Mining ancient microbiomes using selective enrichment of damaged DNA molecules

Abstract

The identification of bona fide microbial taxa in microbiomes derived from historic samples is complicated by the unavoidable mixture between DNA from ante-and post-mortem microbial colonizers. One possibility to distinguish between these sources of microbial DNA is querying for the presence of age-associated degradation patterns typical of ancient DNA (aDNA). The presence of uracils, resulting from cytosine deamination, has been detected ubiquitously in aDNA retrieved from diverse sources, and used as an authentication criterion. Here, we employ a library preparation method that separates molecules that carry uracils from those that do not in a set of samples that includes Neandertal remains, herbaria specimens and archaeological plant remains. We show that this method facilitates the discovery of authentic ancient microbial taxa, as it amplifies degradation patterns that would otherwise be difficult to detect in sequences from diverse microbial mixtures. Main textDNA retrieved from historical or ancient samples is a complex mixture of molecules that contains not only endogenous host DNA, but also DNA from microorganisms that were present ante-mortem or that colonized the tissue post-mortem [1]. Therefore, all ancient DNA (aDNA) shotgun sequencing projects are metagenomic in nature. While earlier aDNA research has mostly focused on the evolution of animals and plants [2,3], a growing number of studies are now centering on the identification and characterization of ancient pathogens and microbiomes . CC-BY-NC 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The past, present and future of ancient bacterial DNA

“…Bacterial aDNA retrieval is prone to false positives, given that many pathogenic bacteria are members of the same genera as environmental bacteria [75]. Authentication via PMD can be thwarted by postmortem colonization at different ages, resulting in a range of damage patterns from ancient to extant that impedes distinction [76]. A key methodological advancement for overcoming the challenge of contamination was the development of mapDamage [77], which is a computational tool that analyses PMD signatures and allows the identification of aDNA within a chronologically heterogeneous genetic sample.…”

New Paradigms for Familiar Diseases: Lessons Learned on Circulatory Bacterial Signatures in Cardiometabolic Diseases

“…Routinely, microbiome studies will usually consist of the following procedures before any analytical pipeline can be implemented: sample collection, DNA extraction, and sequencing library preparation (possibly with or without bacterial enrichment techniques such as culture [39,126,127] or bacterial DNA enrichment), highthroughput sequencing technologies [128] such as amplicon-based sequencing (e. g., 16 S rRNA gene), shotgun metagenomic sequencing [129], as well as RNA sequencing-based approaches [38]. Because procedural controls, which take contamination of samples during collection and cross-contamination during processing and sequencing into account, are rarely implemented, studies in low microbial biomass samples relying on sensitive metagenomic techniques highly confound and inflate the diagnostic implications for microbial contribution to disease [130].…”

Herbaria preserve plant microbiota responses to environmental changes

“…External contaminations are indeed an insidious problem in aDNA research that must be limited by strict laboratory procedures, including dedicated equipment and laboratories. They can also be prevented by protocols that specifically enrich in chemically-modified (uracil-containing) aDNA molecules as presented by Weiß et al [13], who recovered sequences from epiphytic and pathogenic bacteria from herbarium plants. While the PCRbased metabarcoding approach is limited and biased by aDNA fragmentation, microbial sequences can also be identified among shotgun sequences generated from plant-extracted DNA, as reported for Arabidopsis thaliana and Ambrosia artemisiifolia herbarium specimens [14].…”