DOI Seite / Zitierlink:
https://doi.org/10.11588/diglit.53060#0209
208
6 Preliminary analyses of DNA in skeletal material from the Harbor Necropolis
was not observed in this inland Byzantine population. A similar genetic constitution was observed
among the 13 skeletons from Ottoman Ephesos (fig. 84 c), but here, in this later population, one
haplotype of East Asian origin (D) was also found. This haplotype could have been introduced
once as of the invasion of the Seljuk Turks or with successive introgressions over many centu-
ries as to continuous interaction with central Asians belonging to similar language groups as the
present-day Turkish population. Alternatively, it might be just as likely that the lineage is older
in Anatolia and could have been brought here by multiple human migrations over the past mil-
lennia, given the central geographical and cultural position of Asia Minor.
6.4 BONE SAMPLES AND GENETIC ANALYSES OF THE HARBOR NECROPOLIS
The excavations of the Harbor Necropolis started in 2005, several years before the samples for
the DNA-analyses were taken in 2011 and 2013. Thus, the skeletal material had already been
handled by numerous persons, probably leaving modern DNA on the surface of the bones. Since
DNA is easily transferred between objects we touch it is important to decontaminate the bones as
best as possible and carry out proper controls in the lab, to try to identify if contamination occur.
After the skeletons had been examined anthropologically by Kristina Scheelen and Jan
Noväcek, bone samples were taken by cutting a slice of the femur with a hacksaw using protective
gloves and cleaning the equipment with ethanol between consecutive samples, or when available,
by removing a tooth from the jaw. Samples were exported to the University of Oslo for further
processing and DNA analyses. In total, samples from 68 skeletons were collected from four of
the five graves of the Harbor Necropolis (grave 1: n = 24, grave 2: n = 2, grave 3: n = 33, grave
4: n = 9).
In the aDNA laboratory, standards were followed to avoid contamination including protec-
tive clothing. Thorough bone treatment was undertaken to remove contamination from people
handling the skeletal material (fig. 85, 1. 1). For each bone piece, the outer surface layer was
removed, followed by UV irradiation. After the bones were powdered, the bone powder was
bleached. These processes are all implemented to remove potential contamination of bacterial,
fungal and modern human origin. In addition, frequent use of negative controls and physical
separation of the facilities for handling original and amplified DNA, to minimize the risk of
contamination from amplified aDNA copies that could outnumber original aDNA, followed the
standards for working with aDNA.
DNA was extracted and amplified as described by Malmström et al. (2009). In short, a 343-
base pair long sequence stretch of the mtDNA control region corresponding to the positions
16 050-16 392 in the mitochondrial reference sequence was targeted (Anderson et al. 1981),
using five overlapping fragments (fig. 85, 1. 2). All targeted fragments were short, between
120-150 base pairs long, to maximize the possibility to amplify authentic DNA.
Next generation sequencing (NGS) was implemented on the skeletal material from the Harbor
Necropolis, performed at the Norwegian Sequencing Centre along with traditional sequencing
of mitochondrial DNA performed at the ABI facilities at the UiO and Macrogen (Netherlands)
for 20 samples. Several of the samples were sequenced using both methods. A major advantage
with NGS is that a high number - typically hundreds or thousands - of DNA fragments present
in an aliquot of amplified DNA are reported from each sample. The degradation pattern of the
samples could be examined, as shown in figure 85, 1. 3. In ancient samples, DNA degradation is
expected, as decomposition also of the DNA strands start at time of death. For poorly preserved
samples, potentially no original DNA strands could be retrieved, and only degraded DNA-strands
are observed. Thus, based on the degradation pattern the most statistically supported authentic
sequence could be predicted even if it is not present (e.g. c-statistics; Helgason et al. 2009).
Haplotype assignment was based on Vincent Macaulay, mtDNAmanager and Genographic
databases273.
273 <http://www.stats.gla.ac.uk/~vincent/founder2000/motif.html> (accessed 05/07/2016); <http://mtmanager.yonsei.
ac.kr/> (accessed 05/07/2016).
