Tag: Gut

Choose This Year’s Metagenomics Award Winner

Posted on September 3, 2019 by - ProxiMeta

Congratulations to Dr. Ben Tully on winning this year’s Project ProxiMeta: 2019 Metagenomics Award! Read more about his project, 4. The Complete Hydrothermal Microbial Metal Metabolism

This summer, researchers from across the U.S. sent in short proposals for a chance to win a full-service ProxiMeta™ microbiome workup for a sample of their choice. ProxiMeta combines shotgun metagenomics with in vivo proximity ligation (Hi-C) and necessary bioinformatic tools to help researchers assemble high-quality microbial genomes directly from complex microbiome samples.

 

 

HOW TO VOTE

Each project was assessed by a panel of scientists for scientific merit, novelty, impact, and feasibility, and four finalists were selected. Cast your vote on Twitter for your favorite project.

 

 

THE FINALISTS

1. The Gut Microbiome as a Risk Factor for Arsenic-Induced Cancer

Twitter Name: Gut & As-Induced Cancer

It is estimated that ~200 million people worldwide are exposed to arsenic concentrations exceeding current safety standards. Our collaborators have recently demonstrated that mice and human microbiomes can protect mice from arsenic toxicity. While human stool supplementation fully restores protection to arsenic in germ-free mice, researchers were only able to isolate one microbe, Faecalibacterium prausnitzii, that successfully conferred protection to both parent and infant mice. These results are huge because arsenic poses the highest lifetime risk for developing cancer in humans.We will investigate the role of arsenic-transforming bacteria within the gastrointestinal (GI) microbiome as another possible risk factor.

In nature, arsenic-reducing microorganisms are well known for their ability to generate more toxic arsenic products called arsenites, which are typically formed in anaerobic environments like the gut. Past research indicates that ingested arsenic may also be transformed into the toxic product arsenite by gut microbes thus increasing the risk for the host. On the other hand, arsenite-oxidizing microbes may also provide a benefit to the host by lowering arsenite concentrations. The ability of the microbiome to transform arsenic is determined by its genetic composition, therefore ProxiMeta sequencing technology will allow us to immediately analyze our collaborators rodent stool samples for genetic clues regarding this mysterious protection. Our project goals are to expand on this knowledge by: (1) characterizing the genetic basis for protection to arsenic provided by the microbiome (2) identifying, and then isolating, the bacteria-harboring arsenic transforming genes involved in protection.

We predict that differences in the gut metagenome composition will explain the incidences in arsenic susceptibility within a population or even at the family level. This project will provide important insight regarding how gut microbes contribute to cancer and may lead to novel therapies and probiotics that could target the microbiome of arsenic-exposed individuals.

2. Evaluating Antimicrobial Resistance in Backyard Poultry Environments

Twitter: AMR in Backyard Poultry

Approximately 13 million rural, urban, and suburban US residents reported owning backyard poultry (BYP) in 2014, and interest in BYP ownership is nearly four times that amount. BYP ownership has risen recently due to product quality, public health, ethical, and animal welfare concerns of commercial operations. However, BYP ownership and disease treatment is largely under-regulated, unlike commercial poultry production. Lack of regulation poses public health concerns of transmission of antimicrobial resistant (AMR) bacteria, such as AMR strains of Salmonella, Mycoplasma gallisepticum, and Escherichia coli commonly associated with BYP. BYP owners (2014 survey) were largely uninformed about poultry diseases and treatments but were interested in learning more on disease management.

The combination of a lack of regulation and public information warrants further research into the bacterial communities of BYP and their environments. Cloacal and environmental swabs were collected as part of a 2018 citizen science study where BYP owners reported current and historical poultry antibiotic usage. We propose to conduct shotgun metagenomic sequencing and proximity ligation using the ProxiMeta platform, allowing for increased detection of full-length AMR gene alleles compared to that revealed by short-read sequencing. The combination of PacBio reads with HiC intercontig ligation analysis allows for identification of potential gene transfer events of AMR genes within communities and potential dissemination throughout the environment.

