Tag: Metagenomics

A Year in Review: 2024

Posted on December 19, 2024 by - Cytogenomics, Genomics, Hi-C, Metagenomics

image of microscope and dna with Phase Genomics logo

 

Phase Genomics continues to pioneer genomic innovation, driving advancements in human health and life science research over another impactful year. Our efforts range from developing novel tools for detecting chromosomal abnormalities to managing the world’s most extensive phage-host interaction atlas, thereby accelerating genomic research and promoting a healthier future.

Our ultra-long-range sequencing technology is fostering advancements across a wide array of research applications, both at Phase Genomics and in laboratories worldwide. Utilizing our microbial platform, we are driving transformative discovery in metagenomics and ecology while making bounds in human health with cutting-edge approaches to antimicrobial research and oncology.

Thank you to our supporters, collaborators, and clients for their contributions to making this an outstanding year. Here are some key highlights from 2024.

 

Insights in oncology

This year, our cytogenetic platform uncovered novel, clinically-relevant chromosomal aberrations critical for assessing patient care in oncology. Genomic Proximity Mapping™ (GPM) is an upgraded approach to cytogenetic analysis – meeting and  surpassing current risk stratification assessments. Over the summer, researchers at Fred Hutchinson Cancer Center and University of Washington Medical Center published research that used GPM to analyze 48 patient samples, identifying known and novel chromosomal aberrations. Read the MedRxiv preprint to discover the expanding possibilities in leukemia research here » image of DNA

 

Cow burps, super bugs, and our enemy’s enemy–phages

We are actively developing solutions to address the growing threat of antimicrobial resistance and in the same stroke, advancing environmental health efforts with lysin discovery. By leveraging metagenomic data and AI, scientists can harness the evolutionary power of bacteriophages to target and eliminate harmful microbial pathogens with precision. Discover how we are turning the tables using our antimicrobial discovery platform with support from the Bill & Melinda Gates Foundation in our blog here »

 

A novel approach to vaccination

Phase Genomics’ metagenomic deconvolution technology helped crack the code on a potential new vaccine for farmed salmon to defend against sea lice by targeting the parasite’s microbiome. Published in The Economist, researcher Cristian Gallardo Escárate shares results that led to the creation of the groundbreaking invention that could ease global environmental impacts of salmon farming. More here » image of a salmon

Diving into the data 

Two new data analysis tools were made available to ProxiMeta and CytoTerra platform users this year: ProxiMeta™ Explorer and CytoTerra® Curator.  

ProxiMeta™ Explorer is an interactive, cloud-based genome-resolved metagenomic analysis platform for data visualization and exploration. The platform provides fully customizable analyses and reports for tracking genomes across time, conditions, groups, and more with a click of a button. 

CytoTerra® Curator enables users to effortlessly review, revise, and generate reports from cytogenetic data – no prior bioinformatics experience required. From curating calls to constructing circos plots, Curator provides fast, accurate insights for human genomics and oncology research.

 

Tune in to this year’s podcasts

Listen to Phase Genomics CEO, Ivan Liachko, discuss the breadth of applications supported by Phase Genomics’ ultra-long-range sequencing technology in these podcast episodes. Discover the story behind commercializing and implementing biotech innovations and get a glimpse at where this technology is taking us. 

 

Looking Forward

In 2025, we aim to elevate our technology to new heights and broaden our impact across science and medicine. We hope you will follow us on our journey on X, LinkedIn, and BlueSky as we lead genomics innovation to an insightful and healthy future. 

 

Happy New Year from our team at Phase Genomics!

 

 

Phase Genomics’ Technology Powers New Anti-Microbiota Vaccine with the Potential to Ease Global Environmental Impacts of Salmon Farming

Posted on July 23, 2024 by - Uncategorized

Salmon with vaccine and salmon lice swimming away

 

Data shows proprietary technology enables lower cost and higher efficacy through new induced-dysbiosis vaccine targeting a parasite’s microbiome

 

The salmon aquaculture industry has – to put it mildly – a lousy problem. 

 

Sea lice have been sucking salmon dry at facilities across Europe and the Americas at a rate of more than 70 times their wild cousins1, costing the industry a billion dollars annually and wreaking havoc on the natural environment. But researchers at the Universidad de Concepción in Chile recently developed a method to bite back through what could be the world’s first induced-dysbiosis vaccine. A team led by Phase Genomics’ longtime collaborator Dr. Cristian Gallardo Escárate spent the last half-decade creating a vaccine that rids salmon of their lice oppressors by knocking out a key component of the parasite’s microbiome. 

