Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

Area of Science:

• Paleogenomics
• Microbiology
• Ancient DNA Analysis

Background:

• Ancient oral and gut microbiota are typically studied using 16S rRNA gene metataxonomic sequencing.
• The V3 region of the 16S rRNA gene is often targeted for ancient DNA amplification.
• Metataxonomic approaches can lead to skewed microbial taxonomic frequency data.

Purpose of the Study:

• To investigate the causes of skewed microbial profiles in ancient dental calculus.
• To compare amplicon sequencing with shotgun metagenomics for ancient microbiome reconstruction.
• To evaluate the reliability of 16S rRNA V3 amplicon sequencing for ancient microbiome analysis.

Main Methods:

• Shotgun metagenomics sequencing of four ancient dental calculus specimens.
• Comparative analysis of microbial taxonomic counts from paired amplicon (V3 region) and shotgun sequencing datasets.
• In silico analysis of alternative 16S rRNA hypervariable regions.

Main Results:

• Extensive length polymorphisms in the 16S rRNA V3 region cause differential amplification.
• This bias significantly skews taxonomic frequency data in amplicon sequencing-based microbiome reconstructions.
• Shotgun metagenomics provides a more accurate representation of ancient microbial communities.

Conclusions:

• Systematic amplification bias from 16S rRNA V3 amplicon sequencing confounds accurate microbiome profiling.
• Alternative 16S rRNA hypervariable regions likely present similar amplification challenges.
• Shotgun metagenomics is recommended for reliable ancient microbiome reconstructions.