Entering our clean pre-PCR laboratory, the bone samples get a unique identifier, they are registered and photographed. A special isolated cabin serves for the primary mechanical cleaning of the bones. The entire cabin is regularly bleached and UV-C irradiated before and after the scrapings. The samples are also UV-C irradiated on all sides upon entering the lab. The surfaces of the bone samples are removed using a polisher machine. During the polishing, the bone powder by-product is persistently sucked off by a mobile vacuum cleaner. Tooth samples are cleaned with UV-C irradiation and a sandblaster machine. The cleaned and subsequently UV-C irradiated samples are placed into sterile bags until further analyses.

The samples are ground into a fine powder using a mixer mill (Retsch), crushing in ball milling mills, or drilling bone powder. The resulting powder measured in a sterile box and then stored in sterile tubes at 4°C until use. Each batch of samples has a negative control from the milling and later from the DNA extraction. The negative controls are processed parallel with the bone samples.

The DNA extraction takes place in clean laboratory conditions, under a laminar flow box in a separate room. Several extraction methods are applied depending on the state of the sample and the purpose of the analysis. The DNA extraction workflow has been automated in Biomek workstations, making it easier and faster to process samples. The DNA extracts, marked by specific ID numbers, are stored in the freezer. Each series uses separate isolation negative controls, which are also treated in parallel with the sample. This contains only the solutions used in the purification process.

From DNA extractions DNA libraries are prepared with manually or  robotic technology. During the DNA library preparation, the DNA is chemically repaired and the samples are barcoded with individual adapters. After amplification the samples are prepared for next-generation sequencing (NGS), what is performed in-house with Illumina MiSeq Platform. Depending on our research objectives, DNA regions are selected (whole mitochondrial genome, single nucleotide polymorphisms) from the samples using the method of target capture, or random “shutgun” sequencing is performed.

Autosomal DNA

In diploid human cells, the autosomes are composed of 22 chromosome pairs, each containing two variants of an allele or marker. These markers are present in diploid form, that is, maternal and father-based pairs, however, they may carry different alleles and variants of one gene. The examination of autosomal DNA is suitable for determining blood family relationships. The more markers are used, the larger sections are tested, the more likely we can say that the subjects whether were or not a direct family relationship. The autosomes are commonly used in forensic science for the identification and assignment of skeletal elements in disturbing burial sites, reconstruction of historic genealogies, and families.

Nuclear DNA is typed on the Illumia platform. Besides ready-to-use commercial kits, we have been developing in-house protocols for NGS (target capture) analyses of forensically and population genetically relevant markers. Furthermore, we also shotgun sequence the indexed DNA libraries.

The results are evaluated by programs and bioinformatics tools used by international laboratories. Link: Software | David Reich Lab (harvard.edu). After publishing, raw and evaluated DNA data become available in international online databases (ENA, NCBI) and in online supplementary materials of the publications.

Y-chromosomal DNA

In addition to autosomes, there are sex chromosomes (X and Y) in the nuclear genome. The Y-chromosome is inherited through the male line and has relatively few coding regions and a high mutation rate. Most part of it (95%, 24 Megabase pairs long section) does not recombine with the X-chromosome, and therefore analyzing its markers enables the follow-up of paternal lineages and male-specific past population genetic events. The multiplex analyses of Y chromosomal SNPs (single nucleotide polymorphisms) and STRs (short tandem repeats) play significant roles in our studies. Since 2015 we have been using next-generation sequencing techniques in these analyses as well.

Analysis of mitochondrial DNA (mtDNA)

The mitochondria generate the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. The mitochondria have their own genome (mtDNA) with a circular configuration. In contrast to nuclear chromosomal DNA, which is present in each somatic cell as a double set, mtDNA exists only as a single copy. It is inherited through the female line mostly because primarily the female mitochondria contribute to the newly developing individual's genetic makeup, and all members of a maternal lineage belong to the same mtDNA ancestry. Since several hundreds of mitochondria are found in a single cell, their DNA markers can be a useful source of information in maternal relationships, population genetics, and the history of populations. Our primary focus was the Hypervariable Region I (HVR-I) of the mitochondrial genome, which is the most relevant and best-studied part of the mtDNA in population genetic studies. We completed this analysis by typing several polymorphisms in the coding region of the mtDNA, in order to reproduce our data and affirm certain haplogroup classifications. Since 2015, the PCR-based methods have been gradually changed by whole mitochondrial genome sequencing, using target capture protocols and Illumina MiSeq sequencer. The DNA library preparation and capture protocols have also been optimized for Biomek i5 robot, thus increasing the sample processing capacity of the laboratory.