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Journal of Applied Genetics
ISSN 1234-1983
J Appl Genetics
DOI 10.1007/s13353-013-0145-1
First insights into the metagenome of
Egyptian mummies using next-generation
sequencing
Rabab Khairat, Markus Ball, Chun-
Chi Hsieh Chang, Raffaella Bianucci,
Andreas G.Nerlich, Martin Trautmann,
Somaia Ismail, et al.
1 23
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HUMAN GENETICS &ORIGINAL PAPER
First insights into the metagenome of Egyptian mummies
using next-generation sequencing
Rabab Khairat &Markus Ball &Chun-Chi Hsieh Chang &
Raffaella Bianucci &Andreas G. Nerlich &
Martin Trautmann &Somaia Ismail &
Gamila M. L. Shanab &Amr M. Karim &Yehia Z. Gad &
Carsten M. Pusch
Received: 9 July 2012 /Revised: 8 March 2013 /Accepted: 11 March 2013
#Institute of Plant Genetics, Polish Academy of Sciences, Poznan 2013
Abstract We applied, for the first time, next-generation se-
quencing (NGS) technology on Egyptian mummies. Seven
NGS datasets obtained from five randomly selected Third
Intermediate to Graeco-Roman Egyptian mummies (806
BC–124AD) and two unearthed pre-contact Bolivian lowland
skeletons were generated and characterised. The datasets were
contrasted to three recently published NGS datasets obtained
from cold-climate regions, i.e. the Saqqaq, the Denisova hom-
inid and the Alpine Iceman. Analysis was done using one
million reads of each newly generated or published dataset.
Blastn and megablast results were analysed using MEGAN
software. Distinct NGS results were replicated by specific and
sensitive polymerase chain reaction (PCR) protocols in an-
cient DNA dedicated laboratories. Here, we provide unam-
biguous identification of authentic DNA in Egyptian
mummies. The NGS datasets showed variable contents of
endogenous DNA harboured in tissues. Three of five
mummies displayed a human DNA proportion comparable
to the human read count of the Saqqaq permafrost-preserved
specimen. Furthermore, a metagenomic signature unique to
mummies was displayed. By applying a “bacterial finger-
print”, discrimination among mummies and other remains
Ethical standards The experiments performed were in compliance
with the current laws of Germany.
R. Khairat :M. Ball :C.-C. H. Chang :C. M. Pusch (*)
Institute of Human Genetics, University of Tübingen,
Wilhelmstraße 27, 72074 Tübingen, Germany
e-mail: carsten.pusch@uni-tuebingen.de
R. Khairat :S. Ismail :Y. Z. Gad
Department of Medical Molecular Genetics, Division of Human
Genetics and Genome Research, National Research Centre,
El Buhouth Street, Dokki, 12311 Cairo, Egypt
R. Khairat :S. Ismail :Y. Z. Gad
Ancient DNA Laboratory, Egyptian Museum, El Tahrir Square,
11557 Cairo, Egypt
R. Bianucci
Laboratory of Physical Anthropology, Department of Public
Health and Paediatric Sciences, University of Turin, C.so Galileo
Galilei 22, 10126 Turin, Italy
R. Bianucci
Division of Paleopathology, History of Medicine and Bioethics,
Department of Oncology, Transplants and Advanced Technologies
in Medicine, University of Pisa, Via Roma 56, 56126 Pisa, Italy
A. G. Nerlich
Institute of Pathology, Division of Paleopathology,
Academic Clinic München-Bogenhausen, Englschalkinger Str. 77,
81925 Munich, Germany
M. Trautmann
Anthropologie und Osteoarchäologie, Praxis für Bioarchäologie,
Stolzeneckstr. 7, 81245 München, Germany
M. Trautmann
Institut für Forensische Sachgutachten, Dall‘Armistr. 16,
80638 München, Germany
G. M. L. Shanab :A. M. Karim
Department of Biochemistry, Faculty of Science,
Ain Shams University, Cairo, Egypt
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DOI 10.1007/s13353-013-0145-1
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from warm areas outside Egypt was possible. Due to the
absence of an adequate environment monitoring, a bacterial
bloom was identified when analysing different biopsies from
the same mummies taken after a lapse of time of 1.5 years.
Plant kingdom representation in all mummy datasets was
unique and could be partially associated with their use in
embalming materials. Finally, NGS data showed the presence
of Plasmodium falciparum and Toxoplasma gondii DNA
sequences, indicating malaria and toxoplasmosis in these
mummies. We demonstrate that endogenous ancient DNA
can be extracted from mummies and serve as a proper tem-
plate for the NGS technique, thus, opening new pathways of
investigation for future genome sequencing of ancient
Egyptian individuals.
Keywords Ancient DNA .DNA survival .
Egyptian mummies .Embalming material .MEGAN .
Metagenomics .Next-generation sequencing .
Preservation .Temperature
Introduction
Ancient Egyptians believed in the afterlife. Death marked a
transformation from the corporeal transitory life on earth
into a spiritual permanent life in the afterworld. In their
beliefs, the physical body had to be preserved and its integ-
rity was crucial to continue the existence in the hereafter
(Ikram 2003). The will to protect and preserve the integrity
of the physical body into a more perfect and eternal form is
probably the key point which gave rise to artificial mummi-
fication (David 1997;Wisseman2001;Ikram2003;
Lynnerup 2007).
During the course of the Egyptian history, the embalming
priests developed sophisticated methods of artificial mummi-
fication and, for many people, the word ‘mummy’immedi-
ately calls forth images of corpses entombed in sarcophagi.
Until recently, it was assumed that mummification started
during the Dynastic period. It was supposed that the idea of
artificial mummification was given to the Egyptians by
observing the sand naturally desiccated and perfectly pre-
served bodies dating back to the Predynastic period (5000–
3000 BC) (Ikram 2003).
However, excavations carried out at the sites of
Hieraconpolis and Adaima in the south of Egypt have
shown that, during the Naqada II Culture (3500–3150
BC), the first attempts of artificial preservation of the
bodies through the use of resins and bandages were
performed (Ikram 2003).
These discoveries forced egyptologists and scientists to
re-consider their ideas on the origin of artificial mummifi-
cation, which now appears to have developed as early as the
Gerzean Culture (Naqada II) and lasted until the Christian
Era, with several diversifications in the mummification
methods throughout time (Ikram 2003).
Since mummification usually results in excellent human
and animal soft tissue preservation (Pääbo 1985a; David
1997; Lynnerup 2007; David 2008; Corthals et al. 2012),
molecular studies on Egyptian mummies started during the
1980s of the last century (Pääbo 1985b) and were followed
by further reports with different focus and research interests
(Nerlich et al. 1997; Zink et al. 2000; Zink and Nerlich
2003; Zink et al. 2006; Nerlich et al. 2008; Nerlich and
Lösch 2009; Zweifel et al. 2009; Woide et al. 2010;
Donoghue et al. 2010; Hawass et al. 2010; Hekkala et al.
