ASG Project: Microbial-Rich Sponge Metagenomes Insights with Kaowwa

 

Introduction

So, uh, hi everyone. I'm Kaowwa. Today I'll talk to you, um, about some of the first insights that we have about the microbial-rich sponge metagenomes in the ASG project.

Aquatic Symbiosis Genomics Project

Overview

In the Aquatic Symbiosis Genomics project, we are focused on the huge amount of biodiversity that we have in the aquatic ecosystems. In this project, we provide, uh, genetic data for biologists, uh, for them to understand how species evolve and live together. One of, uh, these species is, uh, a microb—don't worry—yeah, that, uh, the square pause, yes, please. Yeah, thank you.

Sample Processing

So, uh, really briefly, how the samples are processed in the ASG project: we extract the genetic material, and then this material moves to sequencing and then to assembly. It's during the assembly stage where the host and the coob genomes are separated. The host usually is a eukaryotic species, so it moves to curation using high-C data, and the coob genomes are released as MAGs. Later, these MAGs are, uh, submitted to public databases such as EBI Magnify and DNA, and later they are published along with the hosts in Genome Notes.

Focus on Sponge Metagenomes

Sponge Microbiota

In ASG, we work with many aquatic species. We have marine species, we have freshwater species, but the sponges are the ones with the richest microbiota that we have. This is, uh, from where most of our metagenomes come from, and possibly this happens because of the nature of the sponges—they are natural, um, filter feeders.

Sponge Structure

So, if you zoom in on a sponge picture, uh, where is the—oh yeah, you can see there are many pores, and the water flows through the pores. This is how the sponge incorporates most of the coob that live inside it. Sometimes the coob contributes up to 130% of the sponge biomass. So, it's really a lot of coob, and sometimes the genome curation of the host in the sponge is quite difficult, quite challenging, because of the great amount of coob.

Curation Process

High-C Map Analysis

Here on the left, we have a high-C map of the sponge, uh, Condr caribensis, and this is a high-C map ready for curation. This is the way that we receive it for curation. For those who are not very familiar with a high-C map, it shows high signals that indicate the affinity between the chromosomes inside the cell.

Misassemblies and Coob Removal

We are expecting to see the highest, the strongest high-C signal only here on the main diagonal, but as you can see, we have some regions which are out of this main diagonal, which possibly are misassemblies that we must correct during curation, or possibly are coob that also need to be removed during curation. We also have here, in the red square, some really small scaffolds which are probably from coob and need to be removed too.

BlobToolKit Plot

Here on the right, we have a BlobToolKit plot. This plot shows us the distribution of our scaffolds in the assembly based on the PacBio coverage and the GC content. Here, we can see we have bubbles in many colors, indicating many taxonomic groups. But if we look at the BTK caption, we see no Porifera. So, our main species, our host, our target species, is not tagged by BTK. Actually, it's there, but it's masked by the great amount of coob.

Comparison of High-C Maps

Before and After Curation

Continuing with the curation stuff, here we have the, the two, actually the same high-C map from the previous slides, but on the left it's before curation, and on the right after curation. In this one, we can see that we have two really different regions. We have the green square, which is the sponge genome—only the host—and the red square is the coob. We can see there's a clear difference in the background between these two regions.

Highlighted Regions and Repetitive Regions

Here, we can see some highlighted regions outside the main diagonal, but that's okay because that's possibly due to the highly repetitive regions from the sponge telomere from the chromosomes, so that's fine. We cannot see this background here, so these are coob that need to be removed. So, us as genome curators, we want to get rid of all metagenomes and all coob. We, we actually don't like them, so we take them off during curation to have a map just like this—with just the host genome on the left side and the right side. In this case, almost more than 50% of the assembly was, uh, coob and was removed during curation.

Insights on Sponge Metagenomes

Current Data

So, what do we have so far in the ASG project about sponge metagenomes? We have 60 sponge species in the project at the moment, 32 of these species with MAGs, 18 species with, uh, with the MAGs already submitted, already public. In total, we have almost 900 metagenomes, where close to 430 are public already.

Microbial Diversity

These are some of the sponge species with the highest number of, uh, MAGs that we have in the project. We got curious about which microbes are these, which bacteria are these. If you consider all the sponge species, all the MAGs that we have for sponges, we have a top five bacteria there, but the number one is the hocoa, or Chloroflexi. They are present in 177% of the metagenomes. This group of bacteria is known to transform many, uh, a wide range of halogenic compounds, usually toxic ones, so they play a really important role in the restoration and conservation of contaminated environments. Usually, the Dehalococcoidia group is the most abundant group in the MAGs that we have. An example of this is the sponge species in the bottom, where 45% of the MAGs are from the hocoa.

Functions of Sponge Microbiota

Nitrogen and Carbon Metabolism

What should be the main functions of this bacteria, this microbial? Usually, they are involved in nitrogen metabolism, especially in sponges' carbon metabolism, and some of them have immunological roles, such as, for example, microorganism selection. They tell the sponge which bacteria or microbes the sponge should keep or, um, remove, eliminate. Sometimes, there is also a cross-talk between the host and the coob, trying to protect the sponge as a whole.

