Cost-effective straightforward method for captured whole mitogenome sequencing of ancient DNA.

Working with mitochondrial DNA from highly degraded samples is challenging. We present a whole mitogenome Illumina-based sequencing method suitable for highly degraded samples. The method makes use of double-stranded library preparation with hybridization-based target enrichment. The aim of the study was to implement a new user-friendly method for analysing many ancient DNA samples at low cost. The method combines the Swift 2S™ Turbo library preparation kit and xGen® panel for mitogenome enrichment. Swift allows to use low input of aDNA and own adapters and primers, handles inhibitors well, and has only two purification steps. xGen is straightforward to use and is able to leverage already pooled libraries. Given the ancient DNA is more challenging to work with, the protocol was developed with several improvements, especially multiplying DNA input in case of low concentration DNA extractions followed by AMPure® beads size selection and real-time pre-capture PCR monitoring in order to avoid cycle-optimization step. Nine out of eleven analysed samples successfully retrieved mitogenomes. Hence, our method provides an effective analysis of whole mtDNA, and has proven to be fast, cost-effective, straightforward, with utilisation in population-wide research of burial sites.

Comparison of Suitability of the Most Common Ancient DNA Quantification Methods.

AIMS: Ancient DNA (aDNA) extracted from historical bones is damaged and fragmented into short segments, present in low quantity, and usually copurified with microbial DNA. A wide range of DNA quantification methods are available. The aim of this study was to compare the five most common DNA quantification methods for aDNA. MATERIALS AND METHODS: Quantification methods were tested on DNA extracted from skeletal material originating from an early medieval burial site. The tested methods included ultraviolet (UV) absorbance, real-time quantitative polymerase chain reaction (qPCR) based on SYBR® green detection, real-time qPCR based on a forensic kit, quantification via fluorescent dyes bonded to DNA, and fragmentary analysis. Differences between groups were tested using a paired t-test. RESULTS: Methods that measure total DNA present in the sample (NanoDrop™ UV spectrophotometer and Qubit® fluorometer) showed the highest concentrations. Methods based on real-time qPCR underestimated the quantity of aDNA. The most accurate method of aDNA quantification was fragmentary analysis, which also allows DNA quantification of the desired length and is not affected by PCR inhibitors. CONCLUSIONS: Methods based on the quantification of the total amount of DNA in samples are unsuitable for ancient samples as they overestimate the amount of DNA presumably due to the presence of microbial DNA. Real-time qPCR methods give undervalued results due to DNA damage and the presence of PCR inhibitors. DNA quantification methods based on fragment analysis show not only the quantity of DNA but also fragment length.