2011; Hawass et al. 2012; Kurushima et al. 2012). In the last
decade, the development of minimally invasive techniques
allowed to gain deeper biological knowledge of mummies
without causing major damage.
The challenge to reveal more genetic information from
ancient tissues without performing massive sampling has
catalysed the application of next-generation sequencing
(NGS) technologies to archaeological specimens (Pusch et
al. 2000; Wisseman 2001). The generation of large volumes
of sequence data is the primary advantage over conventional
methods (Lambert and Millar 2006; Metzker 2010; Pareek
et al. 2011). Recent scientific discoveries that resulted from
the application of NGS highlight the striking impact of these
massively parallel platforms on genetics (Mardis 2008).
These new sequencing methods are particularly suited to
ancient DNA analysis because the generated sequence frag-
ments are up to 400 bp in length, a size comparable to that
found in most degraded ancient genomes (Green et al. 2006;
Poinar et al. 2006; Rasmussen et al. 2010; Reich et al. 2010;
Keller et al. 2012).
Egyptian mummies have never been subjected to NGS
until now. Consequently, our primary aim was not to deter-
mine entire genomes here. Instead, our goal was to deter-
mine the degree of information that can be gained from
mummified tissues when they are analysed by NGS
technologies.
Therefore, five Egyptian mummies and two unearthed
pre-contact Bolivian lowlands skeletons were selected and
characterised. Subsequently, the datasets from those people
who lived in warm climates were contrasted to three recent-
ly published NGS datasets obtained from cold-climate re-
gion specimens, i.e. the Saqqaq, the Denisova hominid and
the Alpine Iceman (Rasmussen et al. 2010; Reich et al.
2010; Keller et al. 2012). The genomes of these three in-
dividuals have been established by two robust methods, the
Solexa and the SOLiD NGS technologies (Liu et al. 2012).
Here, we present the data from a first characterisation of
Egyptian mummies using these new DNA sequencing tech-
nologies and demonstrate that the DNA from mummies can
serve as a proper template for the NGS method. Through an
adequate number of runs, entire genome sequencing of
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ancient Egyptian individuals is likely to become standard in
the not too distant future.
Materials and methods
Samples
Soft tissue biopsies from five human Egyptian mummified
heads were obtained from the Institute of Pre- and
Protohistory and Medieval Archaeology, Department of
Early Prehistory and Quaternary Ecology, Eberhard Karls
University of Tübingen, Tübingen, Germany. Radiocarbon
dating performed with INTCAL04 and CALIB5 protocols
(Reimer et al. 2004) placed the mummies between 806 BC
and 124 AD, which corresponds to a timespan ranging from
the Third Intermediate Period to the Roman Period (Table 1).
Two of these mummies (DMG1 and DMG2) were sampled
twice (equally sized 0.4 cm
2
biopsies locating to the regions of
Musculus sternocleidomastoideus, M. sternohyoideus and M.
trapezius; 0.2 g of muscle biopsies were used in the analyses)
and corresponding extractions/libraries were consequently
termed 1a, 1b, and 2a, 2b. Two long bone samples (0.3 g of
powdered bone) from lowland Bolivian skeletons, one tiny
soft tissue biopsy (0.1 cm
2
) from an undated Egyptian dog
mummy and two further biopsies from human mummieswere
used for comparison (Table 1). The latter were processed as a
pooled sample during the library creation steps (and was
termed sample DMG56) in order to learn more about the
metagenomic spectrum of virus, bacterial, plant, herbal and
protozoan DNA reads harboured within ancient Egyptian
mummified tissues.
In order to differentiate by climate between all samples
used in this study, we applied the term “warm-climate sam-
ples”to the Egyptian mummies and the Bolivian skeletons.
The three remaining datasets taken from the literature were
defined as “cold-climate samples”and shall contrast the
Alpine Iceman, the Saqqaq Palaeo-Eskimo and the
Denisova hominid from the aforementioned individuals.
Assessing the effects of further parameters, e.g. humidity,
pH, salt concentration etc., is not a part of the present study
and will be the topic of future research.
DNA extraction and contamination monitoring
We adopted the previously published criteria for ancient
DNA authentication (Richards et al. 1995;Robertsand
Ingham 2008; Hawass et al. 2010;Kelleretal.2012;
Hawass et al. 2012). DNA extraction work was conducted
in a dedicated facility, physically isolated from the polymer-
ase chain reaction (PCR) technology, the library preparation
steps and the areas for the post-PCR work. Work surfaces
were frequently cleaned with DNase and irradiated with UV
Table 1 Characterisation of samples under investigation, comprising nine samples from warm environments and three from cold environments. Abbreviations used: AD Anno Domini, BC Before
Christ, BP before present, ND not determined, < cold-climate sample, > warm-climate sample
Specimen Habitus Tissue type Excavation location Dating Dynasty/period Average
temperature 15 °C
Reference
DMG1 human Mummy Mummified tissue (muscle) Tomb, Egypt 806–784 BC Third Intermediate Period > This study
DMG2 human Mummy Mummified tissue (muscle) Tomb, Egypt 382–234 BC Hellenistic Period/Ptolemaic Period > This study
DMG3 human Mummy Mummified tissue (muscle) Tomb, Egypt 54–124 AD Roman Period > This study
DMG4 human Mummy Mummified tissue (muscle) Tomb, Egypt 358–204 BC Late Period/Hellenistic-Ptolemaic Period > This study
DMG5 human Mummy Mummified tissue (muscle) Tomb, Egypt 402–385 BC Late Period > This study
DMG20 dog Mummy Mummified tissue (muscle) Tomb, Egypt ND Third Intermediate to Roman Period > This study
DMG56 human Mummy Mummified tissue (muscle) Tomb, Egypt ND Third Intermediate to Roman Period > This study
DMGS1000 human Skeleton Bone Mineral soil burial,
South America
400–1300 AD –> This study
DMGS2000 human Skeleton Bone Mineral soil burial,
South America
ca. 717 AD –> This study
Saqqaq human Hair Hair Cryotic soil,
Western Greenland
4044± 31 BP –<< Rasmussen
et al. (2010)
Denisova SL3003 human Digit Bone Cave soil, Russia 50000–30000 BP Earlier Middle Palaeolithic << Reich et al. (2010)
Iceman human Mummy Bone Glaciers, Italy ca. 5300 BP Old Copper age << Keller et al. (2012)
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light; disposable plastic items were used whenever possible,
non-disposal items were baked at 200 °C, washed with
DNase and irradiated with UV light. If solutions were
bought in pre-made, these were finally prepared on a clean
bench, autoclaved and sterilised by filtration (0.25-μm sy-
ringe filter; Nalgene, Thermo Fisher Scientific, Waltham,
MA, USA). All work was conducted while wearing appro-
priate protective garments. Contamination was monitored
by the use of negative and blank extraction controls, which
were processed along with each sample. Mitochondrial
DNA typing of all laboratory working members was
performed.