 

The new, less expensive and more effective vaccine could produce major economic and environmental benefits. Farmed Atlantic salmon are Chile’s second largest export after copper. In 2021, salmon shipped out of the country were valued at almost $5 billion, according to the USDA. Yet infestations by the sea louse, Caligus rogercresseyi, in salmon aquaculture facilities dampen productivity, spread disease, threaten native fish and reduce profits. Like all sea lice, Caligus are crustaceans, not insects. The parasites attach themselves to their unfortunate fish hosts, feeding on blood and mucus. Lice keep salmon from growing and building muscle. They can also transmit pathogens, including an infectious viral anemia that can spread to native fish. Chilean salmon farms spend hundreds of millions of dollars trying to beat back C. rogercresseyi infestations with increasingly ineffective treatments.

 

The breakthrough from Gallardo Escárate’s lab at Chile’s Interdisciplinary Center for Aquaculture Research, which is nearing commercialization now, could become the first commercial example of a vaccine against a target’s microbiome. And Phase Genomics’ platform for metagenomics unlocked the new dimension of microbiome biology to drive the keystone discovery in salmon sea louse hologenomics. 

 

Slashing costs and scaling back environmental impact with proximity ligation 

Phase Genomics used its proximity ligation metagenomics technology to assemble and annotate the genomes of the entire sea louse, including the myriad microbes living within the parasite. Dr. Gallardo Escárate and his team combed through the never-before-seen data delivered by Phase Genomics to latch onto a key discovery: They realized that one bacterium was providing its louse host with a key metabolite, in this case iron, that the louse could not acquire on its own. The team’s resulting vaccine directly targets that microbe, creating a dysbiosis within the sea louse’s microbiota to turn off the louse’s metabolite tap, killing the pest in the process while saving the salmon.

 

 

Field tests show that salmon administered the anti-microbiota vaccine were essentially free of sea lice. If widely applied, Chile’s salmon aquaculture industry may find itself with extra biomass to export, fewer sea lice to cross-contaminate the natural environment, and more money in the bank. The initial findings, detailed recently in The Economist, show that vaccinated salmon are 90-95% louse free and more effective than fish managed using conventional antiparasitics. The new method also lowers the environmental impact from salmon farming, which accounts for 70% of all salmon consumed2. 

 

Cover image of an issue of The Economist with an image of a globe. Image of an article from The Economist with an image of salmon

 

Phase Genomics’ proximity ligation unlocks anti-microbiota pest control from fish to farm for sustainable

Salmon farms in Canada, Great Britain and Scandinavia also suffer from sea lice, but a different species called Lepeophtheirus salmonis. Dr. Gallardo Escárate cites similar findings around common bacteria core among Chilean sea lice ectoparasites which he believes could present a common vulnerability to the current immune dysbiosis vaccine across species.

 

 

This is not only the first time metagenomic data have been used to create a vaccine against the microbiome of a target, Dr. Gallardo Escárate also believes this proximity ligation approach could serve as a template for the development of vaccines that protect against other parasitic ne’er-do-wells, whether they’re nibbling away at fish in the sea or four-legged farm animals on land. 

 

“Without this detailed knowledge of this parasite and its microbiome, this vaccine would not exist – and we would not have a blueprint to look for this same phenomenon in other species,” says Phase Genomics founder and CEO Ivan Liachko. “This study nicely demonstrates why we need next-generation metagenomics: It has the power not just to solve problems in one or two economically important industries, but to reveal the patterns and leads that will transform dozens of industries. The days of blunt instruments are over. We now can target with surgical precision.”

 

Dr. Gallardo Escárate is currently conducting the largest study to date of the vaccine in one million salmon in the waters off of Chile.

 

1 https://www.captainjacksalaska.com/seafood/pc/catalog/salmon_diseases.pdf

2 https://www.worldwildlife.org/industries/farmed-salmon 

Far and wide: New technology reveals the long arm of viruses in microbial ecosystems

Posted on May 30, 2023 by - Metagenomics

Hydrothermal vent on ocean floor depicting the microbial environment of the featured study

Hydrothermal mat sampling aboard R/V Roger Revelle using ROV Jason. Credit: R/V Roger Revelle, Scripps institute of Oceanography.

 

For decades, biologists largely studied microbes and their viruses in isolation, nurtured in laboratory cultures. Yet, to paraphrase the poet John Donne, no microbe is an island. In recent years, scientists have recognized this by studying microbes not as individual species, but as part of the larger microbiome: the communal ecosystems, each home to many different types of bacteria and archaea, in which most microbes reside. It is in these realms that microbes display their collective might. From guts to geysers, tiny tales of competition and cooperation within microbiomes have big effects on our health and environment — such as the spread of antibiotic resistance and the stability of food webs.