6 Preliminary analyses of DNA in skeletal material from the Harbor Necropolis
was not observed in this inland Byzantine population. A similar genetic constitution was observed
among the 13 skeletons from Ottoman Ephesos (fig. 84 c), but here, in this later population, one
haplotype of East Asian origin (D) was also found. This haplotype could have been introduced
once as of the invasion of the Seljuk Turks or with successive introgressions over many centu-
ries as to continuous interaction with central Asians belonging to similar language groups as the
present-day Turkish population. Alternatively, it might be just as likely that the lineage is older
in Anatolia and could have been brought here by multiple human migrations over the past mil-
lennia, given the central geographical and cultural position of Asia Minor.
6.4 BONE SAMPLES AND GENETIC ANALYSES OF THE HARBOR NECROPOLIS
The excavations of the Harbor Necropolis started in 2005, several years before the samples for
the DNA-analyses were taken in 2011 and 2013. Thus, the skeletal material had already been
handled by numerous persons, probably leaving modern DNA on the surface of the bones. Since
DNA is easily transferred between objects we touch it is important to decontaminate the bones as
best as possible and carry out proper controls in the lab, to try to identify if contamination occur.
After the skeletons had been examined anthropologically by Kristina Scheelen and Jan
Noväcek, bone samples were taken by cutting a slice of the femur with a hacksaw using protective
gloves and cleaning the equipment with ethanol between consecutive samples, or when available,
by removing a tooth from the jaw. Samples were exported to the University of Oslo for further
processing and DNA analyses. In total, samples from 68 skeletons were collected from four of
the five graves of the Harbor Necropolis (grave 1: n = 24, grave 2: n = 2, grave 3: n = 33, grave
4: n = 9).
In the aDNA laboratory, standards were followed to avoid contamination including protec-
tive clothing. Thorough bone treatment was undertaken to remove contamination from people
handling the skeletal material (fig. 85, 1. 1). For each bone piece, the outer surface layer was
removed, followed by UV irradiation. After the bones were powdered, the bone powder was
bleached. These processes are all implemented to remove potential contamination of bacterial,
fungal and modern human origin. In addition, frequent use of negative controls and physical
separation of the facilities for handling original and amplified DNA, to minimize the risk of
contamination from amplified aDNA copies that could outnumber original aDNA, followed the
standards for working with aDNA.
DNA was extracted and amplified as described by Malmström et al. (2009). In short, a 343-
base pair long sequence stretch of the mtDNA control region corresponding to the positions
16 050-16 392 in the mitochondrial reference sequence was targeted (Anderson et al. 1981),
using five overlapping fragments (fig. 85, 1. 2). All targeted fragments were short, between
120-150 base pairs long, to maximize the possibility to amplify authentic DNA.
Next generation sequencing (NGS) was implemented on the skeletal material from the Harbor
Necropolis, performed at the Norwegian Sequencing Centre along with traditional sequencing
of mitochondrial DNA performed at the ABI facilities at the UiO and Macrogen (Netherlands)
for 20 samples. Several of the samples were sequenced using both methods. A major advantage
with NGS is that a high number - typically hundreds or thousands - of DNA fragments present
in an aliquot of amplified DNA are reported from each sample. The degradation pattern of the
samples could be examined, as shown in figure 85, 1. 3. In ancient samples, DNA degradation is
expected, as decomposition also of the DNA strands start at time of death. For poorly preserved
samples, potentially no original DNA strands could be retrieved, and only degraded DNA-strands
are observed. Thus, based on the degradation pattern the most statistically supported authentic
sequence could be predicted even if it is not present (e.g. c-statistics; Helgason et al. 2009).
Haplotype assignment was based on Vincent Macaulay, mtDNAmanager and Genographic
databases273.
273 <http://www.stats.gla.ac.uk/~vincent/founder2000/motif.html> (accessed 05/07/2016); <http://mtmanager.yonsei.
ac.kr/> (accessed 05/07/2016).