Sampling of tissue, DNA extraction and DNA purifica-
tion were performed according to a protocol published pre-
viously (Scholz and Pusch 1997). A slightly modified
version of the aforementioned extraction protocol was also
applied. Here, the DNA was extracted from the DNA-
containing phase according to the protocol of Scholz and
Pusch (1997), but instead of subsequent manual purification
steps, we applied the MagNA Pure Compact System (Roche
Applied Science, Penzberg, Germany) for automated purifi-
cation. The DNA samples were also examined by spiking
reactions to test the effect of inhibition due to the presence
of co-extracted substances (Pusch and Bachmann 2004).
PCR analysis, cloning and Sanger sequencing
Mitochondrial PCR amplification was accomplished
using specific primers designed from the D-loop control
region of the Canis lupus familiaris mitochondrial ge-
nome. The primers used were forward 5-TGCATACAAT
ACTCACAAGCTTTATTT-3 and reverse 5-GACTAC
GAGACCAAATGCGTGT-3, and amplified a DNA seg-
ment between positions 16,572 and 16,672.
Specific primers were designed to detect the gene
sequence of the mitochondrial NADH dehydrogenase
subunit 1 (nad1) (positions 496–603) of the Pinaceae
species. The primer sequences were as follows: forward
5-ATGTCGGTCGACGATGCCGC-3 and reverse 5-
AGGTGCCCAGCGATTCCTTCA-3.
All PCR fragments were amplified in a volume of 25 μl
containing 1× FastStart PCR Master Mix (Roche Applied
Science, Penzberg, Germany), 20 pmol of each primer,
10 mM of each dNTP and aliquots of the extracted DNA.
The cycling conditions using a GeneAmp® PCR System
9700 Thermal Cycler (Applied Biosystems, Foster City,
CA, USA) were 94 °C for 5 min, followed by 45 cycles of
94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, followed
by a final extension of 10 min at 72 °C.
Alternatively, the same protocol for thermal cycling but
with an annealing temperature of 62 °C was applied to
amplify the DNA of the animal mummy, an annealing
temperature of 58 °C was applied to amplify the Pinaceae
species and a temperature of 57 °C was used to amplify
cloned DNA fragments by colony PCR.
The cloning of PCR products was performed with the
CloneJET™PCR Cloning Kit (Fermentas, Thermo Fisher
Scientific, Waltham, MA, USA), according to the manufac-
turer’s protocol. The colony PCR products were cleaned by
ExoSAP-IT (USB Corporation, Cleveland, OH, USA) and
were used for the Sanger cycle-sequencing reactions with
BigDye Terminator v3.1 chemistry (Applied Biosystems,
Foster City, CA, USA). Samples were run on a 3130
Genetic Analyzer (Applied Biosystems, Foster City, CA,
USA).
Library preparation
Both methods, the Solexa (Rasmussen et al. 2010; Reich et al.
2010) and the SOLiD technologies (Keller et al. 2012), are
considered to be robust in their application to ancient DNA.
The Roche 454 protocol was not employed by us, since it is
better suited for nucleic acids with larger fragmentation sizes.
Whole genome libraries were generated for the SOLiD 3
Plus System (Applied Biosystems, Foster City, CA, USA).
Genomic DNA was end-repaired using 1 μl of the end-
polishing enzyme 1 (10 U/μl) and 2 μl of the end-
polishing enzyme 2 (10 U/μl), 4 μl dNTP mix (10 mM),
as well as 20 μl 5× end-polishing buffer, in a total volume of
100 μl. Following incubation at room temperature for
30 min, DNA was purified using the SOLiD™Library
Column Purification Kit (Applied Biosystems, Foster City,
CA, USA). SOLiD™adaptors P1 (5-CCACTACGCCTCC
GCTTTCCTCTCTATGGGCAGTCGGTGAT-3) and P2 (5-
AGAGAATGAGGAACCCGGGGCAGTT-3), each at 2.
5μM, were ligated to purified DNA with 40 μlof5×T4
ligase buffer and 10 μl T4 ligase (5 U/μl), in a total volume
of 200 μl at room temperature for 15 min. After another
purification step, the ligated DNA was eluted in 40 μl
nuclease-free water. No additional size selection was carried
out in order to avoid loss of material. DNA was then incu-
bated with 380 μl Platinum HiFi PCR Amplification Mix
(Life Technologies, Carlsbad, CA, USA) and 10 μl each of
both library PCR primers (primer 1: 5-CCACTACGC
CTCCGCTTTCCTCTCTATG-3 and primer 2: 5-CTGCCC
CGGGTTCCTCATTCT-3) in order to repair the gap in the
double-stranded DNA molecules introduced during adaptor
ligation. The following conditions for thermal cycling were
applied: hold at 72 °C for 20 min plus another hold at 95 °C
for 5 min, followed by two cycles at 95 °C for 15 s, 62 °C
for 15 s, 70 °C for 1 min, one cycle at 70 °C for 5 min and a
final hold step at 4 °C. The PCR product was purified using
the PureLink™PCR Purification Kit (Invitrogen, Carlsbad,
CA, USA). Eluted DNA was again cycled using 100 μlof
2× Phusion HF Master Mix (Finnzymes, Thermo Fisher
Scientific, Waltham, MA, USA) and 8 μl each of both
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library PCR primers 1 and 2 in a total volume of 200 μl.
This mixture was divided into four PCR tubes and cycled
using the following conditions: 12 cycles at 95 °C for 15 s,
62 °C for 15 s, 70 °C for 1 min, followed by one hold step at
70 °C for 5 min. All libraries were purified as described
above and stored at −20 °C until sequencing using the
SOLiD™3 Plus System (Applied Biosystems, Foster City,
CA, USA).
Whole genome libraries were also applied to the Genome
Analyzer IIx (GAIIx) (Illumina, San Diego, CA, USA). The
recommended protocol of the New England Biolabs
NEBNext™DNAsampleprepkitwasused(New
England Biolabs GmbH, Ipswich, MA, USA). About
50 ng/μl of total DNA was end-repaired using 1 μlof
DNA Poly I (Klenow LF) and 5 μl T4 DNA polymerase,
4μl dNTP mix (10 mM) and 10 μl of 10× end-polishing
buffer, in a total volume of 100 μl. Following incubation at
20 °C for 30 min, the DNA was purified using Agencourt
AMPure XP (Beckman Coulter Genomics, Brea, CA, USA),
A-tailed and ligated to Solexa adaptors with the sequences
5-GATCGGAAGAGCTCGTATGCCGTCTTCTGCTTG-3
and 5-ACACTCTTTCCCTACACGACGCTCTT
CCGATCT-3. In a total volume of 50 μl, these adaptors
were ligated to purified DNA using 25 μl of 2× T4 ligase
buffer and 5 μl Quick T4 ligase by incubation at room
temperature for 15 min. After another round of purification
using Agencourt AMPure XP (Beckman Coulter Genomics,
Brea, CA, USA), the ligated DNA was eluted in 10 μl
nuclease-free water. Eluted DNA was cycled using 25 μl
of 2× Phusion HF Master Mix (Finnzymes, Thermo Fisher
Scientific, Waltham, MA, USA) and 1 μl each of both
library PCR primers 1 and 2 in a total volume of 50 μl.