Preliminary Insights

So, so far, our data shows that this is really just an insight, this is really preliminary, we haven't done any analysis about this. But what we could see is that we have the top four MAG species, the sponges with the highest number of metagenomes, they are all high microbial abundance sponges, or A sponges. They vary widely geographically, from Norway to Australia, all of them have a higher number of metagenomes with many microbial groups. We can see, as expected, the Chloroflexi or the Dehalococcoidia only in the A sponges, as expected. Another thing that is expected is a dichotomy between the low microbial and high microbial sponges, which we can also see here, confirmed by our ASG data. These A sponges are expected to have more diverse metagenomes, but on the other hand, the microbes are more redundant. The microbiomes are really similar in composition and function, as can be seen in the deep-sea sponge Parva. This is mostly seen in deep-sea sponges.

Microbial Redundancy in Deep-Sea Sponges

We believe that what's happening is that we can see a large redundancy of microbes working on the same roles in deep-sea sponges, possibly to bring some stability to the inner system of the sponge. Another thing that might be happening is that these bacteria or microbes evolved to live and adapt in a really restrictive environment, such as the deep sea, where there is little light, lower temperatures, and higher concentrations of inorganic compounds, such as nitrogen, for example. They reduce competition and work as a team to explore the resources they have the most in the deep sea.

Future Steps in the ASG Project

Upcoming Sequencing

The next steps in the project: we have already 14 sponge species with their metagenomes sequenced and ready to go to public databases. We have new sponges coming to have their metagenomes sequenced. We are now working on the metagenomes of other groups, such as corals, anemones, and mollusks, which will be public soon.

Conclusion

In summary, the Aquatic Symbiosis Genomics (ASG) project provides invaluable insights into the rich tapestry of microbial life within aquatic ecosystems, with a particular focus on sponges. Our exploration into sponge metagenomes reveals a complex interplay between hosts and their microbial partners, showcasing the significant role that these microbes play in nitrogen and carbon metabolism, as well as in environmental restoration.

The meticulous process of sample curation, including the use of high-C maps and BlobToolKit plots, highlights the challenges faced in distinguishing between host and microbial genomes. Despite these hurdles, our efforts have led to a substantial collection of metagenomic data, with 60 sponge species and nearly 900 metagenomes contributing to our understanding of microbial diversity.

Looking forward, the ASG project will continue to expand, with upcoming sequencing of additional sponge species and new groups like corals and mollusks. We encourage the scientific community and interested readers to engage with our findings, access the public databases, and contribute to the ongoing research in this dynamic field. Together, we can further unravel the complexities of aquatic symbiosis and its implications for biodiversity and environmental health.

Thank you for your interest and support. If you have any questions or would like to learn more, please feel free to reach out.

Q&A Session

1. What is the Aquatic Symbiosis Genomics (ASG) project? The ASG project focuses on studying the biodiversity in aquatic ecosystems by providing genetic data to understand how different species evolve and interact. It involves the extraction, sequencing, and assembly of genetic material to study the genomes of various aquatic species.

2. How are samples processed in the ASG project? In the ASG project, genetic material is extracted from samples, which then undergo sequencing and assembly. During the assembly stage, host genomes (usually eukaryotic) are separated from microbial genomes (coob). The microbial genomes are then released as MAGs and submitted to public databases.

3. Why are sponges significant in the ASG project? Sponges are significant because they possess the richest microbiota among aquatic species. Their natural filter-feeding process allows them to incorporate a vast number of microbes, making them a primary focus for studying metagenomes in the ASG project.

4. What challenges are faced during the curation of sponge genomes? Curation of sponge genomes is challenging due to the high amount of microbial genomes (coob) present. Misassemblies and the presence of coob complicate the process, requiring careful removal and correction to isolate the host genome accurately.

5. What is the role of a high-C map in genome curation? A high-C map helps identify the affinity between chromosomes inside a cell, indicating regions that may be misassembled or contaminated with coob. Genome curators use these maps to correct errors and remove unwanted microbial genomes.

6. What is the significance of BlobToolKit plots in the ASG project? BlobToolKit plots show the distribution of scaffolds in an assembly based on coverage and GC content. These plots help identify different taxonomic groups present in the samples and assist in distinguishing the host genome from microbial genomes.

7. How many sponge species and metagenomes are currently part of the ASG project? The ASG project includes 60 sponge species, with 32 having MAGs. A total of nearly 900 metagenomes have been sequenced, with around 430 made publicly available.

8. What are the main functions of sponge microbiota? Sponge microbiota are primarily involved in nitrogen and carbon metabolism. They also play immunological roles by selecting which microbes to keep or eliminate, and there is often a cross-talk between the host and microbes to protect the sponge.

9. What insights have been gained about microbial diversity in sponges? Preliminary data shows that sponges with high microbial abundance have more diverse but redundant microbiomes. Microbial redundancy is especially observed in deep-sea sponges, where microbes adapt to the restrictive environment and work together to maintain sponge stability.

10. What are the future steps in the ASG project? The ASG project plans to make 14 more sponge species' metagenomes publicly available soon. Additionally, the project will expand to include metagenomes from other groups such as corals, anemones, and mollusks.