Daily Archives: September 9, 2023

Researchers have been focusing on the potential of DNA as a data storage medium due to its capacity to store vast amounts of information in a minuscule space.

In the form of DNA, nature shows how data can be stored in a space-saving and long-term manner. Wrzburgs chair of bioinformatics is developing DNA chips for computer technology.

The hereditary molecule DNA is renowned for its ability to store vast amounts of information over long periods of time in an incredibly small space. For a good ten years, scientists have therefore been pursuing the goal of developing DNA chips for computer technology, especially for the long-term archiving of data. Such chips would be superior to conventional silicon-based chips in terms of storage density, longevity, and sustainability.

Four recurring basic building blocks are found in a DNA strand. A specific sequence of these blocks can be used to encode information, just as nature does. To build a DNA chip, the correspondingly coded DNA must be synthesized and stabilized. If this works well, the information is preserved for a very long time researchers assume several thousand years. The information can be retrieved by automatically reading out and decoding the sequence of the four basic building blocks.

Information can be stored in the form of DNA on chips made of semiconducting nanocellulose. Light-controlled proteins read the information. Credit: Chair of Bioinformatics / University of Wrzburg

The fact that digital DNA data storage with high capacity and a long lifespan is feasible has been demonstrated several times in recent years, says Professor Thomas Dandekar, head of the Chair of Bioinformatics at Julius-Maximilians-Universitt (JMU) Wrzburg. But the storage costs are high, close to 400,000 US dollars per megabyte, and the information stored in the DNA can only be retrieved slowly. It takes hours to days, depending on the amount of data.

These challenges must be overcome to make DNA data storage more applicable and marketable. Suitable tools for this are light-controlled enzymes and protein network design software. Thomas Dandekar and his chair team members Aman Akash and Elena Bencurova discuss this in a recent review in the journal Trends in Biotechnology.

Dandekars team is convinced that DNA has a future as a data store. In the journal, the JMU researchers show how a combination of molecular biology, nanotechnology, novel polymers, electronics, and automation, coupled with systematic development, could make DNA data storage useful for everyday use possible in a few years.

At the JMU Biocentre, Dandekars team is developing DNA chips made of semiconducting, bacterially produced nanocellulose. With our proof of concept, we can show how current electronics and computer technology can be partially replaced by molecular biological components, says the professor. In this way, sustainability, full recyclability, and high robustness even against electromagnetic pulses or power failures could be achieved, but also a high storage density of up to one billion gigabytes per gram of DNA.

Thomas Dandekar rates the development of DNA chips as highly relevant: We will only last as a civilization in the longer term if we make the leap into this new type of sustainable computer technology combining molecular biology with electronics and new polymer technology.

What is important for humanity, he said, is to move to a circular economy in harmony with planetary boundaries and the environment. We need to achieve this in 20 to 30 years. Chip technology is an important example of this, but the sustainable technologies to produce chips without e-waste and environmental pollution are not yet mature. Our nanocellulose chip concept makes a valuable contribution to this. In the new paper, we critically examined our concept and advanced it further with current innovations from research.

Dandekars team is currently working on combining the DNA chips made of semiconducting nanocellulose even better with the designer enzymes they have developed. The enzymes also need to be further improved.

In this way, we want to achieve better and better control of the DNA storage medium and be able to store even more on it, but also save costs and thus step by step enable practical use as a storage medium in everyday life.

Reference: How to make DNA data storage more applicable by Aman Akash, Elena Bencurova and Thomas Dandekar, 15 August 2023, Trends in Biotechnology. DOI: 10.1016/j.tibtech.2023.07.006

The work described is financially supported by the German Research Foundation (DFG) and the Free State of Bavaria. Important cooperation partners are Sergey Shityakov, professor at the State University of Information Technologies, Mechanics and Optics (ITMO) in Saint Petersburg, Daniel Lopez, PhD, from the Universidad Autonoma de Madrid, and Dr. Gnter Roth, University of Freiburg and BioCopy GmbH (Emmendingen).

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DNA Chips: The Billion Gigabyte Storage Solution of Tomorrow - SciTechDaily

DNA's 3D shape its twisted ladders complexly curled into coils and loops and other features beyond its genetic code may influence where "hotspots" of cancer-causing mutations accumulate.

That's according to a new study of how "genomic topography" affects cancer mutations. Genomic topography broadly refers to elements of the genome beyond the sequence of molecules that make up DNA. That includes variations in how tightly our DNA is wound and which genes are "activated" in different cells.

The study, published in August in the journal Cell Reports, catalogs associations between topographical features of DNA and known patterns of cancer-causing mutations across several types of cancer. This granted the researchers new insight into some alcohol-related cancers, and in the future, the vast trove of data could help scientists prevent, understand and treat many different forms of cancer.

"It's the next layer of cataloging of the cancer-specific mutations," said Dr. Katerina Gurova, an associate professor of oncology at Roswell Park Comprehensive Cancer Institute who was not involved in the study. "But we still don't understand for the majority [of the mutations] why DNA topography plays this role or that role."

Related: Bizarre knotted DNA structures linked to cancer in mice

The study looked at mutations embedded within the complete genome sequences of more than 5,000 tumors across 40 cancer types. The team analyzed the influence of 516 topographical features over where these mutations cropped up in the genome.

Some of these features relate to when and where mutations appear during transcription, the process of translating DNA into RNA, which carries genetic information from DNA out into the cell. Others relate to proteins called histones, which DNA molecules wind around like a spool, and the structure of that wound-up DNA. Another feature is related to a protein called CTCF, which regulates the 3D structure of chromatin, the complex formed by DNA and histones. CTCF enables DNA to form into highly compact chromatin loops.