This mixture was cycled using the following protocol: 12–
18 cycles at 95 °C for 30 s, 65 °C for 1 min, 70 °C for 30 s,
followed by 5 min at 70 °C and a final hold at 4 °C. PCR
products were again purified using Agencourt AMPure XP
(Beckman Coulter Genomics, Brea, CA, USA) and applied
to the NGS sequencer model Genome Analyzer IIx.
Furthermore, for a detailed metagenomic analysis of two
mummy DNAs which had been thoroughly pre-characterised
by standard PCRs, they were pooled and additionally used for
library preparation according to the recommended Illumina
protocol and subjected to sequencing using the Solexa plat-
form (Illumina, San Diego, CA, USA) (Table 1).
NGS sequencing
Starting with the 2-ng/μl dilution of the fragment library
preparation, a 60-pg/μl dilution was applied to the emulsion
PCR to perform single-molecule amplification of the SOLiD 3
libraries using the EZBead™system (Applied Biosystems,
Foster City, CA, USA). After a 3′-end modification of the
DNA bound to magnetic beads, the templated beads were
deposited onto chemically modified slides and loaded into
the flow cells of a SOLiD™3 Plus system (Applied
Biosystems, Foster City, CA, USA). Fifty base pairs of
DNA fragments were sequenced using the SOLiD™TOP
Sequencing Kit (Applied Biosystems, Foster City, CA, USA).
According to the Illumina protocol, Solexa libraries were
diluted to a final concentration of 6 pM to be ready for
cluster amplification using the TruSeq PE Cluster Kit v.2.
Subsequently, the Solexa samples were sequenced
according to the manufacturer’s recommendations using
the TruSeq SBS Kit v.5 GA on a Genome Analyzer model
GAIIx (Illumina, San Diego, CA, USA). The read length
using this NGS system was 75 bp/100 bp paired end.
NGS data and metagenomic analyses
NGS reads obtained from the sequencing were mapped
against the human genome assembly hg19 with Bowtie
software (Langmead et al. 2009) or the Burrows–Wheeler
Aligner (BWA) (Li and Durbin 2009) on the Galaxy server.
Visualisation of the mitochondrial output file was accom-
plished with the output file generated by the BWA software.
The BAM files obtained after the mapping steps were
processed and handled using the Integrative Genomics
Viewer (IGV).
Intraspecies comparison was done using one million
reads from each of our datasets and from the three previ-
ously published datasets specific for cold environments
(Saqqaq: Rasmussen et al. 2010; Denisova: Reich et al.
2010; Alpine Iceman: Keller et al. 2012).
Blast alignments were accomplished using either the
Blastn or megablast algorithms against the NCBI nucleotide
collection with seed length 33. Mapping and Blast analysis
was done on the Galaxy main server (http://main.g2.bx.psu.edu/
root), the Tübingen Galaxy server (https://galaxy.informatik.uni-
tuebingen.de/galaxy-local/), the Vienna server at
canis.csb.univie.ac.at or felis.csb.univie.ac.at, and the public
GALAXY@WUR server at http://galaxy.wur.nl:8080/galaxy/
root. Blast results were analysed using MEGAN software
(Huson et al. 2007).
Results
Two closely related protocols for DNA extraction were
applied to seven tissue biopsies from five randomly selected
Egyptian human mummies, an Egyptian dog mummy sam-
ple and two bone biopsies from pre-contact South-American
skeletons (Table 1).
DNA fragments ranging from 75 bp to 300 bp (Fig. 1a)
were obtained when DNA extraction was performed follow-
ing the protocol of Scholz and Pusch (1997). Conversely,
DNA fragments up to ca. 400 bp (Fig. 1b) were yielded when
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the protocol of Scholz and Pusch (1997) was used in conjunc-
tion with the MagNA Pure Compact System (Roche Applied
Science, Penzberg, Germany) for purification. Spiking exper-
iments indicated that the extracted DNA showed negligible or
no inhibition (data not shown).
In order to test for the presence of authentic DNA in ancient
Egyptian mummified tissues, DNA was also extracted from a
small biopsy of an Egyptian dog mummy using the protocol
of Scholz and Pusch (1997). Following the extraction, DNA
was purified and different dilutions were used for the PCR
reaction (Fig. 2a). Amplification of a 100-bp segment of the
mitochondrial D-loop control region yielded a specific PCR
product. Sanger sequencing (Fig. 2b) and Blast results
(Fig. 2c) confirmed the Canis lupus origin. Additional PCRs
carried out in order to test for the presence of human DNA in
the animal sample were negative and, thus, excluded contam-
inant human DNA in the sample.
Since it was expected that the metagenomes of Egyptian
mummies also contain plant DNA originating from the
embalming “recipes”, a further line of experiments was
conducted for the authentication of results.
Specific 107-bp PCR products detecting Pinaceae species
were repeatedly obtained from the extracted DNA belonging to
all the Egyptian human mummies. The PCR products were
sequenced using an ABI Applied Biosystems Sequencer
(Fig. 3a) and sequence integrity was confirmed using Blast
algorithms in the NCBI nucleotide collection databases (Fig. 3b).
NGS libraries were generated for the SOLiD and Solexa
platforms (Fig. 4a). The fragment size range of our libraries
ranged from 200 bp up to 700 bp including adaptors
(Fig. 4a), which coincides with the size range of our extracted
DNA (Fig. 1).
Following the purification steps, the libraries were cloned
into Fermentas CloneJET™PCR vectors (Fig. 4b) and the
obtained clones were sequenced with the Sanger method.
The analysis of the sequences showed the proper composi-
tion of NGS amplicons, with the presence of the two library
adaptors and a short cloned fragment in between the two.
The size range of the small-scale plasmid library was mainly
between 200 bp and 400 bp (Fig. 4b).
Metagenomics
Metagenomic analyses were performed using the MEGAN
software by browsing the Blast hits of the mummy datasets.
Initial screening of metagenomic contents within the pooled
human mummy sample DMG56 revealed infection with at
least two protozoan species. The pathogens Plasmodium spec.
(49 Plasmodium reads, with two hits highly specific for the
species Plasmodium falciparum, i.e. 2.2 %) and Toxo plas m a
gondii (1,270 reads in total) were detected according to the
analysis of 445,557 Blast hits in this Egyptian mummy library
using MEGAN software (Fig. 5a). In other words, within the
taxon of Apicomplexa, 5.4 % of the reads suggested the
presence of plasmodia, while 94.2 % of these reads diagnosed
the parasitosis toxoplasmosis in sample DMG56.
