Posts

Limitations of microbiome measurement: Prof. Gloor shares insights with ISAPP

February 20, 2019

The number of papers published on the human microbiome is growing exponentially – but not all of the studies are equally well designed or reported. Evaluating the latest research requires a basic understanding of the latest approaches to microbiome methods and data analysis.

To help equip scientists not conducting microbiome research with the tools to understand microbiome-focused publications, ISAPP hosted a webinar titled Understanding microbiome experiments: a critical assessment of methods and data analysis. The webinar featured Gregory Gloor, PhD., Professor, Department of Biochemistry, Schulich School of Medicine and Dentistry, The University of Western Ontario, Canada.

A recording of the webinar is available here, and Prof. Gloor’s slides are available here.

Prof. Gloor opened his talk with a sobering perspective: the current body of microbiome publications is fraught with problems. There is a fundamental lack of reproducibility in the microbiome field (Sinha et al. 2017). This is largely due to the large number of tools available and a lack of an a priori established research plan for microbiome analysis, which should be consistently followed throughout a project. At every step of the way, many decisions must be made regarding wet lab methods, bioinformatics toolsets and statistics to use. Different choices lead to different results. Once the biological specimens are assayed, choices for bioinformatics and statistical analyses can greatly influence the conclusions. In short, it’s possible to view the data through so many different lenses that eventually a researcher can find a story worth telling. How close that story comes to the truth is a principle that sometimes is sacrificed for the sake of an interesting story.

Another important challenge to the field is representative sampling. Too few samples are typically taken, often because of cost limitations, so that the samples do not reasonably approximate the truth about the environment being sampled. Conclusions from such studies result in both many false positives and many false negatives.

Prof. Gloor also warned about outsourcing microbiome analysis. Commercial entities often use every metric, hoping the customer will get some outcome they hoped for. Further, their tools are often outdated or proprietary. So caution must be used – there is no substitute for expertise.

Some suggestions for improving outcomes were offered:

  • Each project should stipulate a research approach and outcome a priori, which is consistently followed throughout the project.
  • Methodological consistency is important within a lab, but analytical methods do not necessarily need to be standardized across all labs. If all labs use the same methods, consistent, but incorrect, outcomes may result. So use of different metrics is good, but methods should be consistent within a project. The value of different research groups using different methods to ask particular research questions is that if the same result emerges from different approaches, it increases confidence that the results are true.
  • Gloor cautioned that microbiome datasets are compositional, and compositional data approaches must be used (Gloor et al 2017).
  • Functional readouts have less methodological variation than taxonomic readouts. Therefore, functional analysis of shotgun metagenomics or shotgun metatranscriptomics is typically a more reproducible, and also more informative, readout.
  • Recent advances have significantly decreased the cost of performing shotgun metagenomics for both taxonomic and functional readouts (Hillmann et al 2018).
  • There are now near-complete microbial genomic datasets available for European, North American and Asian populations (Almeida et al 2019) that will make it easier to functionally map datasets.

Prof. Gloor mentioned an interesting aside: prior clinical trial registration, ~60% of large clinical trials showed benefit of the intervention being tested. After the registration process required declaration of primary research outcomes, that number dropped to closer to 10% (Kaplan and Irvin 2015). This suggests that primary outcomes and analysis methods need to be in place to restrict researcher bias. Right now such mechanisms are insufficient in the microbiome field.

Prof. Gloor’s paper, Microbiome Datasets Are Compositional: And This Is Not Optional, provides great background reading for this webinar.

This webinar was developed by ISAPP Industry Advisory Committee representatives as an extension of the annual IAC Learning Forum.

Dr. Gloor is a professor of biochemistry with broad experience in molecular biology, genetics and genomics. His research is focused on the development of tools to examine 16S rRNA gene composition, gene expression of mixed population samples and metabolomic analysis of clinical samples. He is currently working on developing and adapting principled methods to characterize correlation and differential abundance in sparse, high throughput sequencing data as generated in 16S rRNA gene sequencing surveys, meta-genomics and meta-transcriptomics. One of his primary contributions has been the ALDEx2 tool in Bioconductor for the analysis of high-throughput experiments that generate counts per sequence tag: 16S rRNA gene sequencing, metagenomics, transcriptomics and selex-type experiments.

stool sample for lab

Microbiome Analysis – Hype or Helpful?

