Inferring genetic origins and phenotypic traits of George Bähr, the architect of the Dresden Frauenkirche

Abstract

For historic individuals, the outward appearance and other phenotypic characteristics remain often non-resolved. Unfortunately, images or detailed written sources are only scarcely available in many cases. Attempts to study historic individuals with genetic data so far focused on hypervariable regions of mitochondrial DNA and to some extent on complete mitochondrial genomes. To elucidate the potential of in-solution based genome-wide SNP capture methods - as now widely applied in population genetics - we extracted DNA from the 17th century remains of George Bähr, the architect of the Dresdner Frauenkirche. We were able to identify the remains to be of male origin, showing sufficient DNA damage, deriving from a single person and being thus likely authentic. Furthermore, we were able to show that George Bähr had light skin pigmentation and most likely brown eyes. His genomic DNA furthermore points to a Central European origin. We see this analysis as an example to demonstrate the prospects that new in-

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Scientific REPORTS | (2018) 8:2115 | DOI:10.1038/s41598-018-20180-z
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Inferring genetic origins and
phenotypic traits of George Bähr,
the architect of the Dresden
Frauenkirche
Alexander Peltzer
1,2, Alissa Mittnik
2,3, Chuan-Chao Wang
2,4, Tristan Begg2,
Cosimo Posth2,3, Kay Nieselt
1 & Johannes Krause
2,3
For historic individuals, the outward appearance and other phenotypic characteristics remain often
non-resolved. Unfortunately, images or detailed written sources are only scarcely available in many
cases. Attempts to study historic individuals with genetic data so far focused on hypervariable regions
of mitochondrial DNA and to some extent on complete mitochondrial genomes. To elucidate the
potential of in-solution based genome-wide SNP capture methods - as now widely applied in population
genetics - we extracted DNA from the 17th century remains of George Bähr, the architect of the
Dresdner Frauenkirche. We were able to identify the remains to be of male origin, showing sucient
DNA damage, deriving from a single person and being thus likely authentic. Furthermore, we were able
to show that George Bähr had light skin pigmentation and most likely brown eyes. His genomic DNA
furthermore points to a Central European origin. We see this analysis as an example to demonstrate the
prospects that new in-solution SNP capture methods can provide for historic cases of forensic interest,
using methods well established in ancient DNA (aDNA) research and population genetics.
Advances in modern molecular biology methods and the resulting possibility of extracting genetic information
even from ancient specimens, has led to various attempts to reconstruct the genetic legacy of historic individ-
uals. One of the rst attempts was made in 2007 on Sven Estridsen, the last Danish Viking king1, who died
in 1074 AD. Other attempts in reconstructing the genetic legacy of historic individuals include the cases of
Francesco Petrarca2, the identication of the family of Tsar Nicholas II of Russia3, the famous astronomer Nicolas
Copernicus4, King Richard III of England5, the Dark Countess6, a proposed blood sample from King Louis XVI
king of France7 and most recently the Belgian King Albert I8. In all of these cases, (except for King Louis XVI,
where an Exome and shallow WGS approach was performed), either partial mitochondrial information, such as
the hypervariable sequence HVS-I, HVS-II or D-Loop of the mitochondria, or a full mitochondral genome were
sequenced. While this is sucient for investigating maternal ancestry lines, it provides little resolution on genetic
origin. Foremost, when focusing on mitochondrial data only, there is no information on the paternal ancestry
obtained. Additionally, the prediction of disease risks or phenotypic traits such as hair and eye color are not pos-
sible when only mitochondrial information is available. While the availability of cheaper sequencing methods
and ecient mitochondrial capture techniques enabled researchers to move from targeting control regions to
whole mitochondria, the reconstruction of a full high coverage human genome from ancient human remains via
high throughput sequencing still remains costly9. In population genetics, where large cohorts of individuals are
studied, the cost pressure urged researchers to move on to more cost-ecient and large-scale methods. is has
led to the development of specialized in-solution capture methods that target a pre-dened set of SNP positions,
as introduced by Haak et al.10,11. In population genetics of ancient human individuals, these methods are now
widely applied to recover population specic diagnostic markers. While these approaches target up to 3.7 M SNP
positions12 aiming at solely retrieving population diagnostic SNPs in a previously unrivaled resolution, the set of
1Integrative Transcriptomics, Center for Bioinformatics, University of Tübingen, Tübingen, 72076, Germany.