A first analysis of the plant/herbals content in our
mummies indicated that Ricinus communis was one of the
main components found in mummies 1 and 4. Populus spec.
sequences were most frequent in mummy 2, while Pinus
spec. is highlighted in the pooled mummy sample DMG56.
Linum,Olea,Prunus,Abies,Allium and Lotus sequences
were identified to a lesser extent in a number of mummies.
Although mummy 2 (382–234 BC) and mummy 4 (358–
204 BC) originate from similar times, we note differences in
their resin composition with regard to the Populus spec.
sequences. While mummy 2 showed a total of 1,074
Populus DNA reads, there is not a single read count in
mummy 4 for the taxon Populus. None of these plant
DNAs could be identified in the metagenomes of the three
cold-climate specimens.
Comparison between the Blast results of one million
reads from three previously published datasets from cold-
climate specimens (i.e. the Saqqaq, the Denisova hominid
and the Ötztal Iceman) (Rasmussen et al. 2010; Reich et al.
2010; Keller et al. 2012) and our seven newly generated
datasets representing warm-climate samples (i.e. the five
Egyptian mummies and the two lowland Bolivian skeletons)
was performed (Tables 1and 2).
Eukaryotes and bacteria were the two main kingdoms
present in all datasets (Fig. 6). A small portion of DNA
was represented by archea and virus sequences (Table 2).
Fig. 1 a DNA extracted from Egyptian mummies (lanes 2–8) using
the extraction protocol of Scholz and Pusch (1997). Lane 1 is a blank
extraction. bDNA extracted from mummies (lanes 2–8) using the
protocol of Scholz and Pusch (1997) in conjunction with the MagNA
Pure Compact System. Lane 1 is a blank extraction. M is the
GeneRuler™1 kb DNA Ladder
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The viral content in each sample was below 1 %, except for
the Denisova sample, which showed a strongly increased
virus proportion of 9.57 % (Fig. 6).
The archea were even less represented in all samples from
both cold and warm climates (0–0.051 %), except for the
South-American skeletons, which showed an approximately
10-fold increased proportion, both about 0.4 % (Fig. 6).
The eukaryotic reads were abundant in the cold-climate
samples (Saqqaq: 99.7 %, Iceman: 98.7 % and Denisova: 89.
6 %), whereas they strongly varied in our mummy datasets,
ranging between 6.9 % and 96 % (Table 2,Fig.6). According
to the percentage of eukaryotic reads, values were widely
scattered from higher percentages in Egyptian mummy sam-
ples 1a, 2a and 3 (80–96 %) to very low percentages (6.86 %
in mummy sample 4).
More differences could be pinpointed when analysing
different biopsies taken from the same mummy; this concept
is exemplified at its best in sample 1a and 1b taken from
mummy 1 and in samples 2a and 2b taken from mummy 2
(Table 2, Fig. 6).
It is highly likely that the discrepancy seen in the bacte-
rial content between the first (sample 1a from mummy 1 and
2a from mummy 2) and second samplings (sample 1b from
mummy 1 and 2b from mummy 2) is consistent with a
bacterial bloom. This was mainly due to the absence of an
adequate environment monitoring in the new repository of
the Institute of Pre- and Protohistory where the heads have
been recently relocated.
The ratio of assigned reads to the total number of
reads spans a wide range within the datasets of
Egyptian mummies. It is moderately high in, for exam-
ple, mummy samples 2b, 4 and 5 (10.8 %, 12.5 % and
13 %, respectively), but resembles the ratios observed in
the Denisova (26.3 %) and the Iceman (10.8 %) cold-
climate samples (Table 2).
Furthermore, a comparison of cold- and warm-climate
samples’“bacterial fingerprint”was performed. This was
in order to allow comparisons of the bacterial phyla com-
position in warm- and cold-climate specimens independent-
ly from the absolute number of bacterial reads (Fig. 7).
Fig. 2 a Mitochondrial polymerase chain reaction (PCR) products
from an Egyptian dog mummy (lanes 1–3). Lane 4 is the PCR-negative
control. M is the GeneRuler™1kbDNALadder.bSequence
chromatogram of a mitochondrial PCR product amplified from DNA
of a dog mummy. cBlast result of the Canis lupus mitochondrial DNA
sequence
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The relative proportion of the Actinobacteria is the main
difference highlighted in the “bacterial fingerprint”. On this
basis, two major groups of samples could be defined. One
group is represented by the Egyptian mummies and the
glacier mummy Ötzi the Iceman, which display a relative
amount of ≤10 % Actinobacteria in the group of bacteria.
The second group is the so-called “non-mummy group”,
which includes the two South-American skeletons, the
Denisova hominid and the Saqqaq Paleo-Eskimo (Fig. 7).
Here, the relative amount of Actinobacteria is largely in-
creased (≥28 %). Moreover, within the mummy group, an
increased percentage of Firmicutes can also be noted.
Conversely, Proteobacteria show a very divergent range
through all the samples; they span from moderately low in
the Iceman mummy (5.94 %) to high in the Egyptian mum-
my sample 4 (78.44 %). This implies that Proteobacteria
cannot be used as a fingerprint for the unequivocal identifi-
cation of mummies.
Despite a general variability, the Egyptian mummies and
the Iceman show a more homogenous metagenomic picture
and can be easily contrasted both to the warm-climate low-
land South-American samples and to the cold-climate
Saqqaq and Denisova specimens.
Actinobacteria are very common in soil where they decom-
pose organic materials. Firmicutes often build endospores and
can survive in extreme conditions for a long time. Our data
show that no correlation between the environmental tempera-
ture and the “bacterial fingerprint”of a given specimen exists.
Comparisons among the three previously published
datasets of the Iceman, Denisova hominid and the Saqqaq
Fig. 4 a Five exemplary Solexa libraries generated from the ancient
DNA of Egyptian mummies are shown (lanes 1–5), thereby, highlight-
ing the different degrees of efficiency according to the purity and the
concentration of DNA in the starting material. bCloning of a Solexa
library using the CloneJET™PCR Cloning Kit. Lanes 1–8 show the
amplified inserts using vector primers in the PCR. M is the GeneRuler
1 kb DNA Ladder
Fig. 3 a Sequence chromatogram of the Pinus densiflora mitochondrial PCR product. bBlast result using the Pinus densiflora mitochondrial DNA
sequence as the query
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(Rasmussen et al. 2010; Reich et al. 2010; Keller et al. 2012)
and the new datasets from the Egyptian mummies were
performed using the Blast results of a subset of one million
reads from each dataset. This was in order to obtain the
percentage of reads assigned to human DNA in comparison
to the total number of assigned reads.
