Comparisons of aged samples and modern references provide algorithm for mtDNA analysis in challenging material.

Based on results of over 12 years of research, we performed a comparative analysis of haplotypes from 70 to 80 years old bone samples with modern high quality references. Whole mitochondrial genomes were obtained for the purpose of human identification cases conducted by the Polish Genetic Database of Victims of Totalitarianisms-Pomeranian Medical University Research Centre, using Thermo Fisher Scientific's Precision ID line and Ion GeneStudio S5. Converge 2.2 and IGV 2.12.3 were used for secondary sequence analysis and their parameters were altered to construct a new variant calling algorithm. We have found neither a simple change in thresholds, nor removing contaminant reads significantly decreased the number of discrepancies found between haplotype pairs, and conclude that standard analysis settings can rarely be used for poor quality DNA data. The study confirmed some limitations of the analysis of low-quality samples, and of the familial comparisons themselves. Still, the algorithm we developed helps to decide which calls to accept when dealing with difficult material, reducing manual labour, based on Converge-generated Status and EMPOP state of the variants. Additional step for samples with low region coverage is introduced. This protocol can be used in other areas where DNA quantity and quality are low.