It's like "we have a library in every cell, but this library is organized in different manners," Gurova said, adding that these different types of organizational methods are what the researchers mean when they say "topographical" features.

The main goal of the study was to catalog associations between different mutation patterns and these DNA features, but the researchers made some interesting observations about specific cancers.

For instance, they discovered that several mutation patterns linked to alcohol consumption appear early in the process of cell replication, rather than later as most mutations do. This mutational pattern was seen in head and neck, esophageal and liver cancer cells. They also found that, when looking at a type of immune cell cancer, some mutations that result in the same changes to DNA's "letters" can nonetheless be linked to very different topographical features, suggesting they arise for different reasons.

The researchers made their data freely available through a database called COSMIC, which Gurova said might be useful for developing cancer treatments targeted to specific mutations.

That said, the study does have some limitations, including that the data on the topographical features were collected from a different set of patients than the data on the mutations in cancer cells, she said. So it's possible that the results would be somewhat different if data sets were collected from the same cells.

Future research might take the same approach to link other genetic conditions to topographical features of DNA, said Fulai Jin, an associate professor of genetics at Case Western Reserve University. And in the realm of cancer, Jin said future work could further look at patients of different sexes or patients who were exposed to different environments to see how these factors interact with cancer-causing mutations and DNA's topography.

And a major goal of future research will be to determine why the researchers found these particular associations, Gurova said. This would address the questions of why and how DNA's shape influences how cancer arises.

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DNA's 'topography' influences where cancer-causing mutations appear - Livescience.com

An international consortium of scientists, led by Jeff Kidd, Ph.D. of University of Michigan, Jennifer R. S. Meadows of Uppsala University in Sweden, and Elaine A. Ostrander, Ph.D. of the NIH National Human Genome Research Institute, is using an unprecedentedly large database of canine DNA to take an unbiased look at how our furry friends evolved into the various breeds we know and love.

A new paper, published in the journal Genome Biology, outlines what the Dog10K project discovered after sequencing the genomes of close to 2000 samples from 321 different breed dogs, wild dogs, coyotes, and wolves, and comparing them to one reference sample -- that of a German Shepherd named Mischka.

Analyzing more than 48 million pieces of genetic information, they discovered that each breed dog had around 3 million single nucleotide polymorphism differences. These SNPs or "snips" are what account for most of the genetic variation among people and dogs alike. They also found 26,000 deleted sequences that were present in the German Shepherd but not in the comparison breed and 14,000 that were in the compared breed but missing from Mischka's DNA.

"We did an analysis to see how similar the dogs were to each other, and it ended up that we could divide them into around 25 major groups that pretty much match up with what people would have expected based on breed origin, the dogs' type, size and coloration," said Kidd, a professor of Human Genetics and Computational Medicine and Bioinformatics at the U-M Medical School.

Most of the varying genes, he added, had to do with morphology, confirming that the breed differences were driven by how the dogs look.

Relative to dogs, wolves had around 14 percent more variation. And wild village dogs -- dogs that live amongst people in villages or cities but aren't kept as pets -- exhibited more genetic variation than breed dogs.

The data set, which was processed using the Great Lakes high-performing computing cluster at U-M, also revealed an unusual amount of retrogenes, a new gene that forms when RNA gets turned back into DNA and inserted back into the genome in a different spot. The study found 926 retrogenes, the most famous of which, says Kidd, is a retrogene called FGF4, which results in the short leg phenotype seen in dachshunds and corgis.

"Dogs tend to have an increased amount of retrogenes which have resulted in mutations that were selected for, that perhaps people found cute and bred more of," said Kidd. His lab is attempting to figure out why retrogenes and insertions happen so frequently in dogs.

One of the benefits of the Dog10K consortium is its size, which will enable researchers at U-M and elsewhere to examine the genetic underpinnings of other canine characteristics and even common diseases in dogs, such as cancer.

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Dog diversity unveiled by international DNA database - Science Daily

Editors note: The Cap Times has withheld many details about this crime in consideration of the victims privacy and the potential trauma for some readers; however, the story includes information about sexual violence that may still be difficult to read. Caution is advised. The National Sexual Assault Hotline provides 24/7 crisis counseling at online.rainn.org or 1-800-656-4673.

DNA from a man accused of viciously attacking and nearly killing a University of Wisconsin student last weekend contained the most identifiable match possible to evidence found at the crime scene, according to a criminal complaint.

Beyond the biological evidence, Madison police also used business and residential surveillance videos, a Fitchburg officers body cam footage and the suspects own statements about being a monster who saw red to conclude that Brandon A. Thompson stalked and then physically and sexually assaulted the woman sometime between 2:30 and 3 a.m. Sunday in a downtown neighborhood, the complaint shows.

Dane County prosecutors filed three felony charges against Thompson, 26, Thursday in the random attack after police arrested him Wednesday morning.

Thompson, who is a resident of Brooklyn, Wisconsin, was charged with first-degree sexual assault, first-degree reckless injury, and strangulation and suffocation. The woman attacked, whose name has not been released to the news media, was found in critical condition along the 500 block of West Wilson Street around 3:20 a.m. Sunday, according to the Madison Police Department incident report. She is in her 20s and is expected to survive despite suffering injuries so severe she was temporarily in a medically induced coma, according to prosecutors and an update from police on Tuesday.