2Department of Archaeogenetics, Max Planck Institute for the Science of Human History, Jena, 07745, Germany.
3Institute for Archaeological Sciences, University of Tübingen, Tübingen, 72070, Germany. 4Department of
Anthropology and Ethnology, Xiamen University, Xiamen, 361005, China. Correspondence and requests for materials
should be addressed to J.K. (email: krause@shh.mpg.de)
Received: 6 September 2017
Accepted: 11 January 2018
Published: xx xx xxxx
OPEN
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targeted SNPs includes information about various other diagnostic markers as well13. is enables a more detailed
phenotypic and disease specic analysis of historic individuals on a much broader level than before.
Unlike for population genetics studies, the focus within forensic case studies is shied to the identication of
individuals and prediction of phenotypic traits. In the case of the historical gure focused on in this study, George
Bähr, the main goal was to investigate how much information can be retrieved by modern in-solution SNP cap-
ture methods for such studies and whether the approach is generally suitable for characterizing historic individ-
uals. George Bähr is most widely known for his work as architect of several churches and in particular the iconic
Dresdner Frauenkirche, an important monument in German history due to its destruction in the last few weeks
of the Second World War and its recent reconstruction aer the German reunication. Born on the
15th
of March
1666 in the village of Fürstenwalde, south of Dresden, as the son of a weaver14,15, George Bähr moved to Dresden
in 1690 and aer several years of work as a carpenter, he was appointed Master Carpenter of the city of Dresden
in 170516. During his time there, he was responsible for building both general housing and churches, such as the
Orphanage Church in Dresden (1710), the Trinity Church in Schmiedeberg (1713–1716) and several other
churches in Forchheim, Königstein, Hohnstein and Kesselsdorf14. In 1722, he began work on his most ambitious
project, the Dresdner Frauenkirche. In 1730, he was granted the title of Architect for his service to the city of
Dresden over the previous decade, including his work on the Frauenkirche14,15. Unfortunately, Bähr was unable
to see this most prominent piece of work in its full glory, as he died following a pulmonary edema at the age of 72
in 1738, ve years before the church was nished14. His skeletal remains were initially buried in the Johannis
cemetery. However, they were ultimately moved to the crypt of the Frauenkirche in 185414–17, aer the cemetery
was desecrated and moved to a dierent location in the city. Unfortunately, there are no written excerpts or paint-
ings that can be used by historians to gain an impression of the physical and personal appearance of George Bähr.
Unlike for other famous architects, such as Matthäus Daniel Pöppelmann of the same century18, there is almost
no material other than basic family background available for George Bähr. Even the most complete biographical
and historical works, such as the ones by Möllering17, Fischer15 and the most recent biography by Gerlach14,
including intensive archival research, did not reveal any more detailed information on him. Aer the reconstruc-
tion of the Dresdner Frauenkirche, from 1990 to 2005, parts of his skeletal remains were found. In order to obtain
biological information such as physical appearance and potential risk alleles for genetically inherited diseases
from this historic person of interest, we were provided by the George Bähr foundation with bone samples from
his skeletal remains. rough in-solution capture, we were able to obtain high coverage genome wide data from
George Bähr and used that information to reconstruct his genetic ancestry and phenotypic traits such as skin and
eye color. In addition, we found about a dozen risk alleles for medical conditions, including some that might have
contributed to his death.
Results
In total, three independent sequencing experiments were conducted: an initial whole genome shallow shotgun
sequencing to determine parameters such as endogenous DNA content, a mitochondrial DNA capture to obtain a
full mitochondrial genome and a 390 K SNP capture to obtain high density SNP information on George Bähr. e
analysis of the rst shallow whole genome shotgun sequencing (WGS), showed a total endogenous DNA content
of 62.2%. e mitochondrial DNA capture resulted in a 395 X covered mitochondrial genome, accompanied by
two high density SNP in solution capture libraries for population and disease specic SNP detection. On the lat-
ter, a mean depth of 28.19 X coverage on the target dataset of 390 K SNPs published in Haak et al.10 was achieved,
spanning a total of 317,990 SNPs (with ≈80% target eciency of the capture). e rst aim was to authenticate
the analyzed DNA to be of historic origin. In order to authenticate the sequenced fragments, the terminal substi-
tution rates were investigated. Typical double stranded aDNA libraries show cytosine to thymine misincorpora-
tions at the 5′ end and guanine to adenine misincorporations at the 3′ ends19,20. ese characteristic substitutions
accumulate over time and are caused by deamination of cytosine causing miscoding lesions21. As can be seen in
Fig.1, which was created on the intial WGS shallow sequencing run data, up to 7% damage on both 3′ and 5′
ends of the reads can be observed, conrming the presence of ancient DNA.e nuclear 390 K capture libraries
were treated with UDG, following a protocol by Briggs et al.20 to remove damage patterns for improved analysis.
