Wet Lab Protocols Matter: Choice of DNA Extraction and Library Preparation Protocols Bias Ancient Oral Microbiome...

Area of Science:

• Paleogenomics
• Microbiome Research
• Archaeological Science

Background:

• Ancient DNA (aDNA) from dental calculus offers insights into past health and lifestyles.
• Ancient metagenomics workflows are evolving, posing reproducibility challenges.
• Limited research exists on how wet-lab protocols affect ancient oral microbiome recovery.

Purpose of the Study:

• To investigate the impact of DNA extraction and library preparation methods on ancient oral microbiome recovery.
• To assess how sample preservation influences the efficacy of these protocols.
• To understand technical variability in ancient metagenomics.

Main Methods:

• Applied two DNA extraction and two library preparation methods to dental calculus samples from Hungary and Niger.
• Analyzed samples with varying aDNA preservation levels.
• Assessed intra- and inter-sample variability using metrics like DNA fragment length, GC content, clonality, endogenous content, deamination, and microbial composition.

Main Results:

• Both DNA extraction and library preparation protocols significantly influenced aDNA recovery from dental calculus.
• No single protocol consistently outperformed others across all metrics.
• Protocol effectiveness was dependent on the degree of sample preservation.

Conclusions:

• Wet-lab protocols introduce considerable technical variability into ancient metagenomics studies.
• Findings highlight challenges for meta-analyses and the need to consider protocol-induced biases.
• The study prompts discussion on standardizing methods versus optimizing protocols based on sample characteristics and research goals.