Miklós Kásler - Zoltán Szentirmay (szerk.): Identifying the Árpád Dynasty Skeletons Interred in the Matthias Church. Applying data from historical, archaeological, anthropological, radiological, morphological, radiocarbon dating and genetic research (Budapest, 2021)

CHAPTER EIGHT – PCR and NGS investigations

3. Possible

explanation for the differences between PCR and sequencing data

The question arises as to what could have caused the differences shown in Table 12 between the traditional PCR-based A-STR detection and the marker detection via sequencing. When investigating bone samples containing ancient, degraded DNA, several difficulties must be considered.

1. During

amplification of repeating sequences the DNA polymerase can slip which causes allele length differences. This phenomenon is called a “stutter” flaw/error. In our case, this phenomenon can be detected very well via NGS, when we see that two parallel DNA libraries launched from the same template of A-STR markers with longer alleles show different sequencing data. This error probably occurred in every marker investigated with the exception of D2S441.

2. The

phenomena of allele dropout and “null” (silent) alleles is suggested by the analysis of the D7S820 marker from Béla

Ill

’s sample. We know that DNA sequence polymorphism can occur within or in nearby repeating sequences. At the D7S818 marker, on the -3’ end of the sequence motif series a A>G base swap can be seen near the repeating sequences. If the base swap is according to its primer connection area on the DNA template - and this is the case here - then hybridization of the PCR primer either cannot occur or can only occur at lower hybridization temperatures, and thus the marker on the template will either not be detectable at all or be very hard to detect.

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