e same analysis of the (non-UDG treated) mitochondrial capture library showed identical damage patterns as
the initial whole genome shotgun library, as well as minimal mitochondrial contamination as described below,
increasing the condence that the samples indeed contain authentic ancient DNA.
In order to conrm whether the sampled individual was male, a molecular sex determination analysis was
done on the sequencing data of the 390 K capture. e results as shown in Table1 show, that the individual was
indeed male.
To further exclude a potential contamination of the sampled individual with human DNA from other sources,
a mitochondrial contamination test was performed. e estimated mitochondrial contamination was reported to
be very low with levels between 0–2%. Quality and authenticity are a major concern in the eld of ancient DNA.
e last decade has seen a large array of methods to estimate DNA contamination23 as well as reliable criteria for
authenticity such as DNA damage patterns21,24. We followed those criteria strictly and used standard methods to
estimate mitochondrial and nuclear contamination rates based on heterozygocity of the mitochondrial genome
as well as the sex chromosomes. We can show that the DNA extracted from the remains of George Bähr come
from a single male individual that shows damage patterns indicative of at least 100 year old DNA21. We therefore
conclude the authentic ancient origin of the specimens DNA. A total number of 1,163 known SNPs25 on chromo-
some X covered at least twice were analyzed, resulting in a very low X-chromosomal contamination estimate of
0.003% with an estimated error of
.−
E7391683 18
26.
Aer the initial verication and authentication process, the paternal and maternal origin of George Bähr
was determined. For this purpose, a complete 395 X coverage mitochondrial genome of George Bähr was recon-
structed and a quality ltered (q > 30) consensus sequence of his genome was created using schmutzi27. His
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maternal haplogroup was determined to be H35 using Haplogrep 228, which is a common subclade of haplogroup
H in Central Europe29. Furthermore, the Y chromosomal haplogroup of George Bähr was determined to be
R1b1a2a1a2-P312 (Table2). e assigned Y-chromosomal haplogroup is the most common Y chromosome clade
of paternal lineages across much of Western Europe, showing a frequency peak in the upper Danube basin and
Paris area with declining frequency towards Italy, Iberia, Southern France and the British Isles30.
Figure 1. Damage plot for the 5′ and 3′ ends of sequenced reads. Both 5′ and 3′ read ends show
≈ .75%
DNA
damage on the rst respective bases, which is a typical pattern observed for ancient DNA. Since the damage
patterns in the initial WGS screening run and the mitochondrial capture experiment are identical, only the
WGS screening damage plot is shown here for simplicity. Plots have been created with DamageProler.
Sample Coverage on chr X Coverage on chr Y Autosomal Coverage
.cov Ycov Auto()/( )
BährAB 10.84 14.68 38.23 0.38
Table 1. Normalized results of sex determination on the skeletal remains of George Bähr. e last column
describes the fraction of coverage on the Y chromosome versus the coverage on the autosome. Fu et al. reported
that a ratio of
< .005
can be considered a female individual and a Y-rate
> .02
is assured to be a male individual22.
e results therefore indicate strongly that the investigated individual was male.
SNP Haplogroup Other Names for SNP rs ID
Allele Information
Y-Position(hg19) ancestral-derived-Bähr Depth
P312 R1b1a2a1a2 PF6547; S116 rs34276300 22157311 C-A-A 65
L52 R1b1a2a1a PF6541 rs13304168 14641193 C-T-T 1
L151 R1b1a2a1a PF6542 rs2082033 16492547 C-T-T 35
P311 R1b1a2a1a PF6545; S128 rs9785659 18248698 A-G-G 19
P310 R1b1a2a1a PF6546; S129 rs9786283 18907236 A-C-C 19
M412 R1b1a2a1 L51; PF6536; S167 rs9786140 8502236 G-A-A 22
L23 R1b1a2a PF6534; S141 rs9785971 6753511 G-A-A 7
L265 R1b1a2 PF6431 rs9786882 8149348 A-G-G 6
PF6438 R1b1a2 NA NA 9464078 C-T-T 1
L150.1 R1b1a2 PF6274.1; S351.1 rs9785831 10008791 C-T-T 72
M269 R1b1a2 NA rs9786153 22739367 T-C-C 1
L320 R1b1a NA rs2917400 4357591 C-T-T 1
P297 R1b1a PF6398 rs9785702 18656508 G-C-C 1
Table 2. Y-Haplotyping results, determined using the ISOGG database.
