What does this all mean?

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Microbes are vital components of life on Earth. They influence the health and balance of living components including the environments. They are central to many processes, including the preparation of food such as bread, cheese, beverages.

When biologist want to understand and study the diversity and distribution of large organisms such as Eucalypts or Bandicoots, they can easily go our in the field and observe them, though they may sometime require binoculars.

Some of the largest microbes are less than a tenth of the diameter of a human hair, with most nearly only a hundredth of the size. Which means that they are invisible to the naked eye. Even with microscopes which greatly increase the ability to visualise small microorganisms, it is often difficult to identify and study microbes due to their lack of distinctive features and complex interactions. Thus, researchers have had to turn to defining the molecular components of microbes, their genetic blueprint, to truely understand both the diversity of microbes as well as their function.

The resource produced through The Australian Microbiome initiative provides data for the various components of the genetic blueprint of microbes.

How do we study microbial biodiversity?

Living organisms are grouped into units that have similar characteristics either from observable properties such as shape or from their molecular information such as their DNA signatures. However, in many cases, physical features are difficult to use to identify microbes. Historically microbes were classified through shapes, and functional parameters such as their nutrient utilisation (e.g. growth on glucose) or compound degradation abilities (degradation of bile salts). This is quite restrictive to microbes that can be isolated and grown in laboratory conditions, as well as being time consuming. Nowadays, microbial diversity is studied through the various genetic signatures of each organism. Specific genetic markers can be pulled out and allow the differentiation of families, genera or species (amplicons) both from isolates or from whole assemblages in environmental samples. More information can be extracted from studying the full genetic blueprint of each microbe, often analysed as a batch from a particular environment, to understand the functional potential of a microbial community (metagenomes) as well as the actual functional activities shown by microbes under particular conditions, e.g. heat variation or influx of nutrient from stormwater drains after a rain event (metatranscriptomes).

The Australian Microbiome Initiative is creating a large reference of microbial genomic information from thousands of different terrestrial and aquatic (marine and freshwater) locations that can be mined and analysed to define which microbes are present and what they might be doing.

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What are amplicons?

The DNA in all organisms has portions that are similar, with a few specific variations, even shared between all bacteria and/or between all eukaryotes from a small algae to a goat. The more distantly related the organisms, the more variations these portions will have, and each specific organism will have a specific pattern that allows its identification. Amplicons allows researchers to target only a certain informative DNA portion of choice out of each single organism contained within a sample, such as a spoonful of soil or seawater. This portion is then analysed (sequenced) and compared to others, allowing to have a broad view of the identity of all organisms in that sample. The various portions or amplicons have different resolutive power, in the same way as descriptive words, such as “a flower” versus “a dandelion” versus “a white-flowering Japanese dandelion”. The target of the Australian Microbiome Initiative amplicons provide us with a good overview description of all of the organisms present in an environment with medium resolution.

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What are ASVs?

Environments contain complex communities of microbes, meaning that there are many different groups of microbes coexisting. The study of these communities through amplicon sequencing generate thousands of amplicons with varying degrees of similarity (the genetic code from each amplicon can share 100% of the code or just 50% of the code). There are different ways of looking at the resulting information, depending of your objectives:

  • Amplicons can be grouped into batches of similar code (e.g. sharing 97% of their genetic code). The resulting groups will be called OTUs (Operational Taxonomic Units at the defined % grouping) and will give a good overview of the microbial species present but will not be able to provide the fine scale diversity of the microbial community.
  • Amplicons can be grouped into batches of exact code similarity (e.g. only exact matching sequences will be grouped). The resulting groups will be called ASVs (Amplicon Sequence Variants, also named zOTU – zero radius Operational Taxonomic Unit) and will give a refined view of the diversity of microbial species present.
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How do we understand the potential functions of microbes or their actual behaviour?

The study of microbial diversity through targeted genetic markers only helps us understand the composition of communities in terms of what genera and species are there. Any function assigned through these types of studies are made by inference with what may be known from these species, but can be skewed and does not account for variations within species, for example due to environmental adaptation.

The DNA contained in each organism cell(s), i.e. its genome, is a blueprint for that specimen and contains all the information on what that organisms is potentially capable of doing. Thus, to understand the potential functions of a community of microbes in an environmental sample, we need to look at the full genome of all of the organisms present in an environment (see What are metagenomes?).

For an analogy, imagine you are standing in front of a workshop room, with a sign on the door stating workshop. In broad terms, the “targeted genetic marker” could pickup that the room is a workshop and you can infer that that room contains tools and potentially you can further infer what kind of tools from the environment in which that workshop is located, e.g. a car mechanic or a wood factory, but not the exact suite of tools. Now if you open the door, you can look and list all of the tools present in that workshop, and though not in use you can define the potential activities that the workshop would support. This is what “metagenomes” provide, a list of tools available to the organisms present.

Now though having a list of tools is much more powerful, it is still not known if these tools are functional, i.e. some may be broken, or when these tools are being used. For this we need to capture a snapshot of specific conditions when these tools are in use. In cells, the genome are a collection of genes (tools) that the cell can select and express to use in particular conditions, for example, preparing an enzyme to break down a toxic compound. This is done through the generation of RNA which are carbon copies of the genes. The full complement of generated RNA (transcriptome) at a particular point in time will allow you to understand the actual behaviour of a cell at that time. in the same way as for metagenome, the RNA generated from a whole community of microbes can be analysed providing an overall view of what all of the microbes from that community are doing (metatranscriptomes).

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What are metagenomes?

Metagenomes means the analysis of the collection of all genomes in a sample. To study the genetic make up of an organism, for example a koala, researchers can analyse and decode the full genome of one specimen, which will provide the genetic blueprint of that organism. It is the same as going to a library, selecting one book and reading the contained text.

Environmental microbial communities can be very complex with thousands of unique organisms. Picking out each separately to analyse them would be slow and near impossible, so researchers analyse the genome of all of the microbes together, then use a computer to recreate the individual microbe genomes as a giant puzzle.

Again, imagine a library with hundreds of books and a team of researchers wanting to study and understand all of the books and message contained. They could go and painstakingly pick up each book (isolate the organism) and read each book, this is slow and some book might be smaller or harder to work with. Now imagine they take these hundreds of books, place them all together in a giant blender and use a machine to read all of the pieces as a giant puzzle, to digitise the information. This would be faster, ensure all books are included and many more libraries can be looked at.

Using such techniques, enabled by advances in technologies and computing, it is now possible to greatly speed up the quantity of environments looked at and the depth of information retrieved.

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What are metatranscriptomes?

Metatranscriptomes are very similar to metagenomes, however, what is analysed in this case is the full complement of actively used portions of the genome, the expressed RNA. Thus, it is possible to understand what the microbes are actually doing at a location, for example converting nitrogen gas into ammonium, and not only what they have the potential to do.

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See how we put the data produced through the initiative into practice with a few case studies from the consortium as well as from our collaborators.