23 specimens
(for sample details, see Table 1 and supplemental material at
Bone samples were collected, DNA extracted, and PCR
amplified following established aDNA protocols: Using a
Dremel tool, ?1-cm3fragments ?0.5 cm in depth were re-
moved from the bones. A Braun Mikrodismembrator was used
to grind samples. Grinding equipment (stainless-steel balls
and cups, rubber washers) was thoroughly bleached between
each use. Decalcification was done in 5–30 vol of 0.5 m EDTA
(pH 8) overnight at room temperature. The sediment was
collected by centrifugation and digested with 0.25 mg/ml
were extracted twice with phenol and once with chloroform,
and the DNA was recovered and up-concentrated with
Centricon-30 (Amicon, Beverly, MA) devices. The proofreading
used in PCR amplification tominimize thegeneration of DNA
polymerase errors that can mimic errors caused by miscoding
bovine serum albumin (BSA), 10 mm Tris?HCl, 1.5 mm MgCl2,
50mmKCl (pH 8.3), 0.8 mmdNTPs, 1 mm ofeach primer, and
1 unit of DNA polymerase. Thermal cycling conditions were
typically 40 cycles of 95?/52?–66?/68? (30–90 sec each) for
mtDNA amplifications and 40–50 cycles of 95?/52?–55?/68?
(45–60 sec each) for nuDNA amplifications. BSA was added to
734J. Binladen et al.
thePCR toovercome inhibition. Additionally, dilutions(1:10–
1:50) of the DNA extracts were attempted. For primer details
and amplification conditions, see Table 2. The amplification
products were cloned and sequenced following established
procedures (Willerslev et al. 1999).
Strict protocols were followed to minimize the risk of
sample and extract contamination with exogenous sources
of DNA, including the use of aDNA facilities (physically iso-
lated from the laboratories where postamplification manipu-
lation is performed), theincorporation of extraction and PCR
blank controls at ratios of 1:5 and 1:1–2, respectively, and
quantification of a few of the extracts by real time PCR using
SYBR green detection chemistry (Applied Biosystems, Foster
City, CA) (for details see supplemental material at http:/ /www.
genetics.org/supplemental/). Independent replication was
carried out for a subset of results in dedicated aDNA facilities
in Copenhagen and Oxford (Table 1).
As described earlier, either of the complementary DNA
strands can act as templates for PCR, as any single miscoding
lesion event can produce two observable phenotypes post-
PCR. It has been argued that since the chemical events re-
quired to generate direct G / A and T / C transitions are
biochemically unlikely, any G / A and T / C damage that is
observed on a particular DNA strand must have originated on
the complementary strand as C / U and A / H events, re-
spectively (Gilbert et al. 2003b). Others have questioned this
Mp-Wrangel4590 6 50Bone Permafrost
4/556 14/1596 cyt bMicro-satellites
Mp-Alaska8460 13,775 6 145 ToothPermafrost 14/2098i
131/7252 cyt b vWf,a2ab,irib,
Micro-satellites Mp-Siberia26,000 6 1600 BonePermafrost
4/55610/1140 cyt b
43700 6 1000 Bone
19875 6 215
Temperate cave C. antiquitatis 43/7463i
Temperate cave C. antiquitatis 10/1820i
C. antiquitatis 47/11697i
Cropolite Varm cave
12/3552 12S, cyt bjNumt
12,450 6 60
12,090 6 80
54,100 6 1800 Bone
46,200 6 1500 Bone
613 6 90
Temperate cave D. robustus
Temperate cave D. robustus
Temperate cave D. robustus
YBP, years before present.
aData sources: Moa716, Moa237, Moa660, and Moa799 are from Bunce et al. (2003) and data generated in this study; Mp-Wrangel,
Mp-Sibiria, and MP-Alaska8460 are from Greenwood et al. (1999, 2001) and data generated in this study; Ua11835 is from Poinar
et al. (1998, 2003); SC81205 and SC7400 are from Orlando et al. (2003); GL92, GL71, GL55, GL45, and GL76 are from Larson
et al. (2005) and this study; RB41, RB42, RB44, RB46, RB75, and RB91 are from this study; PIN3342-103 and PIN3100-169 are also
from this study.
bAge of specimen based on museum records, accelerator mass spectrometer dating, or on stratigraphy.
cType of specimen.
dType of original preservation environment.
eSpecies name of specimen.
fSampling effort. The numbers of mitochondrial or nuclear clones/the total length of the sequences in base pairs. The average
number of clones is very similar for mtDNA (16; SD 17) and nuDNA (20; SD 33).
gName of the mitochondrial and nuclear sequences studied.
hAge of specimen based on stratigraphy.
iResults were independently replicated.
jResults were quantified by real time PCR.
