TY - JOUR

T1 - Benchmarking a targeted 16S ribosomal RNA gene enrichment approach to reconstruct ancient microbial communities

AU - Eisenhofer, Raphael

AU - Wright, Sterling

AU - Weyrich, Laura

N1 - © 2024 Eisenhofer et al.

PY - 2024

Y1 - 2024

N2 - The taxonomic characterization of ancient microbiomes is a key step in the rapidly growing field of paleomicrobiology. While PCR amplification of the 16S ribosomal RNA (rRNA) gene is a widely used technique in modern microbiota studies, this method has systematic biases when applied to ancient microbial DNA. Shotgun metagenomic sequencing has proven to be the most effective method in reconstructing taxonomic profiles of ancient dental calculus samples. Nevertheless, shotgun sequencing approaches come with inherent limitations that could be addressed through hybridization enrichment capture. When employed together, shotgun sequencing and hybridization capture have the potential to enhance the characterization of ancient microbial communities. Here, we develop, test, and apply a hybridization enrichment capture technique to selectively target 16S rRNA gene fragments from the libraries of ancient dental calculus samples generated with shotgun techniques. We simulated data sets generated from hybridization enrichment capture, indicating that taxonomic identification of fragmented and damaged 16S rRNA gene sequences was feasible. Applying this enrichment approach to 15 previously published ancient calculus samples, we observed a 334-fold increase of ancient 16S rRNA gene fragments in the enriched samples when compared to unenriched libraries. Our results suggest that 16S hybridization capture is less prone to the effects of background contamination than 16S rRNA amplification, yielding a higher percentage of on-target recovery. While our enrichment technique detected low abundant and rare taxa within a given sample, these assignments may not achieve the same level of specificity as those achieved by unenriched methods.

AB - The taxonomic characterization of ancient microbiomes is a key step in the rapidly growing field of paleomicrobiology. While PCR amplification of the 16S ribosomal RNA (rRNA) gene is a widely used technique in modern microbiota studies, this method has systematic biases when applied to ancient microbial DNA. Shotgun metagenomic sequencing has proven to be the most effective method in reconstructing taxonomic profiles of ancient dental calculus samples. Nevertheless, shotgun sequencing approaches come with inherent limitations that could be addressed through hybridization enrichment capture. When employed together, shotgun sequencing and hybridization capture have the potential to enhance the characterization of ancient microbial communities. Here, we develop, test, and apply a hybridization enrichment capture technique to selectively target 16S rRNA gene fragments from the libraries of ancient dental calculus samples generated with shotgun techniques. We simulated data sets generated from hybridization enrichment capture, indicating that taxonomic identification of fragmented and damaged 16S rRNA gene sequences was feasible. Applying this enrichment approach to 15 previously published ancient calculus samples, we observed a 334-fold increase of ancient 16S rRNA gene fragments in the enriched samples when compared to unenriched libraries. Our results suggest that 16S hybridization capture is less prone to the effects of background contamination than 16S rRNA amplification, yielding a higher percentage of on-target recovery. While our enrichment technique detected low abundant and rare taxa within a given sample, these assignments may not achieve the same level of specificity as those achieved by unenriched methods.

KW - Humans

KW - RNA, Ribosomal, 16S/genetics

KW - Benchmarking

KW - Genes, rRNA

KW - Dental Calculus

KW - Microbiota

KW - DNA, Ancient

U2 - 10.7717/peerj.16770

DO - 10.7717/peerj.16770

M3 - Journal article

C2 - 38440408

VL - 12

JO - PeerJ

JF - PeerJ

SN - 2167-8359

M1 - e16770

ER -