Videos submitted by members of the public helped lead police to Thompson, who remained at the scene of the attack and told nearby residents that he found the injured victim, according to the criminal complaint obtained by the Cap Times.

Cash bail is set for $1 million and a preliminary hearing will take place on Sep. 25 in the Dane County Courthouse.

Police Sgt. Daniel Sherrick was the first on the scene, according to the complaint, and said the students condition was "one of the most horrifying things I've seen." She was unable to communicate to police when they arrived and was transported to UW Hospital with life-threatening injuries.

The student had multiple facial fractures, including a broken jaw and broken nose, a missing tooth and a traumatic brain injury and an MRI showed a small brain bleed. She needed eight stitches to repair her upper lip, and nurses in the hospitals emergency department confirmed the student was strangled and sexually assaulted.

The student is currently on a feeding tube and unable to provide a statement as she shows extreme signs of confusion when awake, the complaint said.

The UW student arrived at a friends apartment at 7:56 p.m. on Saturday and left between 2:16 a.m. and 2:18 a.m. Sunday, according to a witness statement described in the complaint. The witness told officers she texted the victim at 2:43 a.m. asking if she made it home safely but the text was never read or responded to. The witness stated the victim is not a person who partied or a drug user.

Initial police reports state that residents in the 500 block of West Wilson Street called 911 after being alerted by a male who identified himself as Brandon. One witness said she was in her house when she heard a male voice yell to her through an open window that a female needed help.

The witness said Thompson claimed he was out for a "high walk" when he came upon the victim and seemed concerned for her wellbeing, according to the complaint. When the witness realized the victim was covered in blood and called the police, Thompson appeared to become "antsy," claimed he did not want to be around police when he was high and left the scene.

A different witness at the scene said he noticed dried blood on the mans hands. The man, whom police later identified as Thompson, told the witnesses he had carried the students bloodied body to that site after finding her nearby in the street. Police investigators said they found blood only in the location where the woman was found by other witnesses, not in the street.

Neighborhood surveillance videos in the area captured a man matching Thompsons description walking behind the victim just prior to the assault, Madison Police Chief Shon Barnes said at a press conference Wednesday. That information was repeated in the complaint filed Thursday. The complaint says Thompson was later located and arrested at Meriter Hospital, although the report does not describe why he sought medical treatment.

Videos from the area also showed a man believed to be Thompson exiting a car parked at Brittingham Park not long before the attack, according to the complaint. The vehicle's description and plate number matched a car Fitchburg Police had pulled over a few hours before the assault for a registration violation. The Fitchburg body camera video showed Thompson, who was driving, wearing clothes that matched those of the man seen in surveillance videos before the assault.

During an interview with detectives, he made incriminating statements, saying he was mad and wanted to hit something, according to the complaint. Thompson also told police the victim "came across a monster" that night.

Thompson admitted to encountering a woman and saw red and didn't know what was going on, the complaint says. Thompson stated the next thing he remembered was the female on the ground in front of him. He also stated, I went into a rage, when I came to, she was on the ground. The only thing I remember is just hitting.

When questioned further about the sexual assault, Thompson said something like, "I don't remember it during the actual assault, I just kind of zoned out during the rage," according to the complaint. When detectives asked Thompson if he could have sexually assaulted the victim, he stated, "I could have."

DNA from a Forensic Nurse Examination of the victim is consistent with the profile of Brandon Thompson, the complaint says, with a probability of one in one quadrillion. The DNA analyst said one in one quadrillion is the highest probability that the Wisconsin State Crime Lab will identify.

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DNA from UW student attack 'one in one quadrillion' match to suspect - The Capital Times

TAG Heuer presents their Motorsports Experience in a popup outside of Ion Orchard. And you are invited to visit!

The two storey popup structure is at the open area outside Ion and will be open to the public daily from 10am to 9:30pm from now till 17 September, 2023. Max Verstappen and Sergio Perez will make an appearance at 5pm on September 14.

In their F1 partnership with Oracle Red Bull Racing, TAG Heuer will showcase the 2018 Red Bulls actual F1 car used in the season.

Also in the booth is a wall to test your reflexes, perhaps in a competition with your friends.

But also, as importantly for us watch enthusiasts, TAG Heuer will also display some of the iconic and historic Monaco timepieces to show the transformation over the years. On display, is a tribute to the first Monaco watch, sports the iconic shade Monaco blue and is skeletonised for the first time in its history. Also on display is the TAG Heuer Carrera collection, which is also closely linked to motorsports. It was first created in 1963 by TAG Heuers legendary former CEO Jack Heuer designed for professional drivers and sports-car enthusiasts.

And as the ultimate challenge, you can step into the shoes of a professional race car driver at the Singapore Circuit Race Simulator. The champion of this simulator will be invited to the TAG Heuer launch event which will be revealed when the countdown clock outside the popup hits 0.