Damage in Ancient Mitochondrial and Nuclear DNA Sequences 735
hypothesis due to the limited knowledge of DNA damage in
fossil remains (Pa ¨a ¨bo et al. 2004). We have therefore decided
et al. (2001a) and not distinguish between the different phe-
notypes of TS1 (A / G/T / C) and TS2 (C / T/G / A).
Total sequence heterogeneity (TSH) was calculated as the
probability of observing transitions in a single position fol-
lowing the formula TSH ¼ l/n, where l is the total number of
quences showing PCR artifacts in the form of ‘‘jumping PCR’’
are marked in the clone data sets (supplemental material S4–
S40 at http:/ /www.genetics.org/supplemental/) as CHI for
chimeric. These sequences were not included in the analysis.
Type 1 and type 2 transitions were calculated in a similar
fashion,resulting inthree values, pTSH, pTS1, andpTS2, foreach
of the 2 3 23 ¼ 46 clone data sets. Transitions were scaled by
multiplying with the AT:GC ratio of the specific gene region
tocompensate for nucleotide composition bias. Subsequently,
pi, i ¼ TSH, TS1, and TS2, were transformed using ri ¼
?log(1 ? pi), where ri¼ aiT, T is the age of the specimen, and
aiis the average damage rate per unit time. The ri, i ¼ TSH,
TS1, and TS2, are referred to as the TSH, TS1, and TS2
Pearson’s correlation coefficient r was calculated between
(i) TSH and TS1 rates; (ii) TSH and TS2 rates; (iii) TS1 and
TS2 rates; (iv) age of specimens andTSH rate; (v) age of speci-
mens and TS1 rate; (vi) age of specimens and TS2 rate; (vii)
age and the ratio of TS2 rate to that of TS1 (TS2/TS1; ex-
cluding cases where rTS1¼ 0); and (viii) TSH rate and TS2/
TS1 (excluding cases where rTS1¼ 0). The correlation analysis
was performed for all 46 clone sets and separately for the 23
nuclear clones and the 23 mitochondrial clones.
A t-test was performed to investigate whether TSH rates
in nuDNA clones were significantly different from those in
mtDNA clones. Data were further divided into three groups
according to the environment of preservation: (i) permafrost
which were originally excavated, although some had sub-
sequentlybeenstored inmuseumsfor years;and(iii)museum
Primers and PCR conditions
Primer sequence (59–39)c
Real time PCR 1
Real time PCR 2
55?/340Tougard et al.
This study 56?/340
Pig / Mt. control
Pig / CD45
56?/340Larson et al.
This study 56?/340
Lion / ATP8 and
56?/340 I. Barnes,
Bunce et al.
Bunce et al.
Bunce et al.
Bunce et al.
Moa / Mt. control
Moa / Mt. control
Moa / KW1 gene
Moa / ADH gene
aSpecimen name and sequence region amplified.
dAnnealing temperature/ no. of PCR cycles.
736J. Binladen et al.
specimens (n ¼ 5), which had always been stored in museums.
Using t-tests it was investigated whether TSH rates, TSH rates
divided by age, and the TS2/TS1 rates ratio differ among the
three groups. Finally, it was investigated (using a chi-square
test) whether the observed patterns of base transitions in
mtDNA and nuDNA sequences were similarly distributed. All
tests were performed at the 1% significance level.
RESULTS AND DISCUSSION
In this study nuclear and mitochondrial DNA were
PCR amplified, cloned, and sequenced from a variety of
clone data to compare the frequencies and types of
miscoding lesion damage. As miscoding lesions are not
the only factor that might influence intraclone hetero-
geneity, it is important to take into account other
sources, such as innate DNA polymerase misincorpora-
tion errors, natural sequence heterogeneity, and varia-
tion in the number of starting template molecules for
PCR (Hansen et al. 2001). We believe these factors are
unlikely to have influenced the data in a significant
fashion for the following reasons:
i.The majority of the clone sequences analyzed were
generated using the proofreading DNA polymerase
enzyme Platinum Taq High Fidelity, an enzyme with
an innate error rate on good quality DNA of 2.0 3
10?6–6.5 3 10?7/nucleotide/cycle (Flaman et al.