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A principal components analysis, conducted on 317,990 SNP positions, revealed that George Bähr’s SNP pro-
le matches with proles commonly found in modern central European individuals as shown in Fig.2. To further
explore the relatedness of George Bähr to European populations, an outgroup
f3
analysis was performed, conrm-
ing the initial PCA results, as shown in Fig.3. To further test whether Africans, South Asians, East Asians, Native
Americans and Oceanians share more anity with George Bähr than with present-day Hungarian, Croatian and
French populations, an
f4
analysis was also performed. e statistics as shown in Table3 imply that there was no
extra ancestry from outside Europe in George Bähr. e results from an unsupervised ADMIXTURE33 analysis
also showed no external genetic components in the genome of George Bähr (Fig.4).
Next, phenotypically interesting SNPs that are considered to be aected by selection were investigated. With
the information obtained by the 390 K SNP capture experiment, George Bähr most likely had brown eyes and
light skin, as shown in Table4. is resembles modern individuals from the same area of Germany, where such a
phenotype is commonly found today34. Furthermore, Bähr was most likely lactose tolerant as he was heterozygous
for the RS4988235 mutation on the LCT gene35,36, again a typical phenotype for central Europeans. e 390K SNP
capture panel does not include SNPs that can be used to determine hair color.
To further elucidate what high density SNP capture methods can provide on such specimen, an extensive
literature survey was performed using SNPedia and the database mining tool Promethease44. e results of this
analysis are shown in detail in Table5. Several potential candidate mutations were found in George Bähr that are
commonly found in modern European populations, such as a variant responsible for the ability to taste bitter-
ness45,46. Interestingly, we also found a large number of SNPs associated with modern diseases like Type-2 diabe-
tes, hypertension and coronary artery disease, which could potentially be related47 to his reported cause of death,
pulmonary edema14. Furthermore, a rare variant responsible for age related macular degeneration48 was found to
be present in George Bähr’s genome.
Discussion
Investigating historic individuals based on genetic data still remains challenging and can only shed light on cer-
tain aspects of an individual, such as eye and hair color and a set of well established disease markers. Previous
studies on historic individuals1–6,8 solely focused on the control region of the mitochondrial DNA and in some
cases on full mitochondrial genomes. Although this enabled the analysis of at least the maternal relatedness of
historic individuals, the analysis of Y-chromosomal data accompanied by a set of autosomal genetic markers per-
mits researchers to recreate a more detailed genetic picture of historic individuals than before.
Within the scope of this project, a complete mtDNA sequence from the skeletal remains of George Bähr and
additionally a set of 317,990 SNPs from his autosomes were retrieved. Standard examination of characteristic
Figure 2. PCA plot generated with EIGENSOFT31,32 with representative modern West-Eurasian populations.
George Bähr is marked with a red triangle, clustering next to Central and Eastern European populations.
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damage patterns on the initial shotgun screening data and the mitochondrial capture data suggest an ancient
origin for the investigated remains. Very low contamination estimates on mitochondrial and Y-Chromosomal
level also showed that the retrieved DNA was authentic and no modern human contamination was found. George
Bähr’s maternal haplogroup was determined to be H35 and the Y haplogroup was determined to be
R1b1a2a1a2-P312, both commonly found in Central European modern populations. Based on phenotypic anal-
ysis, George Bähr had brown eyes, light skin pigmentation and was able to digest lactose in adulthood. e pop-
ulation genetic analysis of ancestry with both
f3
and
f4
statistics as well as an ADMIXTURE analysis on the set of
317,990 SNPs conrmed previous ndings on the mitochondrial level: George Bähr was of Central European
descent and shared no additional genetic components with populations outside Europe.