 

Revealing microbiome mechanics

Traditional, laboratory-based methods struggle to probe the individual components of the microbiome. But “metagenomics” allows us to study the community at large. Metagenomics is the sequencing of DNA from microbial communities, and metagenome-assembled genomes — or MAGs — put together using ever-more sensitive tools and processes, are increasingly able to resolve the inner workings of these complex ecosystems.

Recently, a collaboration between Phase Genomics and a team at Harvard University on a metagenomics project showed that phages — viruses that infect bacteria and archaea — have a surprisingly broad impact on the microbiome of a seafloor hydrothermal vent. Using a technique called proximity ligation (Hi-C), which cross-links DNA strands from the same cell before DNA extraction and sequencing, researchers reconstructed MAGs in this community and found that diverse microbes, including bacteria and archaea separated by billions of years of evolution, sported records of past encounters with the same phages. One explanation is that the phages have an unheard-of level of host diversity — one certainly not predicted by laboratory experiments. Another is that these deep-sea microbes may somehow “share” adaptive immunity across broad and deep evolutionary gulfs.

If phages have similarly broad impacts far above the ocean floor, scientists may have to rethink how communication, cooperation and evolution shape microbiomes — and how they impact the larger creatures, like us, that depend on them.

 

Tapping the archive

Microbiomes teem with phages. But deciphering their reach is no easy task. Thankfully, some bacteria and archaea are hoarders. Their CRISPR-based immune responses record past phage infections by inserting short fragments of phage genomes into a specific region of their own genome. Some studies have even sought to reconstruct the reach of phages in a microbiome by probing the content of these areas — known as spacer regions. Yet, the approach has its drawbacks.

“Spacer regions are rich in repeats, so they don’t get sorted well in the MAG assembly process,” said Yunha Hwang, a doctoral student at Harvard University. “That creates a bias regarding which spacers and phage fragments are ultimately assembled into MAGs.”

Hwang has studied these genetic archives of microbial immunity, and previously reported that, in a desert microbiome, phages may have broad host ranges.

“It was a preliminary result, but very exciting,” said Hwang. “I wanted to see if this was a wider feature of microbiomes, and I wanted to avoid that assembly bias.”

 

Achieving Hi-C depth in deep oceans

Hwang and Peter Girguis, a professor at Harvard, worked with Phase Genomics to employ a metagenomic approach centered on Hi-C, which, by preserving physical linkages between DNA fragments present in the same cell, eases the process of resolving repeat-rich regions like CRISPR spacers.

Hwang collected samples from the microbiome near a hydrothermal vent in the Gulf of California’s Guyamas Basin. Microbial communities like this employ “alternative” metabolic pathways — relying on the plume’s rich geochemical outflow for nutrients, energy and raw materials instead of the sun-based food webs more familiar to surface-dwellers. As soon as she reached port in San Diego, Hwang shipped the microbiome samples to Phase Genomics for cross-linking, DNA extraction, sequencing and MAG assembly.

The spacer regions of the MAGs assembled via Hi-C showed similar profiles of past phage infection compared to conventional spacer-sequencing and assembly. But the higher-quality Hi-C MAGs also eased the search for phage fragments within CRISPR spacers. And, as in Hwang’s study of desert microbiomes, individual phages in the hydrothermal vent microbiome had a broad reach — including bacteria to archaea.

“This was so baffling to us, because these are two separate domains of life,” said Hwang. “The ability for a phage to infect a host depends on fundamental properties of cell biology, and bacteria and archaea are so different — their membranes, their proteins, their genomes. So, what does this mean?”

Another puzzle is that bacteria and archaea that are linked by symbiotic relationships — such as eating one another’s metabolic leftovers — were also more likely to harbor genomic fragments of the same phages in their CRISPR spacers.

 

Spread the word

One theory to explain these findings is that phages within microbiomes, which can be hard-pressed for space in these close-knit communities, have evolved to infect hosts with radically diverse membrane compositions, host defenses and cell biology. But that is not the only possibility. Another is that symbiotic partners, separated by billions of years of evolution but united at the dinner table, may be sharing more than just a meal.

“In symbiotic microbes, when one population or species gets infected by a phage, there could be a selective advantage in sharing that adaptive, genetically encoded immunity with your partners,” said Hwang.