1994; Andre ´ et al. 1997). Although some have spe-
culated that aDNA extracts might increase the mis-
incorporation rates of some polymerases (e.g., Pusch
retain its low misincorporation on DNA amplified
from aDNA extracts (Gilbert et al. 2003b). There-
only minor amounts of the calculated sequence
heterogeneity, although we cannot completely ex-
clude them from the data sets (e.g., errors generated
during bacterial colony growth or sequencing).
ii. It is possible that the number of starting template
molecules in the PCR reactions might influence the
observed sequence heterogeneity. In the most ex-
treme case, a PCR that starts off a single template
molecule will not demonstrate any variation and
data generated in this study we are confident that the
PCR reactions started from considerably more than a
single template molecule, since dilutions up to 1/50
of the extracts prior to amplification still yielded
amplification products for both the mtDNA and the
nuDNA markers, the clones showed variation, and
the PCR products quantified showed starting tem-
plate numbers in the 102–104range (supplemental
material at http:/ /www.genetics.org/supplemental/).
iii. It seems unlikely that single-substitution hetero-
plasmy and recombination in the mtDNA data sets
influence the observed heterogeneity due to both
mtDNA and their apparent absence in complete
et al. 1993; Lopez et al 1996; Xu and Arnason 1997;
Ursing and Arnason 1998; Lin et al. 1999; Cooper
et al. 2001; J. Krause, personal communication).
However, we cannot exclude the possibility that ad-
verse amplifications of nuclear alleles, pseudogenes,
and other gene duplicates might contribute to the ob-
observed heterogeneity in the mtDNA clones can be
explained predominantly by damage, while the varia-
tion observed in the nuDNA data sets may have arisen
from a combination of miscoding lesions and other
factors. Despite this, only aminority of the specimens (8
of 23) exhibited higher TSH in the nuDNA sequences
than in the mtDNA sequences (P ¼ 17%, Figure 1).
However, no significant differences in TSH levels be-
tween nuDNA and mtDNA sequences (P ¼ 17%) were
observed, suggesting that nuDNA miscoding lesion
damage is less than or equal to that of mtDNA, despite
a lower number of cellular copies. Intriguingly, nuDNA
appearstobe morelimitedbyamplification lengththan
represent a simple case of template quantity, whereby
Total sequence heterogeneity
No. of substitutions
125.0033.0021.00 294.7320.0531.58 525.36
No. of substitutions
42.009.00 3.00 218.100.006.35278.44
aThe sum of the different substitutions for all 23 mitochondria and nuclear data sets. Substitutions originating from a C or a G
have been corrected for the AT:GC ratio for each gene region.
Damage in Ancient Mitochondrial and Nuclear DNA Sequences737
the abundance of mtDNA makes itmore likely that long
undamaged molecules exist. Alternatively, the nucleo-
some core (146 bp of nuDNA wrapped around a his-
tone octamer) may represent a key ‘‘preservation unit’’
whereby strand breaks may be common in the linker
chromosomal proteins (e.g., histones) to the DNA in-
creases the chance of forming protein–DNA crosslinks,
structures that could easily act as polymerase blocks
A significant difference was observed in the distribu-
tions of miscoding lesion ‘‘types’’ in the mtDNA and
nuDNA sequences (P , 0.1%; Table 3). Overall the fre-
quency of type 1 transitions (A / G/T / C) is lower
in the nuDNA (15%) than in the mtDNA (24%) and
the frequency of type 2 transitions (C / T/G / A) is
higher (78% vs. 56%). This implies that different types
of damage could occur at different rates in the mito-
chondria and the nucleus. The fact that mtDNA is not
complexed with histone proteins could make it more
susceptible to different types of oxidative and/orhydro-
lytic damage. However, at this stage we cannot rule out
that the observed differences are due to other factors
such as natural variation in the nuDNA.
