- Additionally, the relative enrichment of taxa in the uracil enriched fraction can help to identify bona fide ancient microbial taxa that could be missed using a more targeted approach.. - Conclusions: Our experiments show, that in addition to its use in enriching authentic endogenous DNA of organisms of interest, the selective enrichment of damaged DNA molecules can be a valuable tool in the discovery of ancient microbial taxa.. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.. - The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.. - from hominids has been exploited as a source of ancient microbiomes and analyzed in the context of diet and life- style changes [7–9], whereas coprolites have been used to investigate ecological interactions between animals and microorganisms [10]. - One of the most typical features of aDNA is the presence of uracils (Us) that ori- ginate from post-mortem deamination of cytosines (Cs), especially in single-stranded overhangs at molecule ends [11]. - However, the method seems to be specially suited to study micro- biomes, due to the inherent difficulty to authenticate their ancient origin. - a Relative enrichment (number of reads) in the U-enriched relative to the U-depleted fraction from Vindija Neandertal assigned to the phyla Actinobacteria and Proteobacteria, as well as to Homo sapiens. - b Relative enrichment (number of reads) in the U-enriched relative to the U-depleted fraction from Sidrón Neandertal assigned to the phyla Actinobacteria and Proteobacteria, as well as to Homo sapiens. - The size of the circle represents the amount of reads assigned to the node displayed in the tree or to any taxonomic level below it.. - Assignments to the phyla Actinobacteria and Proteobacteria, as well as the species Streptosporangium roseum and Homo sapiens are named in the taxonomic tree. - from Vin- dija Cave, Croatia, this proportion increased in the Uracil-enriched fraction [15]. - To explore this effect further, we re-analyzed the previously generated Nean- derthal sequence data from both sites by performing taxonomic binning of reads derived from the U-depleted and U-enriched fractions, instead of aligning them only to the human reference genome, as had been done pre- viously. - Reads aligning to the two most abundant bacter- ial phyla (Actinobacteria and Proteobacteria) from the Vindija Neandertals were enriched in the U-depleted fraction, while hominid reads were enriched in the U- enriched fraction (Fig. - In contrast, in reads obtained from the El Sidrón Neandertals, we found enrichment of both hominid and Actinobacteria reads in the U-enriched fraction, whereas Proteobacteria reads were enriched in the U-depleted fraction (Fig. - 1c), which showed almost 50% deamination at the first base in the U- enriched fraction (Fig. - illustrates how U-selection permits distinguishing be- tween ancient bacteria enriched in the U-enriched frac- tion and more recent colonizers enriched in the U- depleted fraction.. - U-selection suc- cessfully enriched for deaminated molecules in all plant samples, as shown by the much higher levels of deamin- ation present in the U-enriched fraction compared with the dsDNA libraries and the U-depleted fraction (Fig.. - The plant samples showed substantial variation in the content of endogenous DNA (2.8–91. - The inability to taxonomically assign the vast majority of reads from sam- ples with low endogenous DNA reflects the incomplete- ness of the reference database compared to the diversity of the microbiomes in those samples. - This is reflected in the higher percentage of reads mapped and assigned from the dsDNA library compared with shorter reads derived from both the U-depleted and U-enriched fraction (Figures S1C,D and S2A-B). - Given the low compositional complexity of microor- ganisms in the samples included in our proof-of- principle experiment, instead of centering our analyses on the compositional assessment of microbial communi- ties, we investigated in detail samples in which a specific Table 1 Provenance of herbarium specimens and archaeological remains. - We identified a large number of reads that were assigned to the bacter- ium Pantoea vagans in a potato (Solanum tuberosum) and a maize (Zea mays) sample (Fig. - vagans with pub- licly available genomes using single nucleotide polymor- phisms (SNPs) ascertained in these modern samples, and genotyped in the U-depleted fraction of either speci- men. - In a potato sample where the pathogenic oomycete Phytophthora infestans was previously identified [6], we found a large portion of reads assigned to the bacterial genus Pseudomonas. - While most reads were classified only to the genus level, some reads were assigned to ei- ther the species Pseudomonas syringae or Pseudomonas rhizosphaerae in different proportions (Figure S3). - Therefore, we performed de novo assembly using reads assigned to the genus Pseudomonas, aiming to generate longer sequences (con- tigs) that allow more detailed taxonomic classification.. - We aligned the contigs to the reference genomes of P.. - 3 Characterization of the bacterium Pantoea vagans identified in Zea mays and Solanum tuberosum samples. - The size of the circle represents the amount of reads assigned to the node displayed in the tree or to any taxonomic level below it. - found different k-mer coverage distributions in contigs aligning uniquely to either genome (Figure S5), an ob- servation that reinforced our confidence in the presence of the two Pseudomonas species in this sample. - Due to the high level of sequence divergence between the Pseudomonas in our sample and the reference genomes present in the database, it is difficult to assess typical de- amination patterns in the dsDNA library (Fig. - vagans, U- selection increases the fraction of molecules carrying a ter- minal C-to-T substitution at the 5′-end 2–3 fold over the li- brary without enrichment, relative to the total number of molecules sequenced. - Reads were aligned to the full non-redundant NCBI nucleo- tide collection (nt) database (downloaded January 2015) using MALT (version in BlastN mode. - The reads were assigned to the NCBI taxonomy using a lowest common ancestor algorithm [37].. - All types of nucleotide substitutions relative to the refer- ence genome were calculated per library using mapDam- age 2.0 (v. - vagans was detected in the metagenomic screening. - The libraries were mapped to the P. - These assemblies were aligned to the reference genome using minimap2 (version 2.10-r764, [44]) and its “asm20”. - strains in a Solanum tuberosum historic herbarium sample, we extracted from this library all reads that were taxonomically assigned to the Pseudomonas genus or to inferior taxonomic levels within it. - Supplementary information accompanies this paper at https://doi.org/10.. - Correlation of the percentage of mapped reads between the U-enriched and the non-selected library D. - Correlation of the percentage of taxonomically assigned reads between the U-enriched and the non-selected library E. - 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