DOI Page / Citation link:
https://doi.org/10.11588/diglit.53060#0210
6.4 Bone samples and genetic analyses of Harbor Necropolis
209
1.1 - Bone treatment
Fragment alignments
Fig. 85 The procedure from bone to DNA sequence is illustrated as sequential steps. Since DNA is on everything we touch with our
bare hands, thorough bone treatment is undertaken to remove contamination from people handling the skeletal material or
from bacteria or fungi from the soil surrounding the material (1.1). For each bone piece, the outer surface layer is physi-
cally removed using a sand blaster, followed by UV irradiation of the bone piece in a crosslinker cabinet. The bone pieces
are then powdered in a Freezer Mill in the presence of liquid nitrogen, and the bone powder is bleached to further remove
contaminants and amplification inhibitors.
DNA is extracted and amplified with frequent use of negative controls (i.e. empty samples including only water and chemical
consumables). The negative controls are included to detect contamination if present. A 343 base pair long sequence stretch
of the mtDNA control region, corresponding to the positions 16 050-16 392 of the mitochondrial reference sequence, was
amplified in five different, but overlapping fragments using PCR (1.2). PCR - Polymerase Chain Reactions - is a method to
make millions of copies of each requested target DNA sequence. All targeted fragments were short, between 120-150 base
pairs, to maximize the possibility to amplify fragmented authentic DNA.
Next generation sequencing (NGS) technology reports a high number (typically hundreds or thousands) of DNA fragments
present in an aliquot of amplified DNA from each sample (1.3). By aligning the sequences underneath each other, the de-
gradation pattern (like deamination, an incident occurring post-mortem where biochemical changes in the DNA make the
instrument read an alternative building block of the DNA strand) of the samples could be examined and evaluated using
networks. When building the networks, identical fragments are grouped together, presented in a circle (A) and the size of
the circle reflects the number of times a variant is observed. In the next step of network building, variants with one observed
difference compared to the main type are added (B), followed by variants with two observed differences (C), and so on.
For poorly preserved samples, potentially no original DNA strands could be retrieved, and only degraded DNA-strands are
observed (D). The degradation pattern of the network could be statistically evaluated and the most likely authentic sequence
predicted (Graphics: G. Bjomstad)
209
1.1 - Bone treatment
Fragment alignments
Fig. 85 The procedure from bone to DNA sequence is illustrated as sequential steps. Since DNA is on everything we touch with our
bare hands, thorough bone treatment is undertaken to remove contamination from people handling the skeletal material or
from bacteria or fungi from the soil surrounding the material (1.1). For each bone piece, the outer surface layer is physi-
cally removed using a sand blaster, followed by UV irradiation of the bone piece in a crosslinker cabinet. The bone pieces
are then powdered in a Freezer Mill in the presence of liquid nitrogen, and the bone powder is bleached to further remove
contaminants and amplification inhibitors.
DNA is extracted and amplified with frequent use of negative controls (i.e. empty samples including only water and chemical
consumables). The negative controls are included to detect contamination if present. A 343 base pair long sequence stretch
of the mtDNA control region, corresponding to the positions 16 050-16 392 of the mitochondrial reference sequence, was
amplified in five different, but overlapping fragments using PCR (1.2). PCR - Polymerase Chain Reactions - is a method to
make millions of copies of each requested target DNA sequence. All targeted fragments were short, between 120-150 base
pairs, to maximize the possibility to amplify fragmented authentic DNA.
Next generation sequencing (NGS) technology reports a high number (typically hundreds or thousands) of DNA fragments
present in an aliquot of amplified DNA from each sample (1.3). By aligning the sequences underneath each other, the de-
gradation pattern (like deamination, an incident occurring post-mortem where biochemical changes in the DNA make the
instrument read an alternative building block of the DNA strand) of the samples could be examined and evaluated using
networks. When building the networks, identical fragments are grouped together, presented in a circle (A) and the size of
the circle reflects the number of times a variant is observed. In the next step of network building, variants with one observed
difference compared to the main type are added (B), followed by variants with two observed differences (C), and so on.
For poorly preserved samples, potentially no original DNA strands could be retrieved, and only degraded DNA-strands are
observed (D). The degradation pattern of the network could be statistically evaluated and the most likely authentic sequence
predicted (Graphics: G. Bjomstad)