Future metagenomic studies of other microbiomes may help resolve these theories, or sire new ones. But the eventual explanations will undoubtedly force scientists to rethink how genetic information flows within microbiomes.

“How do bacteria and archaea build up ‘resilience’ in such closely packed communities?” said Hwang. “Perhaps one way that happens through selective pressure to share records of past phage infections widely. Keeping your neighbor healthy keeps you healthy.”

 

Sounds familiar

Once upon a time, far above the ocean floor, children played a game called “telephone”: passing a phrase from one person to another — in the form of a whisper — to see how the message changed as it is heard by each ear and transmitted by each voice.

It seems that bacteria, archaea and phages play similar games, which is just the latest surprise that metagenomics has revealed about microbiomes. It will certainly not be the last.

Pass it on.

 

 

Unlock the Virome with ProxiPhage

Posted on July 26, 2021 by - Genomics, Metagenomics, ProxiMeta

viruses moving through a net

 

Metagenomic studies are illuminating the diverse array of microbiomes that exist from the ocean floor to our gastrointestinal tracts. Understanding these microbial communities is essential to understanding modern health and the environment; however, outdated lab techniques are laborious, costly, and fail to create a complete picture of the microbiome. This article, posted by Ivan Liachko, describes how advancements in biotechnology are facilitating exciting discoveries with recent tools developed to capture phage and other mobile genetic element dynamics within microbiome samples.

Continue reading to discover how ProxiPhage, a recent addition to the ProxiMeta platform, is helping scientists answer questions relating to microbiome composition dynamics, prophage prevalence, frequency of transient infections, spread of antibiotic resistance, and more.

https://www.linkedin.com/pulse/unlocking-virome-proximity-guided-metagenomics-new-frontier-liachko/

 

 

Better together: long-range and long-read DNA sequencing methods, combined, reach record heights in microbiome discovery

Posted on May 27, 2021 by - Genomics, Hi-C, Metagenomics, ProxiMeta

Microbiome plate and Phase Genomics logo. Reads "Breaking records in microbiome discovery"

 

Click here for an updated blog post.

 

Since its debut, next-generation sequencing has not rested on its laurels. Improved sequencing platforms have reduced error and lengthened reads into the tens of thousands of bases. The debut of long-range sequencing methods that are based on proximity ligation (aka Hi-C) has brought a new order-of-magnitude into reach by linking DNA strands with their neighbors before sequencing.

 

This progress has birthed high-resolution metagenomics, the sequencing and assembly of genomes from environmental samples to study ecosystem dynamics. But metagenomic experiments often undersample microbial diversity, missing rare residents, overlooking closely related organisms (like bacterial strains), losing rich genetic data (like metabolite gene clusters), and ignoring host-viral or host-plasmid interactions.

 

A revolution within a revolution

 

New sequencing platforms and methods can reform metagenomics from within. Long-read platforms, such as the PacBio® Sequel® IIe system, now yield HiFi reads of up to 15,000 base pairs with error rates below 1%. In addition, Phase Genomics created ProxiMeta™ kits to generate proximity-ligated long-range sequencing libraries, which preserve associations between DNA strands originating in the same cell.

 

In a study posted May 4 to bioRxiv, a team — led by Dr. Timothy Smith and Dr. Derek Bickhart at the U.S. Department of Agriculture and Dr. Pavel Pevzner at the University of California, San Diego — employed both PacBio HiFi sequencing and ProxiMeta in a deep sequencing experiment to uncover record levels of microbial diversity from a fecal sample of a Katahdin lamb. Combined, PacBio HiFi sequencing and ProxiMeta eased assembly, recovered rare microbes, resolved hundreds of strains and haplotypes, and preserved hundreds of plasmid and viral interactions.

 

HiFi family trees

 

The team constructed SMRTbell® libraries to generate HiFi data, and ProxiMeta kits to generate long-range libraries. The two datasets, along with the metaFlye and ProxiMeta algorithms, allowed them to assemble contigs and create draft genomes without manual curation.

 

Researchers compared the breadth and depth of HiFi data-derived metagenome-assembled genomes, or MAGs, to control MAGs from assemblies of the same sample made using long, error-prone reads. HiFi data yielded more complete MAGs — 428 versus 335 — from more bacteria and archaea. HiFi data also generated more low-prevalence MAGs, capturing a larger slice of the community’s diversity by picking up more genomes from less common residents.

 

The HiFi MAGs also contained more than 1,400 complete and 350 partial sets of gene clusters for synthesizing metabolites such as proteasome inhibitors, which likely help some of these microbes colonize the gut. HiFi data picked up about 40% more of such clusters than control MAGs, illustrating just how much data is lost when long reads aren’t also highly accurate reads.