Unfortunately, there is not much of a historic record on George Bähr’s private life. us any information that
can be obtained on a genetic level that elucidates and enlarges information on him could be important, given his
contributions to the history of the city of Dresden. Although George Bähr lived a relatively long life given his time
period, his cause of death may have been a pulmonary edema as stated by several authors14. His genetic make
up might have contributed to his death given the detected number of variants found related to obesity, diabetes,
hypertension and coronary artery disease, which are now widely seen as high- risk factors for such a cause of
death63. Although this seems promising in terms of genetic evidence, a direct correlation of such risk factors with
an actual cause of death still remains dicult. We see our results as an example of how genome wide information
can help to reveal more information on historic individuals for whom scarce or incomplete personal details are
available. Written evidence describes that George Bähr’s remains were initially buried at the Johannis cemetery
of Dresden and later moved to the crypt of the Frauenkirche in 185414–17. Unfortunately, given the time period of
the reburial and the demolished condition of the Frauenkirche aer the Second World War, we cannot exclude
entirely the possibility of skeletal mixup. However, our reconstructed genetic prole as well as the historical pro-
venience of the human remains suggest that the analyzed specimens indeed belong to George Bähr.
Figure 3. Outgroup
f3
plot for George Bähr. Dark colored areas highlight more distant populations, white
highlight closer populations with respect to George Bähr (marked with a red box).
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With the rise of cost-ecient in-solution based SNP capture methods, historic samples can now be investi-
gated in a much more detailed way than ever before. In contrast to previous methods that focused on mitochon-
dria or control regions, the additional information obtained using established SNP capture protocols can provide
much more information for researchers or historians to investigate more complex forensic, population genetic
and medical questions. Although genetic methods with respect to phenotype predictions made some progress
in the last few years, one must keep in mind that direct connections between genotype and phenotype are still
challenging. Estimating personal characteristics from genetic data, such as the height or appearance of an indi-
vidual are in their early stages, as shown for example by Mathieson et al.13. For even more detailed predictions,
e.g. facial reconstructions these direct relationships between genotype and phenotype still remain unresolved.
Furthermore, the quality of historic genome data is usually inferior to modern genome data and typically intro-
duces additional error sources, rendering statistically profound statements in the context of phenotypic analysis
even more complicated.
New and updated capture protocols are incorporating more diagnostic positions and thus provide now even
more SNPs for downstream medical and population genetics analysis in the future. We therefore believe that the
current SNP capture methods are just the beginning for studies of historic individuals. For example, Mathieson
et al.13 stated that larger cohort studies, such as the one conducted by Mallick et al.64, could reveal more and more
diagnostically relevant SNPs and associations between SNPs that can hopefully help resolve such questions in
more detail in future.
Methods
Ancient DNA extraction & Initial Screening. Bone samples were taken under standard precaution and
clean conditions from the skeletal remains of George Bähr, which had been placed in the crypt of the Dresdner
Frauenkirche. We performed DNA extraction and library preparation steps in clean-room facilities. Bone powder
was collected using a dental drill and subsequently DNA was extracted using an established protocol65. We pro-
duced indexed libraries using 20 μ aliquot of the generated extract, following the protocol of Meyer et al.66.
Additionally, the libraries were enriched for human mitochondrial DNA in a bead based capture protocol using
long-range PCR products as bait for hybridization as introduced by Maricic et al.67. We included one negative
control for every step of DNA extraction and library preparation to ensure consistency of results. DNA
Worldwide populations Outgroup Europeans Bähr
f4
Z
Mbuti Chimp Hungarian Bähr −0.000108 −0.344
Yoruba Chimp Hungarian Bähr −0.000187 −0.587
Kalash Chimp Hungarian Bähr −0.000089 −0.23
Papuan Chimp Hungarian Bähr 0.00052 1.196
Ami Chimp Hungarian Bähr −0.000055 −0.129
Han Chimp Hungarian Bähr 0.000123 0.297
Karitiana Chimp Hungarian Bähr 0.000451 0.996
Eskimo Chimp Hungarian Bähr 0.000358 0.844
Selkup Chimp Hungarian Bähr 0.000121 0.298
Uzbek Chimp Hungarian Bähr 0.000187 0.488
Mbuti Chimp Croatian Bähr −0.000041 −0.128
Yoruba Chimp Croatian Bähr −0.000065 −0.199
Kalash Chimp Croatian Bähr −0.000078 −0.196
Papuan Chimp Croatian Bähr 0.000558 1.243
Ami Chimp Croatian Bähr −0.000181 −0.415
Han Chimp Croatian Bähr 0.000032 0.076
Karitiana Chimp Croatian Bähr 0.000315 0.671
Eskimo Chimp Croatian Bähr 0.000281 0.648
Selkup Chimp Croatian Bähr 0.00003 0.072
Uzbek Chimp Croatian Bähr 0.000132 0.335
Mbuti Chimp French Bähr −0.000008 −0.024
Yoruba Chimp French Bähr −0.000003 −0.011
Kalash Chimp French Bähr −0.000064 −0.166
Papuan Chimp French Bähr 0.000524 1.232
Ami Chimp French Bähr −0.000225 −0.538
Han Chimp French Bähr 0 0.001
Karitiana Chimp French Bähr 0.000202 0.453
Eskimo Chimp French Bähr 0.000153 0.364
Selkup Chimp French Bähr 0.00002 0.051
Uzbek Chimp French Bähr 0.000189 0.498
Table 3.