September 2017. By Karen Scott, PhD, Rowett Institute, University of Aberdeen, Scotland.

Since we have realized that we carry around more microbial than human cells, and that these microbial inhabitants are important to maintain our health, searching for the bacterial species that are implicated in causing disease has become the holy grail of microbiology. However, to understand which bacteria are unnaturally present or absent in a disease state, we first have to understand what constitutes normal. This is hampered by the fact that we are all different – and our microbial communities are also all different. In fact, the faecal bacterial community in samples taken months apart from one person will be identifiable as coming from that specific healthy adult, but the community will be quite distinct from samples from any other healthy adult. In the same way, the microbial community of two individuals suffering from the same disease will be different.

Despite these differences, scientists have managed to establish some facts over the past 15 years. Too many Proteobacteria, which includes Enterobacteria and E.coli, in your large intestine is not generally good news. Firstly, it means that conditions in the large intestine are probably not as anaerobic as they should be. Secondly, an expansion in these populations usually means a decline in something else – after all food and places to live are finite resources. Bacterial diversity in the adult intestine is also important. The main factor that has been found across many different diseases is that bacterial diversity is lower in diseased individuals than their healthy counterparts. This does not necessarily mean that a low diversity is causing the disease, as various features of the disease (including any antibiotic therapy, inflammation, decreased or increased transit time) may all themselves affect the diversity of the microbiota.

Although scientists have not succeeded in defining a ‘healthy microbiota,’ there is an increasing trend to get your microbiome tested. Entrepreneurial microbiome companies are bombarding us with offers to “send in a small sample and find out about your gut microbiota”. All of course, for a ‘reasonable’ price. So, should you?

This really depends why you want to know, and what level of detail of analysis is being offered. Remember the orders of taxonomy? Kingdom, phylum, class, order, family, genus, and species.  Some companies identify the bacteria in your faeces only to the phylum level. This is a taxonomic level above the level needed to differentiate mammals and fish (these are ‘classes’). If you told someone that there were more fish in the Indian Ocean than mammals would this be a surprise? It would be such an expected fact it would be meaningless. This is similar to describing the microbiota at a phylum level – Bacteroidetes numbers versus Firmicutes numbers. Such numbers are meaningless. However, continuing the fish analogy, if you said that there were more mackerel than tuna in the North Atlantic Ocean this becomes a bit more meaningful. The fisherman immediately knows what type of fish he is more likely to catch, and perhaps even which net to use. The same is true of the microbiome. Telling someone that he/she has a lot of Enterobacteria and few Roseburia is actually useful as we know from studies that this represents an abnormal balance of bacteria and something should be done to redress this. Yet the bottom line health consequence of this abnormal balance of bacteria remains to be determined. So getting your gut microbiome sequenced could be useful – depending on what level of information you will receive, and what you are prepared to do about it.

And so we come to the next problem. Having established what your gut microbiota is, how are you going to make it better? And will that make YOU better? At the moment scientists don’t really have a good answer to these questions. Specific prebiotics can certainly be useful to increase the numbers of some bacteria generally assumed to be beneficial – such as Bifidobacterium, Faecalibacterium prausnitzii and even Roseburia species. But it is not really clear what the exact health benefits of such an increase in bacterial numbers would be. Health claims on prebiotics are currently limited to ‘improve intestinal transit’ and ‘lower the glycaemic response’. If you found out that your microbiota had a low diversity, increasing the variety of foods in your diet, in particular the fibre component, could certainly improve this. Our gut microbiota basically relies on our undigested food to survive, so providing a greater amount and more types of food containing fibre and prebiotics will definitely encourage populations of diverse bacteria to expand. In addition to improving digestive health, fibre fermentation by gut bacteria also results in the production of microbial products that have been shown to have health benefits.

So by all means get your gut microbiome analyzed if you want to, but perhaps instead, save your money and just increase your prebiotic and fibre consumption, which will increase levels of the potentially beneficial bacteria that are already there in your gut.