About ten years ago my former PI (second author in the below paper) did quite a job on this in Burkina Faso, analyzing the composition of the gut in children living outside cities, in cities, and a comparison with European children.
Obligatory disclaimer: I was working in his laboratory at the time, but I wasn't involved in that research.
How does one analyze the gut biome? I imagine it involves running poop through a machine - what kind of machine? I would love to learn more about the technical process.
The paper uses 16S rDNA sequencing, which is a bit old fashioned now but it was a good method when the paper was published. The steps basically involve:
1. Extract all DNA from poop, normally using a kit that basically makes DNA stick to tiny plastic beads. You wash the beads in a bunch of different chemical solutions to isolate DNA from the original sample and purify it. There are a lot of different methods to do this.
2. Amplify a small section of DNA that's universally unique to bacteria and archaea which is used as a barcode. This barcode has some areas that change a lot across different species and some areas that don't change much.
3. Sequence the amplified DNA. The DNA sequencer determines the sequence of nucleotides in each DNA amplicon (an amplicon is a piece amplified piece of DNA). An example DNA sequence is ACCTGGCT
3. The DNA sequencer produces millions of DNA sequences in parallel and stores them and some metadata (e.g. quality and confidence measurements) in text files
4. When this paper was published, a friendly bioinformatician would have taken the text file and clustered the different sequences. Sequences 97% similar were binned together as a rough approximation of a species. Different taxonomic levels have different cutoffs, but it's all quite vague and there are better methods now that involve denoising sequences from quality measurements (e.g. dada2 method)
5. A count for each different bin is generated, and "representative sequences" for each bin are matched against taxonomic databases to see what species are present
6. Normal ecological analysis is done on the count data to calculate alpha and beta diversity or do other types of analysis. Once you have counts, it doesn't matter that the data are from bacteria instead of sheep or penguins
Newer methods involve sequencing every single bit of DNA in a sample, not just a specific region. This is called metagenomics and it's very hard to do and requires very big computers and big DNA sequencers.
Great summary! Although I would argue that 16S is still a perfectly good (and cost-effective) method, especially with DADA2. There are also neat sequencing techniques that like CCS which give you really high resolution of a target region (amplicon) without sequencing a lot of redundant/uninformative DNA.
Very small amounts are sampled, on the order of grams. I'm not sure exactly, I worked on the bioinformatics side of things. I think an Illumina MiSeq requires 50 - 500 nanograms of DNA to work well.
Sampling and storage methods can significantly change the bacterial composition of an environmental sample (in this case, poop). The exact protocols will depend on the aims of the study. The gut microbiome is a gradient and very dynamic. Different parts of the gut will have different bacterial compositions. Some people might prefer to get a locally accurate sample from a biopsy of the intestine, but you won't manage to recruit many participants. Other studies may prefer to use faecal samples as a proxy for overall gut state, which lets you recruit more people. Some protocols may homogenise (blend) the poop before sampling, others might not. Here's a nice review:
> I think an Illumina MiSeq requires 50 - 500 nanograms of DNA to work well.
You can go as low as 10ng depending on the library you use (or so the vendors say), but I'm not sure it's the case for these specific applications (my experience is with other, equally difficult samples, but from a single source).
Really cool, thanks for the details. It's reminiscent of a plot point in a sci-fi I'm reading, Zendegi - the protagonist is hell-bent on cracking the problem of simulating the brain in a computer, but funding is running out. Her coworker excitedly explains that he's awarded a grant for his project: simulating the interactions of microbe species in latrines, for purposes of preventing outbreaks of disease. He wants our protagonist to jump ship on the brain stuff, says it will always be there, that she can do some real good right now, and the skills will transfer over, she won't be wasting her time learning how to simulate the microbial communities in poop.
(Alas, an eccentric billionaire with hopes of uploading himself to a supercomputer swoops in to fund the mind-mapping project...)
Edit: grabbed from the methods section. I was wrong, they didn't culture :)
>Fecal samples were collected by physicians and preserved in RNAlater (Qiagen) at −80 °C until extraction of genomic DNA (28) (details in SI Materials and Methods).
“The person(s) in charge of a clinical trial or a scientific research grant. The PI prepares and carries out the clinical trial protocol (plan for the study) or research paid for by the grant. The PI also analyzes the data and reports the results of the trial or grant research. Also called principal investigator.”
Interesting! In De Filippo et al., they mention that Bacteroidetes sp are absent in urban Europeans, but present in rural Africans; in the OP they mention a similar sounding Bacteroides being present in chimps but not in humans. Is there any similarity between these apart from their similar spelling?
> indicating the importance of preserving this treasure of microbial diversity from ancient rural communities worldwide
This study (and the FA) mention losing/presrving microbial diversity, but don't touch on how we got these microbes in the first place.
I was expecting it comes mostly from what we eat and the environment, including eventually the people around us (and the mother - baby link ?)
I kinda wondered if we hadn't lost these microbes because they disappeared from our surroundings, and we'd get them back if they became prominent again, making the (temporary?) loss less impacting.
Very likely. Almost all probiotics people take are transient. Meaning they just pass through and don't form long term colonies in our guts. There's a few exceptions with some soil-derived probiotics but those are also the least predictable
It seems really likely to me that our bodies have likely adapted to an environment where exposure to certain types of bacteria is very common. For example, there's been a lot of research on the potential of ammonia oxidizing bacteria (AOB) such as Nitrosomonas to reduce body odor when applied topically. There's companies trying to use this to sell you "probiotic shampoo" and other skin care products. But you know where else Nitrosomonas is commonly found? Most soils. These bacteria are also extremely easy to kill off with shampoos and even the small amounts of fluoride added to tap water
Given how deeply we depend on microbes for digestion it doesn't seem far fetched to think our skin microbiome has also adapted to a ready supply of these microbes to help us in certain ways. Besides keeping us less stinky by eating our sweat, skin microbes have also been shown to help with acne,[0] protect us from UV damage,[1] and has even been shown to have deep interaction with our gut and brain health.[2]
Interesting, I discovered once that soil on my socks could make them not smell. I don’t know if the specific soil mattered though, and the circumstances for my discovery were never replicated.
>Almost all probiotics people take are transient. Meaning they just pass through and don't form long term colonies in our guts.
is this also the case for frequent ingestion? say you eat probiotic yoghurt on a regular basis, are you saying the good stuff is in a constant cycle of dying and being replaced?
> Besides keeping us less stinky by eating our sweat
That sounds wrong. Sweat itself doesn't smell until microbes have had a chance to act on it. You can sweat right after a hot shower and you won't smell.
Its fascinating as its not clear between rural African and European urban children, who is healthier. Stereotypically Africans are probably closer to nature, less stress and fewer processed foods, where Europeans have more medicines, variety of foods and less exercise.
Wow is the mobile UX of PNAS bad — the cookie pop-up has no X button and if you click ‘continue’ — which I thought meant continue to the article — it directs you to the terms of service. If you press back, you see the same pop-up again.
Obligatory disclaimer: I was working in his laboratory at the time, but I wasn't involved in that research.
https://www.pnas.org/doi/10.1073/pnas.1005963107