 

The team also used the HiFi MAGs to trace lineages within the community. They computationally resolved 220 MAGs into strain haplotypes, based largely on variations within single-copy genes. One MAG had 25 different haplotypes, which are likely strains of the same genus or species.

 

ProxiMeta’s long-range discoveries

 

The ProxiMeta-generated libraries added flesh to these MAG frames skeletons by unveiling additional rich biological information. Long-range sequencing linked nearly 300 HiFi-assembled plasmids to specific MAGs — revealing the species that hosted them. One plasmid, for example, was found in bacteria from 13 different genera. Long-range data also identified the first plasmids associated with two archaea, Methanobrevibacter and Methanosphaera.

 

Long-range sequencing illuminated the viral burden in this community. The HiFi library included nearly 400 viral contigs, more than half of which came from a single family of viruses that infect both bacteria and archaea. The team identified 424 unique viral-host interactions, including 60 between viruses and archaea, which is a more than 7-fold increase over controls.

 

What’s around the bend?

 

This study has lessons beyond one lamb’s gastrointestinal tract. It shows decisively that the highly accurate long reads generated by HiFi sequencing ideal partners for Hi-C-derived methods like ProxiMeta — together generating increasingly sophisticated metagenome assemblies for biologists to interrogate.

 

Applied to other environmental samples, this platform could illuminate the diversity and complexity of other microbial communities — from the bottom of the sea to mountain peaks, and within the stomach of every human being. It could probe pressing issues of our day, such as antibiotic resistance, soil health, or how microbes can break down pollutants. These endeavors will not just fuel the engines of scientific inquiry. Broader use of this method could generate new insights into pressing problems of our times, including antibiotic resistance.

Breaking the Mold: New Tech Sheds Light on 5 Mysteries of the Fungal World

Posted on February 10, 2020 by - Hi-C, Proximo

 

This month Phase Genomics is celebrating #FungusFebruary by highlighting some of the unique capabilities of our Hi-C technology to solve age-old mysteries in the world of fungal genetics and deliver new potential for researchers to understand fungi, all while helping solve global crop crises and develop new groundbreaking pharmaceuticals.

While we wield the power of genomics to explore the wonders of fungi today, a few centuries ago people dismissed them as just weird plants. Eventually microscopes and anatomical studies revealed fungi as a distinct flavor of life — some varieties quite tasty — but educational experts today continue to bemoan the lack of lessons on fungi in biology curricula, and research on fungi — even those that cause disease — lags.

As a result, scientists lack much basic information on the genetics, life cycles, and reproductive habits of many fungi — even though members of this kingdom could help address a bevy of challenges in food and energy production, illuminate the evolution of complex life and even shelter us on Mars.

Genome studies on fungi of all stripes can resolve evolutionary relationships and ecosystem dynamics, identify metabolites of commercial and medical interest and — for fungi that cause disease — reveal biochemical and genetic targets to help us fight pathogenicity.

Like their animal and plant cousins, fungal genomes also have their challenging parts, including repeats, duplications and structural elements that complicate both sequencing and assembly. Recently, the chromosome conformation method “Hi-C” and advances in next-generation sequencing have helped untangle some of these sticky genomic knots, and show promise in taming genomes across this diverse and neglected kingdom of life.

 

        1. High-resolution mapping of centromeres

 Hi-C’s power lies in its ability to identify regions of the genome that reside in close proximity to one another in the nucleus — information that essentially captures the 3D organization of the genome. But Hi-C doesn’t just identify where particular chromosomes reside within the nucleus. It can also help identify functional elements in genomes that are difficult to identify in other ways.

That is what two groups of researchers (from the Pasteur Institute and the University of Washington) did when they used Hi-C to track down functional elements in yeast genomes — centromeres and rDNA clusters — both of which are typically repeat-rich and difficult to identify without laborious experiments involving functional assays or mapping the binding sites of rare centromere proteins. In fungal species, centromeres are held tightly together at the spindle pole body, and the team used this shared proximity to identify centromere locations in the genomes of numerous yeasts (and subsequently other fungi), despite not knowing their centromeric DNA sequence. Ribosomal DNA clusters similarly congregate in yeast nuclei, which one team exploited to identify their positions in Debaryomyces hansenii.

 

        2. High-quality genomes illuminate biochemical pathways

Fungi harbor a wide array of genes for synthesizing secondary metabolites, which range from harmful toxins to helpful pharmaceuticals. In fungi, genes for synthesizing secondary metabolites tend to occur in clusters, which are also thought to be sites of rapid evolution.