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Invitation: TAG Heuer popup to showcase its DNA and motorsports - - Deployant

Identification and assembly of C. tetani-related genomes from aDNA samples

To explore the evolution and diversity of C. tetani, we performed a large-scale search of the entire NCBI Sequence Read Archive (SRA; 10,432,849 datasets from 291,458 studies totaling ~18 petabytes; June 8, 2021) for datasets potentially containing C. tetani DNA signatures. Since typical homology-based search methods (e.g., BLAST29) could not be applied at such a large scale, we used the recently developed Sequence Taxonomic Analysis Tool (STAT)30 to search the SRA and identified 136 sequencing datasets possessing the highest total C. tetani DNA content [k-mer abundance >23,000 reads, k=32 base pair fragments mapping to the C. tetani genome] (Fig.1a and Supplementary Data1). Our search identified 28 previously sequenced C. tetani genomes (which serve as positive controls), as well as 108 uncharacterized sequencing runs (79 of human origin) with high predicted levels of C. tetani DNA content. Unexpectedly, 76 (96.2%) of these are aDNA datasets collected from human archeological specimens (Fig.1a), with the remaining three datasets being from modern human gut microbiome samples.

a General bioinformatic workflow starting with the 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 natural log of the overall dataset size in megabases (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. The 38 aDNA samples predicted to contain C. tetani DNA were further analyzed as shown in the bioinformatic pipeline on the right. b Topdensity plot of the percentage identities of all BLAST local alignments detected between acBins and reference genomes including C. tetani, C. cochlearium, and other Clostridium spp. Bottomdensity plot of the checkM results for the 38 acBins including estimated completeness, contamination, and strain heterogeneity levels. Completeness and contamination levels are percentage values. c MapDamage damage rates (5 CT misincorporation frequency) for acBins (n=38 biologically independent samples) subdivided by UDG treatment [none (n=27), partial (n=5), and full (n=6)]. Also shown are the damage rates for modern C. tetani genomes (n=21 biologically independent samples). The boxplots depict the lower quartile, median, and upper quartile of the data, with whiskers extending to 1.5 times the interquartile range (IQR) above the third quartile or below the first quartile. d Damage plots for the top five acBins with the highest damage rates, and corresponding mtDNA damage plots. Shown is the frequency of CT (red) and GA (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. Source data for (ad) are provided as a Source Data file.

These 76 ancient DNA datasets are sequencing runs derived from 38 distinct archeological samples, which include tooth samples from aboriginal inhabitants of the Canary Islands from the 7th to 11th centuries CE31, tooth samples from the Sanganji Shell Mound of the Jomon in Japan (~1044 BCE)32, Egyptian mummy remains from ~1879 BCE to 53 CE33, and ancient Chilean Chinchorro mummy remains from ~3889 BCE34 (Supplementary Data2). The 38 aDNA samples vary in terms of sample type (31 tooth, 6 bone and 1 chest extract), burial practices (27 regular inhumation and 11 mummies), sequencing method (26 shotgun datasets and 12 bait-capture approaches), and DNA treatment (6 UDG-treated, 5 partial UDG-treated and 27 untreated samples), all of which needs to be considered for interpretation of downstream analysis (Supplementary Data2).

Although these archeological samples are of human origin, STAT analysis of the 38 DNA samples predicted a predominantly microbial composition (~90% median across samples, Supplementary Fig.1). The predominance of microbial DNA in ancient human tooth samples is expected and consistent with previous studies which have shown microbial DNA proportions as high as 9599%13,17,18,35. C. tetani-related DNA was consistently abundant among predicted microbial communities, detected at 13.8% average relative abundance (Supplementary Fig.1 and Supplementary Data3). A total of 85 species were detected at >= 2% abundance in at least one sample (Supplementary Data4). While 65 of these species have been associated with humans or animals, 20 species have an environment-specific origin, and provide an estimate of possible environmental microbial contamination that could aid in interpretation of results (Supplementary Data4, Supplementary Fig.2). Putative environment-specific microbes make up a low proportion of the microbially classified reads at levels <=10% for 33 samples, and <=5% for 24 samples (Supplementary Data5). The three samples with the highest estimated proportions of reads from putative environment-specific microbes were Tenerife-012-Tooth, Vc-Mummy-Tissue, and Tenerife-013-Tooth (Supplementary Data5). Also noteworthy is that M. tuberculosis and Y. pestis were detected (Supplementary Fig.1) in several datasets associated with bait-capture sequencing of M. tuberculosis and Y. pestis from archeological samples36,37,38.

To further explore the putative C. tetani in aDNA samples, we performed metagenome assembly using MEGAHIT39 for each individual sample and taxonomically classified assembled contigs using both Kaiju40 and BLAST29 to identify those mapping unambiguously to C. tetani and not other bacterial species (Supplementary Data6 and 7). A majority (73%) of the alignments between assembled contigs and reference C. tetani genomes had percentage identities exceeding 99% (Fig.1b). Ninety percent of the alignments had percentage identities exceeding 90%, suggesting that a large fraction of assembled contigs are highly similar to regions of modern C. tetani genomes. Based on mapping of reads to the C. tetani chromosome, the 38 samples had a 1 percent coverage ranging from 28 to 94% (mean of 78.3%) and a 5 coverage ranging from 9 to 93% (mean of 57.5%) (Supplementary Data2). A subset of 16 samples had a 1 C. tetani chromosome coverage exceeding 90%.

For each of the ancient DNA samples, we binned together all C. tetani-like contigs to result in 38 putative, ancient DNA-associated clostridial genome bins or acBins. We then performed QC analysis of each acBin using CheckM41 to estimate genome completeness and contamination (Fig.1b and Supplementary Data8). CheckM estimates genome completeness based on the detected presence of taxon-specific marker genes, and uses duplicated marker genes (if present) to estimate contamination and heterogeneity41. Eighteen acBins were more than 50% complete and 11 were more than 70% complete. Thirty-seven acBins had low (<10%) checkM contamination (Supplementary Data8). acBins with higher genome completeness were associated with datasets produced by shotgun sequencing rather than capture methods, as these datasets had higher levels of C. tetani DNA content (Supplementary Fig.3). We also examined the acBins for potential strain heterogeneity using two independent approaches: CheckM estimation (Supplementary Data8) as well as quantification of per-base heterogeneity from mapped reads (Supplementary Data2). These two metrics had a weak but significant correlation (r=0.38, P=0.019) (Supplementary Fig.4a). Five strains (Sanganji-A2-Tooth, Chinchorro-Mummy-Bone, SLC-France-Tooth, Karolva-Tooth, Chincha-UC12-24-Tooth) were identified as possessing higher estimated levels of strain variation, but all were below 6% (CheckM) and 1.1% (average base heterogeneity).