Twelve datasets were placed in descending order of their
percentage of human Blast hits (Fig. 8). The human content
of the “top group”defined in Fig. 8ranged from 50 % to
80 % in six specimens. Of these six specimens, three are
cold-climate samples and three are Egyptian embalmed
mummies (Fig. 8).
Fig. 5 a Detail of the
phylogenetic MEGAN output
showing the Plasmodium
falciparum and Toxoplasma
gondii hits in the mummy
dataset DMG56. bDetail of the
MEGAN output showing Picea
and Pinus clusters in our
sequenced mummies
Table 2 Typing of Blast hits
after analysing one million reads
of nine new and three
previously published datasets
of the Saqqaq, the Denisova
hominid and the Alpine Iceman
(Rasmussen et al. 2010; Reich et
al. 2010; Keller et al. 2012)
Sample Eukaryota Bacteria Archaea Viruses Ass./total Reference
Mummy 1a 96.03 % 3.60 % 0.00 % 0.16 % 2.55 % This study
Mummy 1b 9.84 % 89.81 % 0.00 % 0.06 % 11.80 % This study
Mummy 2a 88.27 % 10.61 % 0.05 % 0.41 % 0.19 % This study
Mummy 2b 15.16 % 84.39 % 0.00 % 0.05 % 10.80 % This study
Mummy 3 83.16 % 16.20 % 0.00 % 0.29 % 0.33 % This study
Mummy 4 6.86 % 92.70 % 0.01 % 0.05 % 12.50 % This study
Mummy 5 19.19 % 72.45 % 0.03 % 0.08 % 13.00 % This study
S1000 38.13 % 60.00 % 0.40 % 0.10 % 1.76 % This study
S2000 5.20 % 93.24 % 0.41 % 0.03 % 2.30 % This study
Saqqaq 99.70 % 0.07 % 0.00 % 0.00 % 74.72 % Rasmussen et al. (2010)
Denisova SL3003 89.60 % 0.69 % 0.02 % 9.57 % 26.30 % Reich et al. (2010)
Iceman 98.79 % 0.79 % 0.00 % 0.00 % 10.84 % Keller et al. (2012)
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The Ötzi glacier mummy resides in position number one
on this ranking (81 % human content), followed by the
Denisova hominid (70 %) and the Saqqaq (68 %).
Egyptian mummy samples 1a, 2a and 3 showed a similarly
Fig. 6 Comparison of 12
datasets according to their
eukaryotic and bacterial
content. The Saqqaq, Denisova
and the Ötztal Iceman datasets
have been published previously
(Rasmussen et al. 2010; Reich
et al. 2010; Keller et al. 2012)
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high human content ranging from 54 % to 64 %, thus,
resembling the value obtained for the Saqqaq permafrost-
preserved hair. Less abundant human DNA is observed in
the remaining six samples represented by the South-
American skeletons and the remaining four Egyptian mum-
my biopsies.
One Egyptian mummy showed an adequate number of
mitochondrial sequences that specifically map to the human
hg19 genome. The total number of paired-mapped reads to
the hg19 reference is around 150,000 reads, with more than
1,900 reads mapping to the mitochondrial DNA reference
sequence (NC_012920). In other words, this sample has a
proportion of about 1.3 % mitochondrial DNA sequences in
comparison to the total human read count. A 196-bp detail
of the hypervariable region 2 (HVR2), which is covered
about 20-fold, is shown in Fig. 9. The average coverage of
the entire HVR2 is about 12.5-fold and the average coverage
of the entire mitochondrial genome is 11.6.
The coverage of HVR2 helps to detect a number of
diagnostic mutations, which are considered to be authentic,
since all of these single-nucleotide polymorphisms (SNPs)
are absent in the mitochondrial DNA sequences of our
laboratory members. Diagnostic base deviations with a good
segment coverage point to the mitochondrial haplogroup I2,
but further analysis is required in order to consolidate this
tentative result.
Discussion
Mummification reached its apex in the 21st Dynasty (1064–
940 BC), when the embalmers started manipulating the flesh
and “turned the prepared body into a more perfect image of
itself”(Ikram 2003). The embalming priests used sophisticat-
ed “recipes”to prepare the bodies for the afterworld (Buckley
and Evershed 2001; Wisseman 2001; Buckley et al. 2004).
Fig. 7 Characterisation of
bacteria taxa identified in the 12
NGS datasets. The Saqqaq, the
Denisova hominid and the
Alpine Iceman datasets have
been published previously
(Rasmussen et al. 2010; Reich
et al. 2010; Keller et al. 2012)
Fig. 8 Comparison of the 12
metagenomic datasets, ordered
according to their human
percentages. The Saqqaq, the
Denisova hominid and the
Alpine Iceman datasets have
been published previously
(Rasmussen et al. 2010; Reich
et al. 2010; Keller et al. 2012)
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However, apart from rare exceptions in which some steps
of the mummification procedure are reproduced (i.e. the coffin
of Djedbastiuefankh, Pelizaeus Museum, Hildesheim—Late
Period; the Rhind Magical Papyrus—ca. 200 BC; three papyri
in the Cairo, Durham Oriental and Louvre Museums—around
1st century AD), “the Egyptians are curiously silent about the
modes of mummification in both their written and figurative
sources”(Ikram 2003).
The lack of direct evidence was filled with information
derived from numerous written sources. The Ionian Greek
writer Herodotus (5th century AD) provided the earliest
written accounts on mummification (Book II of “The
History”). Coupled and augmented with the records of
Diodorus Siculus (1st century BC) and further completed
by the writings of Porphyry (3rd century AD), these sources
have long provided the basis of current knowledge on mum-
mification techniques (Wisseman 2001; Ikram 2003).
The methods of mummification, defined as the deliberate
act of preservation of the body after death, varied and diver-
sified during the different periods of the Egyptian history
(Wisseman 2001;Ikram2003;David2008; Jeziorska 2008).
The increasingly sophisticated biogeochemical and mo-
lecular techniques, as well as experimental mummification,
allowed scientists to gain further information about how
mummification was practised (Pääbo 1985a; Brier and
Wad e 1995; Brier and Wade 1997; Zimmerman et al.
1998; Barraco et al. 1977; Brier and Wade 1997;
Wisseman 2001; Buckley and Evershed 2001; Aufderheide
2003; Kaup et al. 2003; Ikram 2003; Ikram 2005; Buckley et
al. 2004; Metcalfe and Freemont 2012).
Due to mummies’excellent tissue preservation, studies
aiming to target both DNA and proteins harboured in mum-
mified tissues were successfully carried out (Pääbo 1985b;
Nerlich et al. 1997; Zink et al. 2000; Zink et al. 2001; Zink
and Nerlich 2003; Zink et al. 2006; Kaup et al. 2003; Koller
et al. 2003; Bianucci et al. 2008; Nerlich et al. 2008; Woide
et al. 2010;Hawassetal.2010; Donoghue et al. 2010;
Hekkala et al. 2011; Corthals et al. 2012; Hawass et al.
