The Globe Institute, University of Copenhagen, Copenhagan, Denmark.
Department of Anthropology, Pennsylvania State University, University Park, Pennsylvania, United States.
PeerJ. 2024 Mar 1;12:e16770. doi: 10.7717/peerj.16770. eCollection 2024.
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.
古微生物组的分类特征是快速发展的古微生物学领域的关键步骤。虽然聚合酶链式反应（PCR）扩增 16S 核糖体 RNA（rRNA）基因是现代微生物组研究中广泛使用的技术，但该方法在应用于古微生物 DNA 时存在系统偏差。鸟枪法宏基因组测序已被证明是重建古代牙垢样本分类特征的最有效方法。然而，鸟枪法测序方法存在固有局限性，可以通过杂交富集捕获来解决。当与杂交捕获相结合时，鸟枪法测序和杂交捕获有可能增强对古代微生物群落的特征描述。在这里，我们开发、测试并应用了一种杂交富集捕获技术，以选择性地靶向从与鸟枪法技术生成的古代牙垢样本文库中扩增的 16S rRNA 基因片段。我们模拟了从杂交富集捕获中产生的数据，表明对碎片化和受损的 16S rRNA 基因序列进行分类鉴定是可行的。将这种富集方法应用于 15 个以前发表的古代牙垢样本，我们观察到富集样本中与未富集文库相比，古代 16S rRNA 基因片段增加了 334 倍。我们的结果表明，16S 杂交捕获比 16S rRNA 扩增受背景污染的影响更小，产生的目标回收比例更高。虽然我们的富集技术可以检测到给定样本中低丰度和罕见的分类群，但这些分类群的特异性可能不如未富集方法高。