Using the tools MapDamage242 and pyDamage43, we then examined the 38 acBins for elevated CT misincorporation rates at the ends of molecules, a characteristic pattern of aDNA damage19,20. Since these patterns are known to be affected by UDG treatment, we examined damage rates separately for full UDG, partial UDG, and untreated samples (Fig.1c). As expected, we observed the highest damage rates in the untreated samples, and the lowest damage rates in the full UDG-treated samples, indicating that the damage rates have been suppressed in some samples by UDG treatment. The damage rates calculated by MapDamage and PyDamage were highly similar with a Pearson correlation of r=0.99 (Supplementary Data2). Damage plots for all samples are shown in Supplementary Fig.5 with additional data available in Supplementary Data9 and 10.

Overall, seven acBins possessed a damage rate (5 CT misincorporation rate) exceeding 10%, which is indicative of aDNA21 (top 5 shown in Fig.1d). In addition, all of the acBins except one (Chincha-UC12-12-Tooth) were verified by pyDamage as containing ancient contigs with q values<0.01 (Supplementary Data10). The highest damage rate (17.9%) occurred in the acBin from the Augsburg-Tooth sample, which is the third oldest sample in our dataset (~2253 BCE), despite this sample being partially UDG-treated (Fig.1d). As controls, evidence of ancient DNA damage was also observed in the corresponding human mitochondrial DNA (mtDNA) from the same ancient samples (Supplementary Fig.5 and Supplementary Data2), but not for modern C. tetani samples (Fig.1c). In addition, no damage was detected in the three human gut-derived C. tetani bins identified by our screen.

In general, we observed a significant correlation between damage rates of acBin DNA and corresponding human mtDNA from the same sample (R2=0.38, P=2.8E-03, two-sided Pearson) (Supplementary Fig.6). However, acBin damage rates were generally lower than the corresponding human mtDNA rates, especially for some samples (e.g., Tenerife-004, Tenerife-013, Chinchorro-Mummy-Bone) (Supplementary Figs.5 and6), which may suggest that a subset of the archeological samples have been colonized by C. tetani at later dates (see Discussion). Damage rates were higher for noncapture datasets as these generally received no UDG treatment (Supplementary Fig.7a), and higher for samples associated with regular inhumations than those from mummies (Supplementary Fig.7b). We also observed a significant correlation between acBin damage level and sample age, but only for mummy-derived samples (R2=0.50, P=0.014) (Supplementary Fig.7c). Together, these data suggest that a subset of the acBins display evidence of ancient DNA damage and are plausibly of an ancient origin.

To explore the phylogenetic relationships between the acBins and modern C. tetani strains, we first aligned their contigs to the reference C. tetani genome along with 41 existing, non-redundant C. tetani genomes10, and clustered the genomes to produce a dendrogram (Fig.2a). Five acBins were omitted due to extremely low (<1%) genome coverage (see Methods), which could result in phylogenetic artifacts. We also included C. cochlearium as an outgroup, as it is the closest known related species to C. tetani based on phylogenomic analysis of available genomes44,45. Assessment of the genome-wide alignment for potential recombination showed no difference in estimated recombination levels for acBins compared to modern C. tetani genomes (Supplementary Fig.8).

a Dendrogram depicting relationships of acBins from ancient samples with modern C. tetani genomes. Novel branches are labeled X and Y, which are phylogenetically distinct from existing C. tetani genomes. Shown on the right of the dendrogram are metadata and statistics associated with each acBin including the estimated date of the associated archeological sample. All metadata can be found in Supplementary Data2. b Geographic distribution of ancient DNA samples from which the 38 acBins were identified. Each sample is colored based on the acBin clustering pattern shown in (a). The global map was derived from the Natural Earth [https://www.naturalearthdata.com/] medium-scale data and plotted using the rnaturalearth and ggplot2 R packages. c SNP-based phylogenetic tree of a subset of acBins from lineage 1 and 2 showing high similarity and coverage to the C. tetani reference genome. See Supplementary Fig.9 for more details. Source data for (a, c) are provided as a Source Data file.

The genome-based dendrogram of the acBins and modern C. tetani strains (Fig.2a) matches the expected phylogenetic structure and contains all previously established C. tetani lineages10. Ultimately, the acBins can be subdivided into those that cluster clearly within existing C. tetani lineages 1 or 2 and those that do not, which we have labeled X (8 acBins) and Y (1 acBin). Visualization of the acBin samples on the world map revealed a tendency for geographical clustering among acBins from the same phylogenetic lineage (Fig.2b). For example, lineage 1H acBins originate from ancient samples collected in the Americas, whereas most lineage 2 acBins originate outside of the Americas, and most clade X samples originate in Europe (Fig.2b). Interestingly, some samples from the same region (e.g., Canary Island samples, and Egyptian samples) contain diverse C. tetani lineages, which may be influenced by several factors (see Discussion).