2012; Kurushima et al. 2012).
The general feasibility of PCR-based DNA studies using
different ancient samples originating from warm climates was
evidenced (Lassen et al. 1994;Poinaretal.1996;Fox1997;
Pusch et al. 2003;Poinaretal.2003; Zink and Nerlich 2003;
Zink and Nerlich 2005; Gilbert et al. 2008;Camposetal.2012;
Kurushima et al. 2012). Similarly, ancient human DNA from
Egyptian mummies was retrieved with conventional molecular
genetic methodology (e.g. Pääbo 1985b; Krings et al. 1999;
Rutherford 2008;Hawassetal.2010; Hawass et al. 2012).
We have tested, for the first time, the feasibility and fidelity
of the NGS methods when applied to mummified Egyptian
tissues. Since there is a common fear for contaminant modern
human DNA in ancient specimens, a thorough contamination
monitoring and authentication procedure was applied.
All chemicals and consumables were controlled as
recommended by the ancient DNA guidelines (Richards et
al. 1995; Roberts and Ingham 2008; Hawass et al. 2010;
Keller et al. 2012; Hawass et al. 2012). Extraction blanks
and negative controls were used along with the samples for
PCRs and library preparation, which showed negative
results.
Fig. 9 Details of a 196-bp segment of the hypervariable region 2
(HVR2) of an exemplary Egyptian mummy is given. Reads were
mapped against the hg19 human reference sequence (accession number
NC_012920) and a multi-sequence alignment was established using
the Burrows–Wheeler Aligner (BWA) software. Deviating base posi-
tions from the mitochondrial reference sequence are highlighted by a
four-colour label
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In order to monitor for authentic DNA sequences in
Egyptian mummified tissues, a muscle biopsy from an
embalmed Egyptian dog was also tested by PCR. The re-
sults revealed the presence of authentic Canis lupus ancient
DNA and the absence of human contaminant DNA.
Furthermore, we determined the presence of herbal DNA
from plants, some of which might have been components of
the embalming “recipes”. The DNA results for Pinus,Picea
and Ricinus were confirmed by standard PCRs with subse-
quent Sanger sequencing and served as a further proof of
authenticity (Pinus confirmation is shown in Fig. 3).
The positive results for authentic dog and herbal/plant
DNAs are much less susceptible to contamination than
human DNA and encouraged us to proceed with the library
creation employing ancient Egyptian tissue of human
mummies dating from 806 BC to 124 AD.
A first survey of the NGS-generated metagenomes of the
randomly selected Egyptian mummies indicated further
proof of authenticity by observing the signatures of two
protozoan pathogens, namely, Plasmodium falciparum and
Toxoplasma gondii (Fig. 5a).
The pathogen representation was reproducible among
different runs originating from the same mummy and is
specific to the warm-climate specimens. Furthermore, the
presence of Plasmodium pathogens was confirmed by the
highly specific PCR technology coupled with subsequent
Sanger sequencing.
Four blood-parasite species belonging to the genus
Plasmodium (P. falciparum,P. vivax,P. malariae,P. ovale)
are responsible for different forms of human malaria.
Parasites are transmitted to humans through the bite of
female Anopheles mosquitoes. Plasmodium falciparum
causes the most dangerous and severe form of malaria,
termed malaria tropica. Previous reports have shown the
presence of falciparum malaria in mummies from ancient
Egypt (Nerlich et al. 2008; Bianucci et al. 2008; Hawass et
al. 2010).
Toxoplasma gondii is the other protozoan species that we
identified. The definitive host of Toxoplasma gondii is the
cat. Cats were cult animals in ancient Egypt and an abun-
dance of cats was mummified during the Late and Graeco-
Roman periods (Ikram 2005).
Recent genetic evidence supports the notion that ancient
Egyptians used domesticated cats, Felis silvestris catus, for
votive mummies and imply that taming of the cats occurred
prior to or during Predynastic and Early Dynastic Periods
(Kurushima et al. 2012). Hence, frequent contacts among
domesticated cats and humans can explain the presence of
this specific pathogen in Late and Graeco-Roman human
mummies.
In addition to the human, bacteria, viruses and fungi
DNAs, a fair amount of plant DNA in the metagenomes of
our Egyptian mummies was identified.
Herodotus’accounts mention myrrh, cassia, palm wine,
“cedar oil”and “gum”as the main plant components of the
embalming “recipes”. Previous research (Buckley and
Evershed 2001) showed that, even if salt natron was widely
used as a desiccant, due to the warm environmental condi-
tions inside the tombs, the bodies would have decomposed
without the application of specific organic substances.
Chemical analyses carried out on 13 Egyptian mummies
dating from the mid-Dynastic period (ca. 1900 years BC) to
the late Roman Period (AD 395) suggested that unsaturated
plant oils and animal fats were the key components in
mummification. Subsequently, more exotic substances were
mixed up to this base and applied either on the bodies or on
the bandages (Buckley and Evershed 2001). The peculiar
properties of the unsaturated oils and fats allowed them to
polymerise spontaneously. The polymerisation would have,
in turn, produced a “highly cross-linked aliphatic network,
which would have stabilized otherwise fragile tissues and/or
wrappings against degradation by producing a physico-
chemical barrier that impedes the activities of microorgan-
isms”(Buckley and Evershed 2001).
Buckley and Evershed (2001) showed that beeswax and
coniferous resins were used in the embalming procedures
and that their use increased in importance over time, being
more enhanced in later periods. Those components were
found both on the bodies and on the wrappings.
Components diagnostic for Pistacia resin were also found
in a Ptolemaic female mummy.
In a subsequent study, some components diagnostic for
Pistacia exudates were also chemically characterised in
three further but undated mummies (Nicholson et al. 2011).
Some of the distinctive plant taxa seen in the mummy
MEGAN output coincide with certain ingredients reported
to have been used in the “embalming”recipes (Serpico and
White 2000).
Castor (Ricinus communis), linseed (Linum usitatissimum),
olive (Olea europaea L.) and almond (Prunus dulcis), whose
oils could be used in the moisturising of the body in the
embalming process (Serpico and White 2000), were identified
in trace amounts in our mummies.
Fir (Abies cilicica) and pine (Pinus spp.) (Fig. 5b), which
are considered to be the main components of the embalming
resins (Serpico and White 2000), were well represented in our
mummy datasets. While pine was identified in five mummies,
fir was present only in one mummy. Finally, other ingredients,
which might have been used to influence the smell of a
mummified body, like populus (Populus euphratica), garlic
(Allium sativum) and lotus (Nymphaea lotus), were also iden-
tified. Additional comparisons with forthcoming herbal and
plant DNA datasets obtained from the NGS analysis of other
mummified tissues from Ancient Egypt are pending. This will
show whether the data are due to extraneous DNA from
scattered pollen or if it is endogenous and authentic to the
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mummy as a part of the applied embalming substances. If the
latter holds true, then we will undoubtedly learn more about
the mysterious mummification process from ancient Egyptian
times.