Phase Genomics worked with a University of Minnesota-led team and used Hi-C to generate high-quality genomes of six strains of Tolypocladium inflatum, an insect pathogen that has already given us the immunosuppressant drug cyclosporin. The new assemblies revealed major differences in secondary metabolite production between T. inflatum strains, including novel clusters, transpositions and clusters that may be involved in toxin synthesis. The bevy of discoveries from these assemblies showed how recombination can drive significant divergence even within a single species — and how important it is to build multiple high-quality genome assemblies that can capture that diversity.

 

        3. Fungal dikaryons and the hidden nuclear dance

The genetic differences between strains also apply to pathogenic fungi, like the stem rust, which parasitizes wheat. Phase Genomics partnered with a team led by scientists at CSIRO in Australia to apply Hi-C to stem rust – the particularly deadly scourge Ug99. Like many fungi, stem rust genomes are divided between two haploid nuclei. The team used Hi-C data to assemble complete haplotypes for both haploid genomes of both strains, and discovered that Ug99, a recent arrival that is decimating whole fields of wheat in Africa, has an unexpected origin: The strain arose through “somatic hybridization,” when hyphae from two strains exchange haploid nuclei. This may explain the strain’s sudden rise and deadly wake, and gives scientists new genomic information to understand Ug99’s virulence and identify weaknesses that could give wheat a leg up.

 

        4. Hybrids, beer, and fungal metagenomics

The ability to separate two nuclei from within the same cell can be extended to more complex samples.  Yeasts, which are integral players in brewing, will often hybridize to form new species containing genomes from two organisms at once (the famous lager-producing yeast Sacharomyces carlsbergensis is one example of such a hybrid).  But in a mixed microbial community, such as beer, wine, or a microbiome sample, how can DNA sequencing detect which genomes co-exist within the same cell?  One special power of Hi-C is that it traps sequences that are within touching distance of each other, and therefore must come from inside the same cell.  The Dunham lab at the University of Washington used this property to analyze an open-fermentation beer from a local brewery.  The exciting result was that they were able to discover a new hybrid yeast, later named Pichia apotheca, using Hi-C data to identify it as a hybrid bearing two genomes from related organisms.  This new hybrid species has since been used by home-brewers to ply their craft and gives beer a very unique flavor.

 

        5. The Epigenetics of Symbiosis

Nature has plenty of examples of plants and fungi getting along. One of them is Epichloë festucae, a filamentous fungus that has evolved a symbiotic relationship with certain grass species. When Phase Genomics worked with a Massey University-led team, they discovered that E. festucae’s genome carries hallmarks of this symbiosis. The analysis of Hi-C data revealed that important genes are clustered into blocks separated by repeat-rich regions. Hi-C and RNA-seq data together showed that genes within the blocks have similar expression patterns — indicating that genes needed for symbiosis with their grass hosts tend to cluster together in the same blocks.

 

Looking Forward

Cutting edge genomic technologies like Hi-C have the potential to keep making up for lost time and reveal even more intimate details of the hidden lives of fungi. This #FungusFebruary, it’s worth asking: What other mysteries about this long-overlooked kingdom are worth solving?

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

 

Project ProxiMeta: 2019 Metagenomics Award

Posted on June 3, 2019 by - Metagenomics, ProxiMeta

Win a Free Proximity-Ligation Metagenomics Project

Win a chance to collaborate with Phase Genomics on a metagenomics research project. The grand prize winner will receive a full-service ProxiMeta Metagenome Deconvolution project, including proximity-ligation and shotgun library prep, sequencing, and analysis. Characterize a microbial community of your choice and assemble hundreds of bacterial and eukaryotic genomes, associate plasmids and phage with hosts, and discover novel microbial life.

Submit your proposal by August 8, 2019 The four project finalists will be announced on September 5, 2019 via Twitter based on scientific merit, novelty, and impact. After a week of public voting, the project with the most votes will be named the 2019 Metagenomics Award winner and will receive a full ProxiMeta service project.

With ProxiMeta, you can explore the microbiome with confidence. Only high-quality microbial genomes can provide true insights into the dark matter of the microbiome. Submit your proposal for the 2019 Metagenomics Award today!

KEY DATES

8 August                                      Deadline for Entries

4 September                               Finalists Announced

5-12 September                          Vote for Projects @PhaseGenomics Twitter

12 September                             2019 Metagenomics Award Announcement

 

Help Us Choose the Winner!

