Sanger sequencing of mitochondrial hypervariable region of ancient samples for DNA authentication and screening before high-throughput sequencing

• We present a study of ancient samples from the Bronze Age sequenced by Sanger sequencing. • Sanger sequencing is used here as an authentication step of ancient DNA before high-throughput sequencing. • Sanger sequencing can be used as a screening of interesting haplogroups. • This approach shows the importance of Sanger sequencing in the time of high-throughput sequencing methods. Working with ancient DNA is challenging and requires an authentication step. We analyzed eleven badly preserved samples from the Nitra culture with the influence of Únětice culture dated to the 4000 BP Bronze Age. We present estimated mitochondrial haplogroups of four samples based on Sanger sequencing of HVR1 and a part of HVR2. Sanger sequencing has strong limitations in haplogroup prediction, especially when working with ancient DNA. However, Sanger sequencing in this study is featured as a control step to provide us with quality results of mitochondrial DNA and as a screening of present haplogroups. DNA quality estimation is further supported by sample concentration and fragment analysis. The presence of mitochondrial DNA is confirmed by PCR. However, the Sanger sequencing of mitochondrial DNA can confirm its presence in good quality for further sequencing. Therefore, only suitable samples were selected for further high-throughput sequencing of the whole mitogenome.