Twenty-four acBins fall within the C. tetani tree and possess average nucleotide identities (ANIs) of 96.4% to 99.7% to the E88 reference genome (Supplementary Data2), which is within the range considered to be the same species46. These include new members of clades 1B (1 acBin), 1F (1 acBin), 1H (9 acBins), and 2 (9 acBins), expanding the known genomic diversity of clade 1H which previously contained a single strain and clade 2 which previously contained five strains (Fig.2a). Four additional acBins clustered generally within clade 1 but outside of established sublineages (Fig.2a).

In addition, we used Parsnp47 to construct a more stringent, core SNP-based phylogeny from a reduced set of 11 acBins that aligned to the reference C. tetani genome and passed several criteria (see Methods) (Fig.2c and Supplementary Fig.9). Only acBins from established C. tetani lineages 1 and 2 passed these criteria, and their phylogenetic positioning is consistent with their clustering pattern (Fig.2a). The reads associated with the core SNP alignment also showed reduced per-base heterogeneity when mapped to contigs (Supplementary Fig.4b). Notably, acBins from the Sanganji, Tenerife, Chinchorro, and Chincha samples do not show evidence of branch shortening in the tree indicative of ancient genomes, and instead cluster with modern strains. These acBins tend to have higher rates of strain variation, which could affect branch lengths, or low damage rates potentially indicative of a more recent origin (Supplementary Data2).

We also assembled a novel strain of C. tetani from a human gut sample (SRR10479805) which phylogenetically clustered with strain NCTC539 (98.7% average nucleotide identity; Supplementary Data11) from lineage 1G. The other two identified human gut samples were removed from further analysis as they predominantly matched C. cochlearium based on BLAST analysis.

Nine acBins clustered outside of the C. tetani species clade. Eight of these cluster together as part of a divergent clade (labeled X) (Fig.2a). These samples span a large timeframe from ~2290 BCE to 1787 CE, are predominantly (7 of 8) of European origin (Fig.2b and Supplementary Fig.10), and come from variable burial contexts including single cave burials, cemeteries, mass graves and burial pits37,48,49,50,51,52,53 (Supplementary Data2). Two of the samples from sites in Latvia and France are from plague (Y. pestis) victims37,53, and another is from an individual with tuberculosis38. The highest quality clade X acBin is from sample Augsburg-Tooth (~2253 BCE), with 53.9% estimated completeness and 4.11% contamination (Supplementary Data8). Comparison of clade X acBins to other Clostridium species revealed that they are closer to C. tetani and C. cochlearium than any other Clostridium species available in the existing NCBI database, but are divergent enough to be considered a distinct species. On average, based on fastANI54 analysis of orthologous sequences54 Clade X genomes have 86.5+ 1.7% ANI to C. tetani strain E88, and 85.1+ 1.3% ANI to C. cochlearium (Supplementary Fig.11a and Supplementary Data12). Based on ANI analysis of the whole genome alignment, clade X genomes have 90.8+ 0.22% ANI to strain E88 (Supplementary Data2). These similarities were confirmed by analysis of BLAST alignment identities between clade X contigs and reference genomes (Supplementary Fig.11b). As in the genome-wide tree, individual marker genes (rpsL, rpsG, and recA) from clade X acBins also clustered as divergent branches distinct from C. tetani and C. cochlearium (Supplementary Figs.1214). Finally, we re-examined the damage patterns according to phylogenetic clade, and found that clade X genomes possess the highest mean damage; 6/8 clade X genomes have a damage level exceeding 5% and 3/8 exceed 10% (Supplementary Fig.7d and Supplementary Data2). These analyses suggest that clade X may represent a previously unidentified lineage of Clostridium, including members of ancient origin. We designated this group Clostridium sp. X.

One sample (GranCanaria-008-Tooth from the Canary Islands dated to ~935 CE) also formed a single divergent branch (labeled Y) clustering outside all other C. tetani genomes (Fig.2a). Based on CheckM analysis, this acBin is of moderate quality with 74% completeness, and 0.47% contamination (Supplementary Data8). A comparison of the GranCanaria-008-Tooth acBin to the NCBI genome database revealed that it is closely related to C. tetani and more distant to other available Clostridium genomes (Supplementary Data13). Based on fastANI54, it exhibits an ANI of 87.3% to C. tetani E88, and 85.1% to C. cochlearium, below the 95% threshold typically used for species assignment (Supplementary Data13). Based on ANI analysis of the whole genome alignment, it has a 91.2% ANI to strain E88 (Supplementary Data2). To further investigate the phylogenetic position of this species, we built gene-based phylogenies with ribosomal marker genes rpsL, rpsG and recA (see Supplementary Figs.1214). Each of these three genes support the GranCanaria-008-Tooth lineage as a divergent species distinct from C. tetani. The damage level for this acBin is relatively low (~4.0%), whereas its human mtDNA damage level is ~11.6% (Supplementary Fig.5). We designated this acBin Clostridium sp. Y.

We next carried out a comprehensive comparison of genome content and structure between the acBins and modern C. tetani strains. We first clustered protein-coding sequences from all modern genomes and acBins into a set of 3729 orthologous groups, and compared their presence/absence across all strains (see Methods and Supplementary Data14). Based on this analysis, we observed considerable overlap in gene content between the acBins versus the modern reference genomes, with the greatest overlap observed between acBins from C. tetani lineages (1 and 2) and the smallest overlap observed for Clostridium sp. X (Supplementary Fig.11c). For instance, plasmid genes from the E88 reference genome were on average detected in 61% of the most complete acBins from Fig.1c (comparable to 69% in modern C. tetani genomes), and only 35% of other acBins (Supplementary Data15). Twenty orthogroups from the E88 plasmid were found in all of these acBins, including the plasmid-specific genes repA, colT, and tent (Supplementary Data15). In addition to these genes, sporulation-related genes are also highly conserved across the most complete acBins. Of 80 identified sporulation-related genes present in strain E88, 52 of these were detected in 100% of the most complete acBins, and 69/80 were present at over 90% frequency (Supplementary Data16). Thus, we conclude that key C. tetani functions, including plasmid replication, collagen degradation, neurotoxin production, and sporulation, are conserved in a subset of acBins (i.e., those in Fig.1c) for which enough genomic data was available to assemble genomes with moderate-high completeness.

