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1) Product Images from "Ancient Clostridium DNA and variants of tetanus neurotoxins associated with human archaeological remains"
Article Title: Ancient Clostridium DNA and variants of tetanus neurotoxins associated with human archaeological remains
Figure Legend Snippet: Petabase-scale screen of the NCBI sequence read archive reveals C. tetani related genomes in ancient human archeological samples. ( A ) Analysis of 43,620 samples from the NCBI sequence read archive. Each sample is depicted according to its C. tetani k -mer abundance (y-axis) versus the overall dataset size (x-axis). A threshold was used to distinguish samples with high detected C. tetani DNA content, and these data points are colored by sample origin: modern C. tetani genomes (red), non-human (light blue), modern human (blue), ancient human (black). The pie chart displays a breakdown of identified SRA samples with a high abundance of C. tetani DNA signatures. ( B ) Geographical locations and timeline of ancient DNA samples. The 76 ancient DNA datasets are associated with 38 distinct samples (BioSample IDs), which are represented as individual data points. Four samples lack date information and are absent from (B). ( C ) C. tetani chromosomal coverage and; ( D ) plasmid coverage detected for 38 a ncient DNA associated c lostridial m etagenome- a ssembled g enomes (acMAGs) using the C. tetani E88 genome as a reference. Samples are numbered in order of their average percent sequence identity to the reference (E88) strain, from ‘1’ (closest to reference) to ‘38’ (most dissimilar from reference). See table S2 for information on associated BioSample IDs and sample names. The tent and colT genes are indicated on the plasmid in red and blue, respectively. ( E ) Average persample coverage of C. tetani chromosome, plasmid, and key virulence genes, tent and colT . Also shown is the estimated completeness and contamination of C. tetani related MAGs assembled from aDNA samples. MAG quality estimates were performed using CheckM.
Techniques Used: Sequencing, Ancient DNA Assay, Plasmid Preparation
Figure Legend Snippet: Phylogenetic analysis reveals known and novel lineages of C. tetani in ancient DNA samples, as well as a previously unidentified Clostridium species (“X”). ( A ) Whole genome phylogenetic tree of acMAGs from ancient samples and modern C. tetani genomes along with previously labeled phylogenetic lineages. Novel lineages are labeled “X” and “Y”, which are phylogenetically distinct from existing C. tetani genomes. ( B) Geographic clustering of newly identified lineage 1H acMAGs in ancient samples from the Americas, and ( C ) of newly identified clade X species in ancient samples from Europe. ( D ) Average nucleotide identity (ANI) between clade X MAGs recovered from ancient DNA and genomes of modern Clostridium species. Clade X MAGs show the highest ANI to C. tetani and C. cochlearium at a level that is sufficient to classify them as a novel Clostridium species. Note that one sample (Vac-Mummy-Tissue) was removed due to insufficient data required for fastANI. See table S10 for ANI values and genome IDs. ( E ) Distributions of damage levels for acMAGs from each phylogenetic group.
Techniques Used: Ancient DNA Assay, Labeling
Figure Legend Snippet: C. tetani DNA from a subset of ancient samples show hallmarks of ancient DNA. ( A ) MapDamage misincorporation plots for five acMAGs displaying the highest damage levels. The plot shows the frequency of C→T (red) and G→A (blue) misincorporations at the first and last 25 bases of sequence fragments. Increased misincorporation frequency at the edges of reads is characteristic of ancient DNA, and this pattern is not observed in a representative modern C. tetani genomic dataset ( B ). ( C ) Correlation between damage levels of acMAGs and corresponding human mtDNA from the same sample.
Techniques Used: Ancient DNA Assay, Sequencing
Figure Legend Snippet: Analysis and experimental testing of a novel TeNT lineage identified from ancient DNA. ( A ) Maximum-likelihood phylogenetic tree of tent genes including novel tent sequences assembled from ancient DNA samples and a non-redundant set of tent sequences from existing strains in which duplicates have been removed (see Methods for details). The phylogeny has been subdivided into four subgroups. Sequences are labeled according to sample followed by their associated clade in the genome-based tree ( Fig. 3A ), except for the Barcelona-3031-Tooth sequence (*) as it fell below the coverage threshold. ( B ) Visualization of tent sequence variation, with vertical bars representing nucleotide substitutions found uniquely in tent sequences from ancient DNA samples. On the right, a barplot is shown that indicates the number of unique substitutions found in each sequence, highlighting the uniqueness of subgroup 2. ( C ) Structural model of TeNT/Chinchorro indicating all of its unique amino acid substitutions, which are not observed in modern TeNT sequences. Also shown is a segment of the translated alignment for a specific N-terminal region of the TeNT protein (residues 141-149, uniprot ID P04958). This sub-alignment illustrates a segment containing a high density of unique amino acid substitutions, four of which are shared in TeNT/El-Yaral and TeNT/Chinchorro. ( D ) MapDamage analysis of the tent /Chinchorro gene, and associated C. tetani contigs and mtDNA from the Chinchorro-Mummy-Bone sample. ( E ) Cultured rat cortical neurons were exposed to full-length toxins in culture medium at indicated concentration for 12 hrs. Cell lysates were analyzed by immunoblot. WT TeNT and TeNT/Chinchorro (“ch”) showed similar levels of activity in cleaving VAMP2 in neurons. ( F - G ), Full-length toxins ligated by sortase reaction were injected into the gastrocnemius muscles of the right hind limb of mice. Extent of muscle rigidity was monitored and scored for 4 days (means ± se; n=3). TeNT/Chinchorro (“ch”) induced typical spastic paralysis and showed a potency similar to WT TeNT.
Techniques Used: Ancient DNA Assay, Labeling, Sequencing, Cell Culture, Concentration Assay, Activity Assay, Injection, Mouse Assay