This analysis is especially important considering the public health concerns of AMR persistence in backyard environments. Additionally, investigation of lytic and prophage presence would allow investigation of phage-mediated bacterial regulation that would not be possible with short-read sequencing alone. ProxiMeta analysis of these samples would provide the most comprehensive insight of AMR presences and persistence in BYP environments to date. These findings will be critical for new regulation and disease management for the increasing number of BYP flocks, which currently pose a potential health risk.

3. Unraveling the Metagenomics of Contamination

Twitter: Steel Site Contamination

We propose a metagenome characterization of contaminated Munger Landing sediment located in the St. Louis River, Duluth, MN USA. Seasonal samples are already collected and stored; of which one will be sequenced. Munger landing, is located downstream from the U.S. Steel Superfund site and contaminants include PAHs, dioxins, PCBs, and heavy metals.

Soil condition is integral to high productivity and ecosystem balance at all trophic levels. Human activities erode soil condition through agriculture, mining, sewage outflows and/or chemical/waste disposal into waterways. These practices alter the chemical structure of the soil and break down the microbial community processes responsible for ensuring the balance of biogeochemical cycling patterns in the soil. We hypothesize the activity of these pathways involved in cycling of nitrogen, phosphorus and carbon are altered in contaminated soil systems.

Metagenomic profiling of Munger Landing will provide data to examine microbes, metabolic pathways, and contaminant-processing genes present in the community that can be characterized further using qRTPCR. This project will be presented within a community college microbiology course module. Curriculum utilizing real-world data and the sequencing technology from Phase Genomics will teach students experimental design, troubleshooting, hypothesis testing, data analysis and how to communicate the broader impacts of a study to society, the field of environmental microbiology or conservation.

In the future, this data will assist in designing a longitudinal metagenomic and metatranscriptomic study to assess the ability of remediation to ‘recover’ bacterial community function at the Munger Landing site; slated to start in 2020-2021 as compared to two uncontaminated control sites. Ten sites, slated for remediation, have been identified as having high chemical and heavy metal contamination for the St. Louis River Estuary. The Munger Landing project will establish a workflow that can be applied to other contaminated sites.

4. The Complete Hydrothermal Microbial Metal Metabolism

Twitter: Hydrothermal Microbiome

Hydrothermal vents replenish the oceans with much-needed micronutrients, spewing iron, magnesium, nickel, and other metals from the earth’s crust. These metal micronutrients are used as biological cofactors for organisms throughout the marine food chain. Boiling, sterile hydrothermal fluids quickly cool and are colonized by highly specialized microorganisms that begin to cycle the metal species mixing with the seawater. Though regularly sampled, rarely have hydrothermal plumes been tracked through the water column to establish how microbial colonization occurs through time and space. We lack understanding regarding the replicability of colonization to what extent stochastic processes shape microbial community structure.

While on station at the East Pacific Rise hydrothermal vent field, size-fractionated samples (0.2, 3.0 and 5.0-μm) were collected in the hydrothermal plume emanating from Bio Vent. Samples fluids were collected from the source through the first 1-km of dispersal – the key distance for colonization – and this effort was repeated over the course of 10-days – to determine the replicability of natural colonization events. The application of standard metagenomics sequencing and microbial genome reconstruction through binning would provide novel insight into the cycling of metals within the plume but the use of cross-linked DNA techniques would deliver an unprecedented understanding of how strain diversity impacts colonization and how microbes interact with extrachromosomal elements in the environment.

While some microbes are poised to take advantage of reduced metal species for lithotrophic growth, microbes from the water column that become entrained in the plume will need metal-resistance adaptations to alleviate stress from the elevated metal concentrations present. Metal-resistance genes dispersed through the viral and plasmid pools are essential elements for understanding the functioning of the microbial community in this globally important source of metals to the oceans and effective interpretation of the community can only be achieved through cross-linked DNA metagenomic techniques.