We then examined genome similarities by visualizing the alignment of each genome to the reference E88 chromosome and plasmid (Fig.3a). Several low-coverage acBins can be seen in C. tetani lineages 1 and 2 (Fig.3a), which is expected given their low completeness estimates (Fig.2a). However, the divergent GranCanaria-008-Tooth genome (branch Y) and Clostridium sp. X consistently have a low alignment coverage, similar to that of C. cochlearium (Fig.3a), which we suspected may be due in part to these species being more distantly related to C. tetani. Consistent with the idea that clade X represents a distinct species from C. tetani, we identified fourteen genes present in four or more clade X members and absent from all other C. tetani genomes. The genomic context of four of these genes (labeled by orthogroup) is shown in Supplementary Fig.15. Although these genes are unique to clade X, their surrounding genes are conserved in other C. tetani genomes, implying that genome rearrangements may have resulted in these genes being either gained in Clostridium sp. X or lost in C. tetani.

a Visualization of the chromosomal and plasmid multiple sequence alignment. Orthologous blocks are shown in black and the missing sequence is colored white. The reference gene locations are plotted above the alignments. b Gene neighborhoods surrounding the repA gene (left) and tent gene (right) in modern strains versus acBins. Selected unique differences identified in acBin gene neighborhoods are highlighted. The boxed region shows the assembled tent locus in two clade X acBins. Comparison reveals a putative deletion event in the clade X strains that has removed the majority of the tent gene along with five upstream genes, leaving behind conserved flanking regions. See Supplementary Fig.18 for more information. c Per-clade coverage of the tent gene normalized to the coverage of repA. The data include n=33 biologically independent samples, including acBins from clade 1 (n=3), 1B (n=1), 1F (n=1), 1H (n=8), 2 (n=9), X (n=7), Y (n=1), and acBins whose clade affiliation could not be determined (N.D., n=3). The coverage was calculated as the average depth of coverage based on mapped reads to each gene. The boxplots depict the lower quartile, median, and upper quartile of the data, with whiskers extending to 1.5 times the interquartile range (IQR) above the third quartile or below the first quartile. See Supplementary Fig.17 for the associated read pileups. Source data for (ac) are provided as a Source Data file.

To examine differences in plasmid gene content and structure directly, we then compared the gene neighborhoods surrounding the plasmid-marker genes repA and colT (Fig.3b, expanded data shown in Supplementary Fig.16). In several acBins from C. tetani lineages 1 or 2, the gene neighborhoods surrounding these genes are similar to that in modern strains (Supplementary Fig.16). However, particularly in Clostridium sp. X and Y, we identified unique gene clusters distinct from those in modern strains. For example, in two Clostridium sp. X genomes and the Clostridium sp. Y genome, we identified a conserved toxin/antitoxin pair and a phage integrase flanking the repA gene (Fig.3b). In Clostridium sp. Y, these genes were found on an assembled 53.6kb contig (SAMEA104281224_k141_98912), which was indeed predicted as a plasmid by the RFplasmid program with a 70.4% vote using the Clostridium model55. We also observed a unique gene arrangement surrounding colT that is conserved in two clade X genomes (Supplementary Fig.16). Additional differences were identified in a few lineage 2 acBins; for example, Tenerife-004-Tooth contains unique genes neighboring repA, and the Tenerife-013-Tooth acBin uniquely encodes the repA gene adjacent to its tent and tetR gene (Fig.3b).

We then performed a detailed comparison of the plasmid-encoded neurotoxin gene, tent, and its gene neighborhood (where possible) across the strains. As shown in Fig.3a as well as based on mapped read coverage to these regions (Fig.3c, Supplementary Fig.17, and Supplementary Data17), the tent gene was detected at a relatively high depth of coverage in acBins from C. tetani lineages 1 and 2. The tent gene neighborhood structure from lineage 1 or 2 acBin strains is also similar or identical to that in modern strains, with the exception of Tenerife-013-Tooth (as it encodes the repA gene nearby) (Fig.3b).

However, in the acBins from lineage X and Y, the tent gene was either missing or was fragmented, suggesting a possible gene loss or pseudogenization event (Fig.3c). This pattern can be seen clearly in read coverage plots (Supplementary Fig.17) and when normalizing tent depth of coverage to that of the plasmid-marker gene, repA (Fig.3c). The tent locus in the two Clostridium sp. X genomes for which assembly data is available over this region appears to have undergone a deletion event resulting in the deletion of over 90% of the tent sequence as well as 3 neighboring genes (Fig.3b and Supplementary Fig.18). This analysis further supports the idea that the tent fragment may be a nonfunctional pseudogene in these clade X strains.

Ultimately, our comparative genomic analysis of gene content and neighborhood structure demonstrates that the plasmids in several of the ancient samples (particularly those of Clostridium sp. X) are distinct from modern C. tetani plasmids, while the plasmids of acBins from lineages 1 and 2 are similar to those of existing C. tetani strains. This reinforces our earlier phylogenetic analysis indicating that clade X and branch Y represent new Clostridium species that are closely related to but distinct from C. tetani.