f4
statistics results between worldwide populations, Chimp, Europeans and Bähr.
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sequencing was performed in an initial screening run for the enriched library pools on the Illumina Genome
Analyzer IIx platform with
×+276 7
cycles, following the instruction manual for multiplex sequencing
(FC-104-400x v4 sequencing chemistry and PE-203-4001 v4 cluster generation kit). In contrast to the manual, the
raw reads were aligned to the PhiX 174 reference sequence to obtain training data for a modied base calling
Figure 4. ADMIXTURE graph created with K = 7 for the set of elaborated reference populations. George Bähr
can be found within the variance of Central European populations, here highlighted with a red rectangle.
SNP
Gene
LCT SLC45A2 SLC45A5 EDAR HERC2
rs4988235 rs16891982 rs1426654 rs3827760 rs12913832
Ancestral G C G A A
Derived A G A G G
Coverage
×39
×114
8
×
×43
46
×
Derived allele
frequency 50% 100% 100% 0% 57%
Table 4. Phenotyping results of George Bähr. To ensure consistency, the analysis was limited to high quality
bases (q > 30) and duplicates were removed aer merging of both sequencing libraries. e SNP RS4988235 in
LCT is responsible for lactase persistence in Europe37,38. Both SNPs at SLC24A5 and SLC45A2 are considered to
be responsible for light skin pigmentation39, whereas the SNP in HERC2 is the primary determinant of light eye
color in present-day Europeans40,41. e SNP in the gene EDAR aects tooth morphology and hair thickness42,43,
and was not found to be derived in the investigated sample. All these results were obtained on the 390K SNP
capture dataset.
Potentially pathogenic and phenotypically relevant SNPs
rs ID Eect Citation
rs1333049 Associated with coronary artery disease 49–51
rs2383206 / rs10757274 Associated with coronary artery disease 52
rs5186 7.3x increased risk of Hypertension 53,54
rs1061170 5.9x increased risk of age related macular degeneration 48
rs1121980 Early onset obesity 55
rs1421085 Obesity 56,57
rs9939609 Obesity / Diabetes 58
rs13266634 Diabetes 59
rs4506565 Associated with Diabetes 60
rs17817449 Associated with Body weight & increased BMI 61,62
rs10246939 Able to taste bitterness 45,46
Table 5. Potentially pathogenic and phenotypically relevant SNPs found in George Bähr.
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application called Ibis68. e reads were then ltered according to their individual indices and went into RAW
data processing.
Nuclear 390 K capture. In the clean room facilities of the Institute for Archeological Sciences in Tübingen,
Germany, two further libraries from
μl20
extract each were produced in a similar fashion to the screening
library, but additionally implementing a UDG and endonuclease
VIII
damage repair treatment20, to remove
deaminated bases. e libraries were amplied to reach an amount of about
ng1,000
DNA for each which was
subsequently used in an in-solution hybridization capture approach11, targeting a set of
394,577
SNPs10. DNA
sequencing was performed on a HiSeq 2500 with
×2101
cycles.
RAW data processing and authentication. General RAW data processing for the initial shallow whole
genome sequencing (WGS), mitochondrial capture dataset and the 390 K SNP capture data was done using the
EAGER pipeline69. In all cases, sequence adapters were clipped with Clip&Merge with default settings and the
paired end reads were merged respectively. For the initial WGS and the 390 K SNP capture data, the read mapping
procedure was performed with BWA70 0.7.15 and reads were mapped against the hg19 human reference genome.