*All finalists projects are owned by verified researchers at U.S. academic institutions.

 

RESOURCES

 

Hi-C solves the problem of linking plasmids to hosts in microbiome samples

Posted on August 8, 2018 by - ProxiMeta

Plasmids are hard!

Plasmids are an important part of microbial biology. Plasmid-borne genes can have serious public health consequences by conferring virulence traits or resistance to antibiotic drugs, and can be readily shared among bacterial cells through cell-cell conjugation or other means. In principle, any gene that gives bacterial cells a selective advantage is likely to be shared via plasmids among related cells. For example, so-called “epidemic resistance plasmids” have been instrumental in the rise of multi-drug resistance in pathogenic E. coli and Klebsiella pneumoniae.

However, determining the bacterial hosts of any given plasmid in a sample can be difficult. The classic approach is to isolate host and plasmid together and culture them in the lab. However, in complex samples with numerous organisms, many of which cannot be cultured readily or even where culturing may alter the selection pressure on the organisms of interest, this approach is often impossible. Alternatives like statistical metagenomic approaches also have difficulty with plasmid-host association, as plasmids do not necessarily resemble their host genomes in either abundance or nucleotide composition and single-cell sequencing approaches are expensive and have a limited range of samples and species they can be used on.

Hi-C to the rescue

Fortunately, recent developments in genomic technology have yielded some novel tools that allow us to circumvent this limitation. Hi-C is a method that allows us to measure 3-dimensional distances between sequences inside intact cells and was originally developed to model 3D folding of genomes inside cells. These structural measurements include a clear signal about which sequences originated inside the same cell simply because the cell membrane generally prevents inter-cellular sequences from coming into contact. Hi-C therefore provides direct physical evidence of DNA sequences originating from the same cell.

Phase Genomics has developed the ProxiMeta™ Hi-C metagenome deconvolution method, which is specifically optimized for metagenomic applications (Figure 1). At Phase Genomics we use ProxiMeta Hi-C to reconstruct whole genomes from a variety of complex samples such as human fecal, wastewater, soil, and co-culture communities (for more information, see our paper about ProxiMeta).

 

Figure 1. Schematic of ProxiMeta Hi-C. (a) Hi-C crosslinking junctions will form only between sequences in the same cell. (b) Proximity-ligation creates chimeric Hi-C junctions between adjacent DNA molecules which can be directly observed by paired-end sequencing. (c) clustering methods can be used to infer the starting genomes based on the Hi-C junction information. Originally published here.

 

As a necessary part of their life cycle, plasmids need to pass through their bacterial host cells to replicate. Therefore, plasmids typically form Hi-C links to their host genomes simply by virtue of being inside the same cell as their host genome. So, to find the hosts of a given plasmid,  one only needs to find these plasmid-genome links. Our analysis of metagenomic Hi-C data bears this conclusion out repeatedly through multiple publications, as described below.

Hi-C links plasmids and hosts

A pair of early publications showed that using this method we could correctly associate several plasmids with their bacterial hosts in an artificial community using and early version of the Hi-C  method.

In our more recent paper, we have demonstrated that Hi-C links between plasmids and hosts in a complex human fecal sample link described plasmids to their known hosts. Excitingly, in a single experiment we can now assemble numerous novel microbial genomes, complete with plasmid content, from a complex sample with hundreds of different species present.

An exciting finding from this complex community is that we can directly visualize how plasmids are shared between bacteria in a community (Figure 2). Recall from above that the sharing and spread of plasmids is a serious problem in the epidemiology of antibiotic resistance and infectious disease. For example, the sequence marked with “*” in Figure 2 shows substantial similarity to a plasmid called pBUN24, in addition to other plasmids with unknown hosts. It is clear that this plasmid shows contacts with a variety of genome clusters corresponding to different organisms, suggesting that all of these organisms can act as hosts for this plasmid.