Although a number of aDNA studies attribute the
presence of type 1 transitions to postmortem damage
(e.g., Hansen et al. 2001; Gilbert et al. 2003b), other
studies (e.g., Hofreiter et al. 2001a; Pa ¨a ¨bo et al. 2004)
argue that their existence is an artifact of regular DNA
this study between TSH and the number of type 1 (r ¼
0.48; Figure 2A) and type 2 transitions (r ¼ 0.66; Figure
2B) and between TS1 and TS2 rates (r ¼ 0.45; Figure
2C) make it difficult to explain the type 1 transitions
solely on the basis of DNA polymerase errors. Further-
more, we find no obvious correlation between the rate
org/supplemental/). However, we do observe some
tions compared to previous observations (Gilbert et al.
2003b). In this study, we observe a total of 167 and 512
(?1:3) type 1 and type 2 events (Table 3), respectively
(counts are adjusted for base composition), which is
lower than the 177 and 366 (?1:2) type 1 and type 2
events observed by Gilbert et al. (2003b). Additionally,
only 7 of the 46 data sets investigated (both mtDNA
and nuDNA) show more type 1 than type 2 transitions
(supplemental material at http:/ /www.genetics.org/
supplemental/) compared to 26 of 65 human mtDNA
observed type 1:type 2 ratio is higher than that reported
by Hofreiter et al. (2001a). When distinguishing be-
tween consistent and singleton substitutions, Hofreiter
et al. (2001a) report consistent changes to be only type
2 events, and the ratio for the remaining singletons to
be 44 type 1 to 282 type 2 (?1:6) (M. Hofreiter, un-
The correlation analyses show a clear overall bias
toward type 2 transitions (in both nuDNA and mtDNA
templates) with increasing levels of total sequence
heterogeneity (r¼ 0.42; P ¼ 0.7%; Figure 2D). Thispat-
tern has previously been reported for human D-loop
sequences (Gilbert et al. 2003b), although it has been
argued that in this case the pattern could be partially or
completely explained by contamination, which is espe-
cially problematic in studies of human aDNA (Pa ¨a ¨bo
et al. 2004). Our results therefore confirm the general
presence of a type 2 damage bias in mtDNA and dem-
onstrate that this observation also holds true for nuDNA.
As such, the results corroborate the previously noted
observation (Gilbert et al. 2003b) that, under equal en-
rate than type 1 transitions. This in turn indicates that
Figure 1.—Total sequence heterogeneity observed in the mtDNA and nuDNA clone sequences from the 23 specimens (see
Table 1). Solid bars, mtDNA total sequence heterogeneity. Shaded bars, nuDNA total sequence heterogeneity.
738J. Binladen et al.
deamination of cytosine and its homolog 5-methyl cyto-
sine to uracil and adenine is the dominant type of mis-
coding lesion in both mtDNA and nuDNA sequences
from fossil remains.
The observed lack of correlation between DNA dam-
0.29, P ¼ 2.3%; TS1: r ¼ 0.15, P ¼ 16%; TS2: r ¼ 0.14,
P ¼ 17%; TS2/TS1: r ¼ ?0.15, P ¼ 2.1%; supplemental
material at http:/ /www.genetics.org/supplemental/) as
numerous studies have demonstrated that preservation
conditions rather than age determine rates of DNA
degradation (Pa ¨a ¨bo 1989; Lindahl 1993; Ho ¨ss et al.
1996; Poinar et al. 1996; Kumar et al. 2000; Hofreiter
et al. 2001a; Smith et al. 2001; Gilbert et al. 2003b;
Pa ¨a ¨bo et al. 2004; Willerslev et al. 2004a; Willerslev
and Cooper 2005). More surprising is the apparent lack
of significant differences among the three groups
(permafrost, cave, and museum) for TSH rate and the
ratio of TS2/TS1 (P-values .20%; Table 4), because
these environments represent different temperature re-
gimes of storage that are known to influence damage
rates (Ho ¨ss et al. 1996; Smith et al. 2001; Willerslev
etal.2004a). However,the museum group differed from
the other two groups when comparing TSH rate/age
(permafrost, P , 1%; cave, P , 1%), while there was no
difference between the permafrost and the cave groups
(P ¼ 12%). This is likely due to long-term storage at
room temperature after excavation of most of the mam-
tantly, the museum samples (all museum samples are
from pigs ,130 years of age) show significantly higher
overall sequence heterogeneity than the environmental
samples, suggesting that the given museum storage con-
museum/herbarium material is an important source
of aDNA, it is concerning that little research has been
conducted to investigate how to maximize biomolecular
accumulates as the result of suboptimal storage condi-
tions in museums (as has been observed empirically by
many researchers), then alterations to current sample
storage practices should be investigated.