For the mitochondrial capture data, reads were mapped against the rCRS reference genome. e CircularMapper
approach as implemented in EAGER was used with default settings to increase mapping qualities towards the
ends of the utilized mitochondrial reference genome. In all three datasets, WGS, 390 K and mitochondrial cap-
ture, DNA damage authentication was performed using our in-house tool DamageProler to determine whether
characteristic misincorporation patterns of aDNA are present in the investigated datasets21. In addition, the mito-
chondrial data was tested for potential contamination in the EAGER pipeline using schmutzi27. On the 390 K cap-
ture data, the “MoM” estimate from “Method 1” as well as the “new_llh” X-chromosomal authentication method
in ANGSD26 was used to quantify potential autosomal contamination on the X chromosome. Furthermore, a
molecular sex identication of the remains of George Bähr was performed using the method previously described
in Fu et al.22. is approach calculates the number of reads mapping against the target SNPs on the Y chromosome
and compares this to the total number of reads mapping against the target SNPs on the autosome. An empirical
threshold from the literature (see71) was then used to determine whether the investigated individual was male or
female.
Y-chromosomal analysis. e Y chromosomal haplogroup was determined by examining a set of diagnos-
tic positions on chromosome Y using the ISOGG database version 11.228 (August 19, 2016), utilizing all available
positions on the 390 K capture dataset. In order to perform this analysis, the analysis was restricted to reads with a
mapping quality higher than 30. Further detailed investigations revealed that mutations separating George Bähr
from upstream Y haplogroups such as R1b1a2a1a (see Table2) are present. For potential haplogroups within the
clade investigated R1b1a2a1, R1b1a2a and R1b1a2 (see Table2) characteristic mutations were found, which made
the placement of George Bähr in Y haplogroup R1b1a2a1a2-P312 most likely.
Population Specic analysis. Principal components analysis. A principal components analysis using the
smartpca method available in EIGENSOFT31,32 was performed using default parameters and the options lsqpro-
ject: YES and numoutlieriter: 0. e investigated sample was projected onto the variation of 777 present-day West
Eurasians with
317,990
SNPs10.
Admixture. An ADMIXTURE33 analysis was performed aer pruning the data for linkage disequilibrium in
PLINK72 with the parameters–indep-pairwise 200 25 0.4 retaining 181,529 SNPs of the 390 K capture dataset10.
ADMIXTURE was executed with default 5-fold cross validation, varying the number of ancestral populations
between K = 2 and K = 15 in bootstraps of 100 with dierent random seeds. Again, 777 modern West Eurasians
and individuals from worldwide representative populations such as Mbuti, Yoruba, Han, Papuan, Karitiana,
Eskimo, Uzbek, Amim, Selkup and Kalash were used for the analysis. e lowest cross-validation errors were
observed with K = 7.
Outgroup
f3
/
f4
statistics. Additionally,
f3
statistics of the form
̈fBX(Mbuti;ahr,)
3
were calculated to test which
West Eurasian populations share the most genetic dri with George Bähr. is analysis was performed using
ADMIXTOOLS73 with the parameter settings inbreed: YES, computing standard errors with a block jackknife.
For the computation of
f4
statistics of the form
̈fB(Worldwidepopulations, Chimp; Europeans, ahr)
4
ADMIXTOOLS73 was applied and again standard errors were computed with a block jackknife.
Phenotypic analysis. Aer uploading a VCF le74 to the respective web service Promethease, a more detailed
report is created stating potential causes for diseases as well as phenotypic traits. To ensure that found variants
are indeed trustworthy, the IGV tool was used to manually conrm the ndings of the method before reporting75.
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Acknowledgements
We want to thank Kajo Kusen and the George Bähr Stiung Dresden for their help in getting access to the skeletal
remains of George Bähr. Furthermore, we wanted to express our deepest gratitude to Dr. Siegfried Gerlach for his
personal help in collecting literature and written information on George Bähr. We wanted to furthermore thank
Judith Neukamm for proof reading. C.C.W was supported by the Max Planck Society and Nanqiang Outstanding
Young Talents Program of Xiamen University.
Author Contributions
J.K. designed the experiments. J.K. carried out the skeletal sampling. A.M. and C.P. performed the ancient DNA
experiments. A.P. and T.B. researched literature and archival data on George Bähr. A.P., A.M. and C.C.W. analyzed
the data. A.P. wrote the manuscript with contributions from all co-authors. All authors read and approved the
manuscript.
Additional Information
Competing Interests: e authors declare that they have no competing interests.
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