 

Figure 2. Heatmap representing quantitative Hi-C links between plasmids (columns) and genome clusters (rows) in a human fecal metagenome. For scale see top right key (blue=no contact). Columns where more than one cell shows signal are possible instances of plasmid sharing. All genome cluster rows are near-complete genomes, e.g. have >90% completeness and <10% redundancy according to CheckM analysis.

 

In a more recent collaboration with Mick Watson’s group at the Roslin Institute, we applied ProxiMeta Hi-C to the cow rumen microbiome, a very complex microbial community. In this peer-reviewed paper, we were able to not only discover scores of novel genomes in this community, but also to profile plasmid-genome linkages for these genomes. Thus, Hi-C linkages of plasmids to genomes are robust even to very high complexity of the community.

Looking to the future: Plasmid Biology conference and more.

We have multiple exciting ongoing collaborations using Hi-C to understand the host range and biology of plasmids and other mobile elements; the best is yet to come! To see several examples of our Hi-C technology applied to this problem, you need only read the abstracts for the 2018 Plasmid Biology conference, August 5-10 at the University of Washington in Seattle.

We will be writing more about the uses of ProxiMeta and metagenomic Hi-C on this blog in the future, so stay tuned.

Lil BUB Aids in Discovery of New Bacteria

Posted on August 1, 2018 by - Hi-C, Metagenomics, ProxiMeta, Random, Science

Published author, talk show host, movie star, musician, and philanthropist—Lil BUB has now also helped to discover novel microbial life living in her gut in collaboration with AnimalBiome, KittyBiome, and Phase Genomics. Enter to sequence your cat’s microbiome in our #Meowcrobiome twitter raffle!

 

We live in an era of discovery, especially as it relates to the microbiome and how microbial diversity influences our world, our health—and even our pet’s health. To better understand the microbial life of our feline friends, Lil BUB volunteered to sequence her gut microbiome. Thanks to a recent collaboration with AnimalBiome, KittyBiome, and Phase Genomics, Lil BUB helped discover 22 new microbes living in cats which, in time, could reveal new insights into cat health and happiness.

When KittyBiome started back in 2015 with an intent to understand the cat microbiome,  Lil BUB’s owner Mike “Dude” Bridavsky provided a sample of her poop to be analyzed. Because of Lil BUB and over 1,000 other cats, KittyBiome’s microbial census will help us identify what microbes are associated with healthy cats and work towards helping cats with Inflammatory Bowel Disease (IBD), diabetes and other ailments likely to be associated with the microbiome.

 

USING GENOMICS TO FIND MICROBES

Late last year, Phase Genomics offered to analyze samples from Lil BUB and another cat, Danny (belonging to Jennifer Gardy—a microbiologist at the University of British Columbia and science TV host), using our ProxiMeta™ Hi-C Metagenomic Deconvolution platform to obtain complete microbial genomes from their samples.  This method solves a huge problem in microbiome research—how to tell apart different species when their DNA is all mixed up in one sample (imagine a thousand jigsaw puzzles mixed together).

ProxiMeta Hi-C revealed about two hundred different species of microorganisms living in Lil BUB and Danny’s poop, many of which have never been seen before. The genome sequences of the microorganisms found in these samples were analyzed using our software and other microbiome analysis tools to measure the quality of the different assembled genomes and to see if those genomes matched any known microbes (Lil BUB’s and Danny’s data are available for free on our website). Without using our ProxiMeta Hi-C platform to extract these genomes, many of them would have been undetectable and gone unseen.

Lil BUB and Danny the Cat

Phase Genomics sequenced both Lil BUB (left) and Danny’s (right) poop samples.

 

OVER 20 NEW BACTERIAL GENOMES DISCOVERED

Lil BUB being heldTogether, Lil BUB and her buddy Danny carry 22 previously undescribed bacterial species in their guts.  Lil BUB’s poop sample had 13 species and Danny’s sample had 9 species that have never before been fully sequenced or characterized.

