Optimizing analysis parameters and sample input is crucial in forensic genetics methods to generate reliable results, and even more so when working with muti-copy mitochondrial DNA (mtDNA) and ...low-quality samples. This study compared mitotypes based on next-generation sequencing (NGS) results derived from the same samples at two different sequencing library concentrations—30 pM and 0.3 pM. Thirty femur samples from the Second World War were used as a model for poorly preserved DNA. Quantitative PCR (qPCR) method targeting 113 bp long fragment was employed to assess the quantity of mitogenomes. HID Ion Chef™ Instrument with Precision ID mtDNA Control Region Panel was used for library preparation and templating. Sequencing was performed with Ion GeneStudio™ S5 System. Reference haplotypes were determined from sequencing samples at 30 pM library input. Haplotypes were compared between optimal (30 pM) and suboptimal (0.3 pM) library inputs. Often the difference in haplotypes was length heteroplasmy, which in line with other studies shows that this type of variant is not reliable for interpretation in forensics. Excluding length variants at positions 573, 309, and 16,193, 56.7% of the samples matched, and in two samples, no sequence was obtained at suboptimal library input. The rest of the samples differed between optimal and suboptimal library input. To conclude, genotyping and analyzing low-quantity libraries derived from low-quality aged skeletonized human remains therefore must be done with caution in forensic genetics casework.