We need your help choosing which project to sequence! Below are our four finalists, read through the project proposals and choose your favorite; voting is open to the public and will take place on Twitter September 5, 2019 for one week.

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

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

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

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.

 

 

 

RESOURCES

Earth’s Wine Cellar: Digging into the Microbiome of Vineyards

Posted on September 6, 2018 by - Hi-C, Metagenomics, ProxiMeta, Uncategorized

 

Phase Genomics partnered with Browne Family Vineyards to begin to understand, the microbiome makeup of soils within different vineyards across the state of Washington. The findings were unveiled at the Pacific Science Center’s “STEM: Science Uncorked” winetasting event.

There are many different factors that contribute to soil composition, such as parent material, topography, climate, geological time, and the thousands of different and undiscovered microbes living in the soil—the least understood factor. In April of 2018, Browne Family Vineyards staff visited five of their vineyards, filled a bag with soil from each site, and sent it to Phase Genomics to analyze the microbiome in each of the soil samples.

SYMBIOSIS BETWEEN PLANTS AND MICROBES

Plants rely heavily on their microbiome to live, grow, and protect themselves from pathogens. One example of this symbiotic relationship is that plants release chemicals into the soil in order to attract microbes. These microbes bring nutrients such as nitrogen, iron, potassium, and phosphorus to the plants in exchange for sugar, which the microbes require to survive. Microbes also play an important role in nitrogen fixation, organic decay, and biofilm production to protect the plant roots from drought. It is evident that this symbiotic relationship between microbes and plants is critical to the health and survival of both, but further research into this complex community is inhibited by two main problems: It is impossible to isolate microbes in such a complex mix and most of the microbes have never been discovered before.

THE DARK MATTER OF THE MICROBIOME

Microbes live in communities where they rely on each other. This makes it difficult to isolate or culture (i.e. grow) microbes without killing them or altering their genetic makeup. Moreover, there can be millions of microbes living in a single teaspoon of soil, making these samples extremely complex environments. This causes most of the microbial world to be unknown, sometimes referred to as the “Dark Matter of the Microbiome”.

The most effective way to identify the microbes in the community is to look at the genetic makeup of the microbiome to try to classify microbial genomes present. Standard practices include sequencing of 16S (a hypervariable genomic region) and shotgun sequencing.  By combining these standard practices with Hi-C, researchers are now able to fully reconstruct genomes from a mix because Hi-C captures the DNA within each microbe to exploit key genetic features unique to each individual in the community. The Phase Genomics Hi-C kit and software, ProxiMetaTM, uses this information to capture even novel genomes straight from the sample without culturing—illuminating the dark matter of the microbiome.

THE PROCEDURE

Shotgun Sequencing Procedure and Difficulties

Figure 1: Shotgun Sequencing Procedure and Difficulties

Once the soil samples were collected from the five vineyards, Phase Genomics produced shotgun libraries to obtain DNA from all of the microbes in each sample (Figure 1)—essentially taking the soil sample, breaking open all of the microbial cells then purifying the DNA (1.A). Since DNA is fragile, most of it gets broken into smaller pieces during this process, leaving a mix of many DNA fragments from all of the microbes that were present in the original soil sample. The fragmented DNA is then sequenced and the “sequence reads” are uploaded into a database of known microbial genomes (1.B). This database then searches for matches or “hits” to see if the reads are similar to anything in the database (1.C).

A problem with relying on shotgun data is that it’s unclear which DNA fragments belong to which microbe, thus relying heavily on computational techniques and the accuracy of the reference database for classification. This results in little improvement or clarity on the makeup of the sample, again, leaving the microbiome in the dark. Though shotgun sequencing only provides a glimpse into the microbial community, this data allows scientists to differentiate the taxonomy (phyla, genera, species) of the microorganisms living in the soil.

THE RESULTS

Shotgun sequencing identified over 10,000 different species from each of the vineyard soil samples; however, it is impossible to know if this is the true number of species because only ~ 20% of the reads matched the database, indicating ~80% was either incomplete or undiscovered (see table below).

Table 1: Vineyard Read Classification
Vineyard Total Reads Percent of Reads Classified Number of Organisms Found Percent of Unknown Organisms
Canyon 19,001,222 15.95% 10,726 73.32%
Canoe Ridge 21,214,190 17.66% 11,721 55.55%
Waterbrook 19,469,954 19.6% 10,782 50.58%
Skyfall 63,850,810 16.17% 15,101 80.08%
Willow Crest 43,941,026 17.13% 13,914 71.84%

 

Moreover, of assigned reads, >50% did not match to a genus or species—hinting that many of the organisms found are novel. Without digging too deep into the microbiome analysis, it is evident that the microbial makeup is different for each of the samples. Varying levels of reads from each vineyard were able to be classified (Table 1), and among the classified reads, the vineyards have 3-4 microbes that vary in abundance in common. These microbes, such as Proteobacteria, Rhizobacteria, and Actinobacteria, generally, are very common in soil.