These new bacterial species mostly belong to the order Clostridiales, and the team is currently analyzing the genomes to better characterize them. This discovery will help continue to build a database that contains cat bacteria that are new to science, so we can better identify the contributions of the microbiome to various health conditions.

This cool discovery, made with the help of Lil BUB and Danny, highlights that there’s a  universe of undiscovered microbial life out there. If we found 22 potentially novel species in only two cats, just imagine what else is out there, and what the implications might be for new ways to support and improve the health of our pets.

 

WHO ARE OUR HERO CATS?

Lil BUB is a one of a kind critter, made famous on the Internet due to her adorable genetic anomalies. She is a “perma-kitten”, which means she will stay kitten-sized and maintain kitten-like features her entire life. She has an extreme case of dwarfism, which means her limbs are disproportionately small relative to the rest of her body. Her lower jaw is significantly shorter than her upper jaw, and her teeth never grew in so her tongue is always hanging out. Lil BUB is also a polydactyl cat, meaning she has extra toes – 22 toes total!  Lil BUB and Her Dude travel all over the country raising hundreds of thousands of dollars for animals in need.

Danny, an exotic shorthair with a face much like Grumpy Cat, is equally adorable.  He is the companion cat of one of KittyBiome’s original researchers, Jennifer Gardy, and was one of the very first cats to lend his poop profile to the KittyBiome initiative.  He is a very healthy cat and his microbial profile has helped us learn what a balanced gut in cats looks like.

WHAT’S NEXT?

Phase Genomics and AnimalBiome are eager to learn more about these newly-discovered bacterial species. They hope to work with the scientific community to analyze, identify, characterize and publish these genomes, starting with exploring their identities based on 16S rRNA and other marker genes.

HOW TO GET INVOLVED

  • Help characterize the new bacteria: If you know of a researcher, scientist or cat-lover who would like to help us, we are soliciting input on the analysis that needs to be done to properly characterize and publish these genomes. Participants who contribute in a substantive manner to the project will be co-authors on the publication. All data associated with the project will be deposited into publicly available databases and we will publish the findings in open access journals, so all pet lovers can read them. We will hold a raffle to award one lucky contributor a free Hi-C sample kit from Phase Genomics. If interested, contact us at team@animalbiome.com to learn more.
  • Name the new bacteria: We’re looking for input from the community on what we should name these 22 new bacteria, so if you have any fun ideas, please drop us an email at team@animalbiome.com. The format should follow standard practices of scientific nomenclature, so it should be constructed like this: “Clostridium _________.”
  • Submit your pet’s sample for genomic research: If you don’t win the raffle and still want your pet to contribute to scientific knowledge through the identification of new bacterial species, please contact us at team@animalbiome.com. We can provide you with the details and pricing involved for us to identify new species in your cat or dog through in depth analyses like we did for Lil BUB and Danny using the Hi-C approach pioneered by Phase Genomics, which would also result in a publication.

Improving databases of the microbiome of cats (and dogs) with new bacteria like this could help us learn more about how the gut microbiome helps support the digestive health of all pets.

ENTER YOUR CAT IN OUR TWITTER RAFFLE

Phase Genomics, AnimalBiome and KittyBiome are hosting a twitter raffle where you can enter to sequence your cat’s microbiome! All you have to do is go to either the Phase Genomics’ or AnimalBiome’s original tweet of this blog, retweet it with a picture and introduction of your cat with the hashtag #Meowcrobiome. On August 8th 2018, we will randomly draw one (1) winner whose cat poop will be scientifically analyzed by Phase Genomics with ProxiMeta Hi-C to search for novel microbes, and three (3) additional winners whose cat poop will receive a Kitty Kit to have their cat’s poop analyzed by Animal Biome to compare their cat’s gut to healthy cat guts.  Send in your cat’s poop, and you too can help discover new microbial life!

LIL BUB AND DANNY’S STORY FEATURED ON GEEKWIRE PODCAST

GeekWire discussed Lil BUB, Danny, and the new bacteria found in their poop in their weekly Week in Geek podcast. Check out the full podcast on their website (the segment begins around 22:58), or play just the segment about Lil BUB and Danny below.