For further metagenomic analyses, two warm-climate
specimens were processed along with the Egyptian
mummies. The skeletons S1000 and S2000 were unearthed
from the Llanos de Moxos eastern plain (Bolivian
Amazonia), a vast seasonally savannah region. The climate
is characterised by two marked seasons: the rainy season
from October to April, when precipitation can reach
500 mm per month, and the dry season, with monthly pre-
cipitations of less than 50 mm. Temperatures are high
throughout the year (Lombardo and Prümers 2010).
For comparison purposes, three previously published
datasets of cold environment samples (i.e. the Denisova
hominid, the Saqqaq and the Ötztal Iceman) were included.
The Blast results of all NGS datasets showed distinct
differences among the metagenomes of the cold-climate
samplescomparedtotheEgyptianmummiesandtothe
two samples from South America.
All cold-climate specimens yielded a high proportion of
Eukaryota DNA within the total DNA (89.6–99.7 %). One
of the analysed samples, the Egyptian mummy 1a, reached a
top value of 96 %, therefore, displaying a similarity to the
cold-climate specimens (Fig. 6).
We speculate that the abundance of Actinobacteria DNA
in the Saqqaq, the Denisova hominid and in the two
Bolivian skeletons is due to the overall contact with soil.
The soil-living Actinobacteria are present in cold as well as
in hot or arid regions. Therefore, this fingerprint is indepen-
dent of the environmental temperature parameter.
Conversely, Actinobacteria are scarcely represented in all
warm and cold-climate mummies (i.e. the Egyptian mummies
and the Iceman), since they were not buried in soil. The
Iceman was embedded within glaciers and ice-preserved until
its discovery in 1991. The Egyptian mummies were
embalmed, bandaged and buried in sarcophagi placed inside
tombs. Hence, the bacterial fingerprint allows discrimination
between mummified tissues and non-mummified tissues in-
dependently of the parameter temperature.
The human DNA content in the analysed Egyptian
mummies was variable. Human DNA was abundant in the
mummy extracts 1a, 2a and 3. Lower amounts were present
in extracts 1b, 2b, 4 and 5 (Fig. 8).
Since two different biopsies from specific mummies were
processed, a sample-dependent effect with regard to the
amount of human DNA was identified. For example, samples
1a and 1b taken from mummy 1 and samples 2a and 2b taken
from mummy 2 differ in the human DNA content as well as in
the individual metagenomic composition (Figs. 6and 8). The
first sampling was performed prior to the unfortunate bacterial
bloom that tookplace in the entire repository of the Institute of
Pre- and Protohistory (Tübingen, Germany), and included the
biopsies termed 1a, 2a and 3 (Fig. 6). Consequently, an excess
of bacterial DNA is noted in all the other samples taken from
mummies whose preservation condition had macroscopically
worsened due to inappropriate environmental conditions with-
in the mummy collection.
Finally, three of five randomly selected Egyptian mummies
showed a representation of the human DNA content compara-
ble to the recently published ancient genomes from cold-
climate environments like the Saqqaq (Rasmussen et al.
2010), the Denisova hominid (Reich et al. 2010) and the
Alpine Iceman (Keller et al. 2012)(Fig.8). These data show
that temperature is not the decisive parameter influencing
human and animal DNA survival in archaeological samples.
The mummy datasets were additionally mapped against
the hg19 reference genome and showed low absolute num-
bers of mitochondrial reads in relation to the total number of
human reads (0 to 1 %).
This low amount of mitochondrial reads serves as further
proof for authenticity. In the case of modern human DNA
contamination, significantly higher amounts of extraneous
DNA in the observed metagenomes should have been ob-
served. Furthermore, all NGS-generated mitochondrial data
were cross-checked against the haplotypes of our laboratory
staff. Identical SNP patterns were never detected.
Even on the small-scale level of NGS sequencing for an
initial characterisation of our samples and displaying only a
mitochondrial read count of 1 %, we showed that one
Egyptian mummy yielded a well-covered HVR2 region of
the mitochondrium and gave an indication for haplogroup I2
(Fig. 9). Moreover, it is believed that haplogroup I2 has its
phylogenetic origin in the Near East/West Asia (Derenko et
al. 2007; Saunier et al. 2009; van Oven and Kayser 2009;
Palanichamy et al. 2010; Terreros et al. 2011; Fernandes et
al. 2012; Behar et al. 2012).
On the basis of all the data obtained so far, the possibility
of retrieving authentic DNA from Egyptian mummies has
been highlighted.
We conclude that warm temperature does not cause a
complete degradation of nucleic acids and that the mummifi-
cation appears to be a benefit with regard to DNA survival and
preservation. Moreover, we emphasise that the human DNA
content can reach sufficient amounts to obtain a good cover-
age not only for the mitochondrial genome, but also for the
entire nuclear genome, when sufficient NGS runs are applied.
Conclusions
The analysis of next-generation sequencing (NGS)-generated
mummy metagenomes reveals different DNAs. This variety
of DNA can provide new insights in both the retrospective
diagnosis of clinically relevant pathogens (e.g. bacteria,
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Author's personal copy
viruses and protozoa) and the chemical characterisation of the
ingredients used during the embalming process. Furthermore,
it can be used in the characterisation of the mitochondrial and
nuclear haplotypes and, in turn, may give clues about the
individuals’phylogeny or even kinship. Hence, modern
NGS technology appears to be a powerful aid to mummy
research and strengthens the new discipline of Molecular
Egyptology (Hawass et al. 2010).
To sum up, a good proportion of eukaryotic DNA in a
specimen does not automatically imply that it has to come
from a cold environment. On the other hand, a distinct
difference within the warm-climate specimens can be
pinpointed: DNA from mummified tissues can be differen-
tiated from DNA from unearthed skeletons originating from
warm-climate regions. Furthermore, a high bacterial content
in a specimen does not speak against a good recovery of an
endogenous human genome.
The bacterial fingerprint cannot predict experimental
NGS success or failure in the specimen under investigation,
but it can be used for sample typing.
We show that a “bacterial fingerprint”can be applied for
the identification of mummified tissue signature as
mummies independently from the climate in which they
have been preserved. On the basis of this “bacterial finger-
print”, both the Egyptian mummies and the glacier mummy
of the Alpine Iceman can be grouped together, even though
they experienced very different temperatures over thousands
of years.
Acknowledgements We are grateful to Heiko Prümers, Iris
Trautmann and Andreas Keller for their technical support and
for providing sample/library access. Financial support was provid-
ed by the Graduiertenförderung Tübingen to R.K., M.B., C.-
C.H.C. and C.M.P. Further support was obtained from the
DAAD-GERLS program.
Conflict of interest The authors declare that they have no conflict of
interest.
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