In summary, this study demonstrates that there is no
significant evidence for nuDNA sequences being more
Figure 2.—Correlation analyses. Colors indicate the preservation environment: blue, permafrost; red, cave and swamp; and
green, museum samples. 1 denotes mtDNA sequences and s denotes nuDNA sequences. (A) Type 1 transitions (TS1) as a func-
tion of TSH. (B) Type 2 transitions (TS2) as a function of TSH. (C) TS1 as a function of TS2. (D) TS2/TS1 as a function of TSH.
Results of t-test
Permafrost vs. caves
Permafrost vs. museum
Caves vs. museum
aThe total sequence heterogeneity for specimens preserved
under different conditions (permafrost, caves, or museum
storage) is compared.
Damage in Ancient Mitochondrial and Nuclear DNA Sequences 739
prone to miscoding lesions than mtDNA sequences
despite the large discrepancy in cellular copy numbers.
The data also suggest that deamination of cytosine (and
5-methyl cytosine) is the most frequent type of miscod-
ing lesion in mtDNA and nuDNA sequences from fossil
remains, although the rate at which they are affected
may differ. Additionally, a bias toward deamination of
cytosine relative to type 1 transitions with an overall in-
crease in damage appears to apply to both ancient
mtDNA andnuDNA sequences.Finally,wenote thatthe
findings reported in this study are limited by the sample
size, and as more relevant data accumulate, future
studies might be able to focus on other important ques-
tions, such as whether correlations exist between spec-
imen type and state of DNA damage.
will increasingly focus on nuclear genes from fossil and
archival material. However, as the aDNA community
embarks in this new direction, considerable care needs
to be exercised to ensure that authentic sequences are
generated. In the past two decades, many aDNA articles
have been published in which the mtDNA data have
turned out to be contaminated, pseudogenes, and/or
modified by damage (for recent reviews see Pa ¨a ¨bo et al.
2004; Willerslev and Cooper 2005). Therefore, we
find it important to highlight briefly the variety of fac-
tors that need to be considered when amplifying and
analyzing ancient nuDNA.
spectrum in both ancient nuclear and mitochondrial
DNA, further studies should attempt to generate PCR
products of the same length with an equal number of
starting template molecules. Additionally, when design-
ing PCR assays for ancient nuclear loci, restricting
amplification and maximize the number of starting
template molecules. Importantly, sequence heteroge-
interpreted as damaged might indeed represent real
in the pig data). For the moa data, however, one of the
low starting template numbers can also lead to cases of
‘‘allelic dropout’’ whereby one allelic form is amplified
preferentially over another, which calls for reproduc-
ibility of results (Taberletet al. 1996; Morin et al. 2001).
drial phylogenies (see Willerslev and Cooper 2005),
nuclear pseudogenes and gene duplications can cause
problems in the interpretation of nuDNA sequences.
Complete genomes are now available for a variety of or-
duplicated genes. However, in cases where a genetic
background is not well characterized, there is a consid-
erablerisk ofcoamplifyingafunctional duplicatedgene
or a nonfunctional pseudogene.
Finally, contamination has been a major problem in
the field of aDNA (Pa ¨a ¨bo et al. 2004; Willerslev and
Cooper 2005). When amplifying mitochondrial tem-
plates, a contaminatingsequencecan often beidentified;
for example, a D-loop sequence from contemporary hu-
mans is readily distinguishable fromthat of Neanderthals
(e.g., Serre et al. 2004b). When amplifying highly con-
served nuclear targets, contamination will not be so easy
consequently it might be difficult to distinguish between
exogenous and endogenous DNA sequences.
We advocate that as the field of aDNA moves into am-
plifying nuclear targets, factors such as damage, ampli-
con size, allelic variation, low template copy numbers,
and contamination need to be taken into account.
We are grateful to Hendrik Poinar, Johannes Krause, Tina Brand,
and Martin B. Hebsgaard for help and discussion; Andrei V. Sher and
Ross Macphee for providing the woolly rhinoceros and mammoth
samples; and Michael Hofreiter for providing unpublished sequence
data. J.B. and E.W. were supported by the Carlsberg Foundation of
Denmark, the National Science Foundation of Denmark, and the
the Carlsberg Foundation. R.B. was supported by the Biotechnology
and Biological Sciences Research Council and Natural Environment
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