Given the considerable scientific and biomedical importance of clostridial neurotoxins, we next focused on tent and reconstructed a total of 18 tent gene sequences (all from lineage 1 and 2 acBins) from aDNA using a sensitive variant calling pipeline (see Methods). Six tent sequences have complete coverage, and 12 have 75-99.9% coverage (Supplementary Data18). Six partial tent sequences were also reconstructed but had lower average depth of coverage as shown in the read pileups (Supplementary Fig.17). Four of the reconstructed tent sequences are identical to modern tent sequences, while 14 (including two identical sequences) are novel tent variants with 99.199.9% nucleotide identity to modern tent, comparable to the variation seen among modern tent genes (98.6100%). We then built a phylogeny including the 18 tent genes from aDNA and all 12 modern tent sequences (Fig.4a). The tent genes clustered into three subgroups with modern and aDNA-associated tent genes found in subgroups 1 and 2, and aDNA-associated tent genes forming a novel subgroup 3 (Fig.4a). All three of the tent sequences in the novel tent subgroup 3 are from clade 1H aDNA strains.

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 three subgroups. Sequences are labeled according to sample followed by their associated clade in the genome-based tree (Fig.2a), 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 3. 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 141149, 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 the indicated concentration for 12h. Cell lysates were analyzed by immunoblot, and the image shown is a representative of four independent experiments. WT TeNT (uniprot accession # P04958) 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. The extent of muscle rigidity was monitored and scored for 4 days (meanss.e.; n=3 per group, 9 total). TeNT/Chinchorro (ch) induced typical spastic paralysis and showed a potency similar to WT TeNT. Source data for (a, b, d, e, g) are provided as a Source Data file.

We then visualized the uniqueness of aDNA-associated tent genes by mapping nucleotide substitutions onto the phylogeny (Fig.4b and Supplementary Fig.19), and focusing on unique tent substitutions found only in ancient samples and not in modern tent sequences. We identified a total of 46 such substitutions that are completely unique to one or more aDNA-associated tent genes (Fig.4b, Supplementary Fig.20, and Supplementary Data19), which were statistically supported by the stringent variant calling pipeline (Supplementary Data20). The largest number of unique substitutions occurred in tent/Chinchorro from tent subgroup 3, which is the oldest sample in our dataset (Chinchorro mummy bone, ~3889 BCE). tent/Chinchorro possesses 18 unique substitutions not found in modern tent, and 12 of these are shared with tent/El-Yaral and 10 with tent/Chiribaya (Fig.4b). The three associated acBins also cluster as neighbors in the phylogenomic tree (Fig.2a), and the three associated archeological samples originate from a similar geographic region in Peru and Chile (Supplementary Fig.21). These shared patterns suggest a common evolutionary origin for these C. tetani strains and their unique neurotoxin genes and highlight tent subgroup 3 as a distinct group of tent variants exclusive to ancient samples (Fig.4a).

We then focused on tent/Chinchorro as a representative sequence of this group as its full-length gene sequence could be completely assembled. The 18 unique substitutions present in the tent/Chinchorro gene result in 12 unique amino acid substitutions, absent from modern TeNT protein sequences (L140S, E141K, P144T, S145N, A147T, T148P, T149I, P445T, P531Q, V653I, V806I, H924R) (Supplementary Data21). Seven of these substitutions are spatially clustered within a surface loop on the TeNT structure56 and represent a potential mutation hot spot (Fig.4c). Interestingly, 7/12 amino acid substitutions found in TeNT/Chinchorro are also shared with TeNT/El-Yaral and 5/12 are shared with TeNT/Chiribaya (Supplementary Data21). As highlighted in Fig.4c, TeNT/Chinchorro and TeNT/El-Yaral share a divergent 9-aa segment (amino acids 141149 in TeNT, P04958) that is distinct from all other TeNT sequences. Reads mapping to the tent/Chinchorro gene show a low damage level similar to that seen in the C. tetani contigs from this sample, and their damage pattern is weaker than the corresponding damage pattern from the associated human mitochondrial DNA (Fig.4d).

Given the phylogenetic novelty and unique pattern of substitutions observed for the tent/Chinchorro gene, we sought to determine whether it encodes an active tetanus neurotoxin. For biosafety reasons, we avoided the production of a tent/Chinchorro gene construct and instead used sortase-mediated ligation to produce limited quantities of full-length protein toxin (Supplementary Fig.22), as done previously for other neurotoxins57,58. This involved producing two recombinant proteins in E. coli, one constituting the N-terminal fragment and another containing the C-terminal fragment of TeNT/Chinchorro, and then ligating these together using sortase. The resulting full-length TeNT/Chinchorro protein cleaved the canonical TeNT substrate, VAMP2, in cultured rat cortical neurons (Fig.4e), and can be neutralized with anti-TeNT anti-sera (Supplementary Fig.22). TeNT/Chinchorro induced spastic paralysis in vivo in mice when injected to the hind leg muscle, which displayed a classic tetanus-like phenotype identical to that seen for wild-type TeNT (Fig.4f). Quantification of muscle rigidity following TeNT and TeNT/Chinchorro exposure demonstrated that TeNT/Chinchorro exhibits a potency that is indistinguishable from TeNT (Fig.4g). Together, these data demonstrate that the reconstructed tent/Chinchorro gene encodes an active and highly potent TeNT variant.

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