Variation exists in microbiome sequencing and analysis

Microbiome sequencing and analysis is at the heart of microbiome science.  While established protocols do exist in order to identify the bacteria in samples, there is still a lot of variability between labs, individuals, reagents, analytics techniques, and instruments.  This makes comparing data across groups difficult.  The microbiome quality control project (MBQC) was carried out in order to determine the sources of variability that exist.  During the project identical samples were sequenced by various labs, and the results were compared.  The project’s conclusions were published this past week in the journal Genome Biology.

There was a lot of variation from many of the sources during experimentation.  Overall, DNA extraction technique proved to be a major source of error.  On the other hand, sample storage protocols, such as length of time the sample spent in the freezer, appeared to only play a small role in variability. Another encouraging result was that the the bioinformatics pipelines, i.e. the software programs used to determine the bacteria from the raw data, displayed consistent results.  The project also included negative controls that should have contained no bacterial DNA at all, however many labs reported seeing non-trivial sequences.  In addition, the samples with known compositions often times had spurious DNA from bacteria that should not have been there.  Sometimes the results showed upwards of 7x more bacteria organizational taxonomic units (OTUs) than expected.

This initial MBQC project accomplished two of its major goals, and therefore should be considered a success.  First, it demonstrated a need for quality control within microbiome scientists.  Second, it helped narrow down the variables that need to be studied more robustly in future MBQC projects.  For now though, we must just acknowledge that error does exist in microbiome studies, and to keep that in mind when interpreting results.

Stool consistency should be considered during microbiome research

Stool sampling remains the most common method of measuring the microbiome of the GI tract.  Researchers are well aware of its limitations, but its ease and convenience for both scientists and donors makes it nearly irreplaceable at the moment.  The most common issue regarding stool samples that is often pointed out is that it is not representative of the GI tract, and that it only samples the lower colon and not the more proximal GI tract.  In addition, it does not account for bugs that are attached to the mucous linings of the intestine rather than those that transiently pass with our feces.  Related to this point, last week Jack Gilbert and John Alverdy, professors from the University of Chicago, published a piece in the journal Gut regarding stool microbiome sampling and stool consistency.

Professors Gilbert and Alverdy argue that stool consistency greatly affects the stool microbiome populations.  The stool consistency is normally a function of intestinal transit time, with the shorter the duration between eating and passing stool being associated with watery stool, while a longer duration is associated with a more solid stool.  They point to studies that that show different bacteria have evolved to either grow rapidly when the stool is quickly moving through the lumen, in order to proliferate with the shorter duration access to nutrients, or to grow slowly and more completely utilize the available nutrients when the stool is accessible for longer periods.  Measurements of stool consistency are hardly ever performed during normal sampling, and these same studies tend to make generalizations about different phyla, like Bacteroides and Ruminococcacea, when in fact these different can be explained by stool consistency. 

In a time where microbiome diagnostics are starting to be considered as helpful indications for varous diseases, this type of quality control needs to be established.  Stool sampling is not perfect, but it is necessary, and for that reason steps should be taken now to improve and control its usefulness, especially in a clinical setting. 

Freezing fecal samples preserves the microbiota

Editors note: Happy St. Patrick's day to all our readers!  We hope you all enjoy some tasty fermented beverages today, (always in moderation based on yesterday's blog), and instead of corned beef and cabbage, how about corned beef and kimchi!

The microbiome field has exploded over the past few years in large part due to the advent of high-throughput sequencing technologies.  These technologies give scientists the ability to sequence the bacteria in a sample at a fraction of the cost with much greater accuracy than prior methods.  With the growth of this new field, there are more research teams conducting microbiome research with each lab doing things slightly differently. It’s important for scientists to understand the multiple factors that influence the results of experiments, and one of those variables is the storage condition of samples prior to DNA extraction. 

A research team from Ireland published a paper in the Proceedings of the National Academy of Sciences (PNAS) that investigated the impact that storage techniques had on the microbial communities within samples using a MiSeq from Illumina. While it is likely that immediately extracting the DNA from a sample is the most ideal method for research, this is often not feasible due to sampling locations as well as collaborations between investigators at various sites. 

In this study, samples were collected from 7 individuals and each sample was separated into three groups, fresh samples that were processed within 4 hours of sampling, samples that were “snap frozen” and immersed in dry ice for 4 minutes before being stored for a week at -80°C, or samples that were frozen immediately at -80°C.  The researchers found that there were no significant differences between the three experimental groups. The samples that were sampled fresh, snap frozen using dry ice, and those frozen only at -80°C had similar numbers of total bacteria as well as bifidobacteria which was sampled due to its sensitivity to freezing as well as its low abundance in fecal microbiomes. 