Proteobacteria

Proteobacteria

There are obvious differences in the biodiversity of the soil samples both in number of species and relative abundance. For example, Canoe Ridge and Waterbrook samples were >20%, Delftia, while the microbes in the other vineyards were more evenly distributed, with abundance closer to 1-5%. Interestingly, Delftia, a rod-shaped bacterium, has the ability to break down toxic chemicals and to produce gold.

Actinobacteria

Actinobacteria

There are two main components that influence microbe classification in these samples: the desired taxonomy level, and the statistical threshold, or minimum number of reads, set to define it. Much like zooming in and out, the most “zoomed out” analysis is achieved by a stringent threshold and will reveal phylum, while the most “zoomed in” analysis is achieved by a more lenient threshold and will reveal genus and species

If the data is “zoomed in” further, about 37% of the microbes in each community can be identified by genus. On average, 63% of the communities do not match to a genus at all, hinting that these microbes may have never been sequenced. The most abundant microbe genera present in these samples are Bradyrhizobium, Streptomyces, and Nocardiodes.

As discussed earlier, this data highlights the issues that are present with shotgun data and the corresponding analysis: there is still far too much that is unknown. In order to better understand these samples, we also performed Hi-C on two of the samples which will be discussed in further detail in the next section.

 

HI-C AND FINDING NOVEL GENOMES

One thing all these soil samples have in common is that they are composed of numerous novel species. To obtain more information on the microbes present in these samples, and solve the issue discussed earlier surrounding shotgun data, Hi-C was performed on two of the soil samples, Skyfall and Willow Crest. Essentially, Hi-C assigns DNA fragments from shotgun sequencing to the correct species by connecting DNA while the cells are still intact.

Hi-C enables clustering of shotgun assemblies and subsequently yields complete genomes from a microbiome, even if the genome has never been sequenced before. With complete microbe genomes, it becomes easier to classify organisms down to the strain-level—a step even further than species. By having the genome, we can essentially read a microorganism’s blueprint and learn more about its genes, evolution, and even function once the genome is annotated.

For example, preliminary data from the Willow Crest soil sample yielded 400 different genome clusters. When compared to known bacterial genomes in the RefSeq database, which aggregates all published microbial genomic data, over half of the extracted genomes are unable to be identified at a genus level and thus likely represent newly discovered bacterial organisms.

SCIENCE UNCORKED

When the microbiome data from the vineyards were presented to the public at the Pacific Science Center, two questions consistently arose: How does this influence wine taste, and how can growers select for a healthy microbiome? These very forward-thinking questions unfortunately cannot be answered—yet.

Scientists do know that soil plays a big role in plant health, and this could in part be due to the plants’ symbiotic relationship with microbes, as discussed earlier. It has also been shown that biodiversity can benefit plants because of the diverse functions individual microbes have, i.e. with more microbes, there are more potential functions being served versus 1 microbe serving one function. However, nailing down answers to these questions will take a lot of research. With emerging technologies, like Hi-C, the answers have become much more obtainable.

Though the term “microbiome” may not be household vocabulary, many of the attendees were very aware about the role that microbes play in human health, and how they influence the world around us. It goes to show that the rapid developments in the microbiome field are reaching beyond just research and becoming more tangible for the general public. Relevant stories—like looking into the microbiome of vineyards— are helping them understand the intricate concept of microbial life.

Learn more about ProxiMeta Hi-C and the microbiome by visiting our website www.phasegenomics.com and connect with us on twitter by following @PhaseGenomics

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.

 

 

New Video: From Contigs to Chromosomes

Posted on March 15, 2018 by - Genomics, Hi-C, Metagenomics, ProxiMeta, Proximo, Webinar

Phase Genomics CEO and Founder Ivan Liachko, Ph.D. offers an inside look at our ProxiMeta™ Hi-C and Proximo™ Hi-C technology. He explains in this 40 minute presentation how Hi-C is revolutionizing genome and metagenome assembly. Watch “From Contigs to Chromosomes” now and reach out to http://phasegenomics.com/contact-us/ with any questions.

Thanks to IMMSA for hosting this webinar.

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