 

 

Uncovering the microbiome: What will you do with metagenomics?

Posted on March 1, 2018 by - Hi-C, Metagenomics, ProxiMeta, Science

In this Nature Microbiology blog post, Mick Watson shares his journey into the depths of the rumen microbiome. Read more here to learn how Phase Genomics ProxiMeta Hi-C Metagenomic Deconvolution techniques are helping investigators advance their metagenomic research in complex samples. This study successfully assembled 913 genomes and will help to improve our understanding of the microbial population in cow rumen in an unprecedented way using these new metagenomics techniques. We look forward to seeing what else comes from Microbiome 2.0. and are proud to be a part of this impressive piece of work.

Hundreds of Genomes Isolated from Single Fecal Sample with Hi-C Kit

Posted on October 18, 2017 by - Hi-C, Metagenomics, ProxiMeta, Science

 

Hi-C Kit Microbiome

A Phase Genomics Hi-C kit for any sample type are now available!

Phase Genomics recently launched its ProxiMeta™ Hi-C metagenome deconvolution kit + software
product, enabling researchers to bring this powerful technology (previously only available through the ProxiMeta service) into their own labs. A new paper posted to biorxiv describes the results of employing ProxiMeta technology to deconvolute a human gut microbiome sample.

 

In the paper, ProxiMeta was used on a single human gut microbiome sample and isolated 252 individual microbial genomes or genome fragments, with 50 of these genomes meeting the “near-complete” threshold typically used as the standard according to the CheckM tool (>90% complete, <10% contaminated). Examining the tRNA and rRNA content of the genomes found 10 to meet “high-quality” and 75 to meet “medium-quality” thresholds. Additionally, 14 of the genomes represent near-complete assemblies of novel species or strains not found in RefSeq, showing that even after many years of research, there remain numerous unknown microbes in the human gut that are discoverable with new approaches.

 

ProxiMeta’s results were compared to those achieved with MaxBin, a common tool used to perform metagenomic binning based on heuristics such as shotgun read depth and tetranucleotide profiles. MaxBin was able to create 29 near-complete genomes (cf. 50 for ProxiMeta), with only 5 meeting high-quality (cf. 10) and 44 meeting medium-quality (cf. 75) thresholds based on tRNA and rRNA content. In terms of ability to construct similar sets of near-complete genomes, ProxiMeta and MaxBin constructed 27 of approximately the same genomes, with ProxiMeta constructing an additional 32 genomes that MaxBin did not, and MaxBin constructing 9 genomes that ProxiMeta did not. ProxiMeta’s assembled genomes also exhibited a much lower amount of contamination than MaxBin’s assembled genomes, with 43% of MaxBin’s assemblies exceeding the 10% contamination limit that is the typical standard for genome quality, compared to only 2% of ProxiMeta’s assemblies.

 

Other results unique to ProxiMeta include the discovery of near-complete genomes for 14 novel species or strains and various associations of plasmids with their hosts. Of the 14 novel genomes, 10 appear to be of the class Clostridia, a common group of gut microbes that are poorly characterized due to their difficulty to culture.  ProxiMeta also assigned 137 contigs containing plasmid content to a cluster and identified candidate plasmid sequences as being present across multiple, distantly related bacteria. For example, ProxiMeta placed a known megaplasmid into an assembly for Eubacterium eligens that included homologous plasmid sequences placed into several other genomes, suggesting either the presence of the megaplasmid into other species, or variants of the megaplasmid being found on other mobile elements spread through the metagenome.

 

The depth of the resulting data and results offers the opportunity to learn much more about this microbial niche and research continues to unlock new discoveries about this community. Phase Genomics is thrilled to be able to offer all researchers the same new power to dig deeper into their mixed samples than ever before, especially now with a product that puts the power of discovery in their hands.

 

To learn more about ordering our kits or services, just send us an email at info@phasegenomics.com