This study has shown that immediately freezing fecal samples should appropriately preserve them for use in research. This type of study is incredibly valuable in order for the greater scientific community to understand the impact that important variables such as storage techniques can have on microbial sampling.  There are many variables that play a role in microbiome data and it is important for studies like this as well as initiatives like the Microbiome Quality Control Project to lead the way in allowing us to better understand these factors.  

Current challenges in microbiome research

Scanning electron micrograph of E. Coli bacterium.

Curtis Huttenhower, Rob Knight, Owen White, Jacques Ravel (all members of our SAB), and others recently published a commentary titled "Advancing the Microbiome Research Community" in Cell.  The paper was a 'State of the Microbiome' address and outlined the current challenges and the future outlooks of the microbiome field.

Among the many challenges they outlined include how to design causality studies, and how to attribute causality of phenotypes to specific bacteria, proteins, or metabolites.  The paper also discussed the bioinformatics bottleneck that is occurring in the field, as the necessary expertise required to properly analyze sequencing data is severely lacking.  There also lacks a proper centralized online repository for all sequencing data that is generated.  This sequencing data needs to include metadata that would describe how the data was generated.  Furthermore, the authors describe the need for guidance on how to properly define and comprehensively describe phenotypes, such as inflammatory bowel disease (IBD), in microbiome studies.  Without this standardization, proper comparison between studies can not take place.  Finally, institutional review boards (IRBs) need guidance on how to properly regulate human microbiome studies in order for scientists to properly prepare and carry out their projects.

While the above areas are only a few of the challenges facing the field, each of them reinforces the mission and programs of the AMI.  The MBQC is standardizing techniques and creating rigorously tested analytical protocols by investigating the sources of variation in microbiome testing.  In addition, the AMI is focusing on future data repository efforts.  These are just a few of the areas we hope to serve the field in the coming years.  We believe a nonprofit, specifically the AMI, can help fill these voids and address many of the issues outlined in this paper.

Quality control is critical to microbiome research

We just got back from two days in Rockville, MD for a meeting of the Microbiome Quality Control Project (MBQC).  The MBQC is a project that seeks to understand variability in microbiome data from sampling to analysis and make recommendations as to best practices.  Its steering committee includes some of the leading scientists in the microbiome field, Rob Knight, Curtis Huttenhower, and Owen White (all members of the AMI Scientific Advisory Board), along with epidemiologists from the National Cancer Institute Rashmi Sinha and Christian Abnet.  

Labs from around North America signed up to participate in two different aspects of the study, microbiome handling and bioinformatic data analysis. The labs that were involved in the handling aspect of the study received anonymized samples from a central repository and were tasked with extracting DNA from the samples and sequencing them.  They then made their data available (still anonymized) for the groups who signed up for the bioinformatics phase of the projects to analyze the data. Because all of the groups were handling and sequencing the same samples, differences in results meant that the variability was being caused by some aspect of the handling process or differences in the analysis.

At the meeting the steering committee presented the results of the study followed by a day and a half of discussion of the results and what steps could be taken to develop more standardized results, protocols, and reference materials for future microbiome studies. The results were exciting to see but even forgetting the results, the overall initiative was a huge success. This was a project that took place for over a year and all of the project's participants volunteered an enormous amount of time and conducted their research studies without receiving any funding.  It was wonderful to see a group of the world's leading scientists to come together like this with the sole goal of bettering future microbiome research.  

The AMI supports the MBQC and looks forward to being involved in the future phases of this work. As we learned at the meeting, there is so much left to be done to improve microbiome quality control and this was just the beginning.  More information about the results of the study and the meeting will be published in the coming months.

Excitement over the microbiome needs to be kept in check.

On August 20th Nature published a piece asking readers to curb their collective enthusiasm over advancements in the microbiome field.  This is a lesson that we at the AMI have heard over and over again from the scientists we talk to.  When talking about linking diseases and phenotypes to the microbiome Owen White told us, and I am paraphrasing, "The way the science is currently performed by many researchers, I would be more surprised if an investigator DIDN'T find a connection between the microbiome and whatever he or she was studying!"  

This is an important lesson to keep in mind, and it is the reason why so many of the leading microbiome scientists are supporting projects such as the MBQC standardization project and the HSPH microbiome core.  These scientists realize that for the microbiome to make a true impact on human health the early stages are critical, and good science needs to be done.  It also speaks to the need for high quality journals with rigorous reviewers.

These goals are shared by the AMI.  We hope to support the MBQC as it continues to advance and grow.  We also will start our own microbiome core (AMICAR) to help researchers ensure the quality of their studies from study design through data analysis.