Oligotyping to Achieve Increased Taxonomic Resolution of the Oral Microbiome

The development of novel bioinformatics tools has allowed for researchers to glean increasing amounts of information from next generation sequencing of 16S rRNA gene data. Current methods of sequence analysis have been limited to taxonomic identity up to genera or operational taxonomic units (OTUs). However, within these taxonomic classifications, numerous species can reside and play key roles in health and disease. One example of this can be seen within the oral microbiome where numerous Streptococci species cohabitate and are functionally diverse while sharing similar 16S rRNA sequences that are not distinguished between in routine taxonomic classification.

This week’s journal club at 3:30pm on Friday, July 31st at West End pub will discuss “Oligotyping Analysis of the Human Oral Microbiome” by Eren et al. This publication demonstrates the usage of oligotyping, a supervised computational approach designed to provide increased taxonomic resolution in the oral microbiome (such as distinguishing between the aforementioned Streptococci), to achieve a better understanding of the human microbiome.

I look forward to discussing:

  1. Their results on the spatial location of various oligotypes
  2. The application of oligotyping to other microbiome data
  3. Limitations of oligotyping

Relevant/ Related Publications:

For the original methods paper on oligotyping, click here.

For information on minimal entropy decomposition, or MED (‘unsupervised oligotyping’), click here.

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Summing up: Exploration of the Virome in Inflammatory Bowel Disease by Norman et al.

Last week we drew our attention to the virome as discussed in “Disease-specific alterations in the enteric virome in inflammatory bowel disease” (Norman et. al). Continuing on the thread of ‘other’ components of the microbiome from our discussion last month on the mycobiome, this paper’s exploration into the virome was particularly eye opening. Their demonstration of numerous correlations between virome and microbiome diversity and size in ulcerative colitis and Crohn’s disease enforced and echoed the role non-bacterial components of the microbiome play in health and disease.

In addition, numerous elements of the methodology used were of particular interest. The multi-centre approach utilized in this study – involving cohorts from Cambridge, Los Angeles and Chicago – strengthened and added depth to their findings. In addition, the authors’ use of household controls was appreciated by the group as it took into account various elements, such as the built environment, diet, and human behaviours, that regular healthy controls or familial controls might not.

One area of interest that was highlighted, however, was the usage of the number of Operational Taxonomic Units (OTUs), as the number by which samples were rarefied for alpha diversity, as we had not seen this done before. Furthermore, many were surprised that only 15% of sequences were identifiable although this did highlight the need for a comprehensive viral database if future work on the virome is to occur.

Conversation then transitioned into a discussion on whether the field at large was ready to foray into the virome. Challenges such as how one might capture and identify the incredibly large diversity of the virome without a viral analog to the bacterial 16S rRNA gene or the fungal ITS regions was discussed. For example, this paper’s methodology and findings largely focused on bacteriophages, although many other types of viruses exist and are part of the virome. Acquiring a complete virome could be difficult due to the challenge of extracting all viral genetic material (as viruses differ in nucleic acids, structure, etc. unlike bacteria or fungi). This challenge was further compounded by the possibility that current methods for viral genetic extraction protocols (such as the virus-like preparations in this paper) may either be losing genetic material or be skewed by contamination due to an apparent lack of benchmarking.

Overall, this month’s meeting of the HMJC was impressed by much of the findings and methods utilized by the authors and found their study served as an excellent and needed foray into the world of the virome that highlighted its potential and its infancy.

June 2015 Journal Club Paper: http://www.sciencedirect.com/science/article/pii/S0092867415000033

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“Disease-specific alterations in the enteric virome in inflammatory bowel disease” [Norman et al, 2015]

Is IBD going viral? Pursuant to last month’s journal club discussion on the importance of examining the fungal mycobiome, a closer look at the intestinal virome seemed like the logical next step. Eukaryotic viruses have the ability to interact with IBD risk genes to potentially alter disease (Basic et al, 2014, Cadwell et al, 2010). Various environmental stimuli (nitric oxide, antibiotics) can modulate the relationship between virus and host, including bacteriophages and their hosts. Changes such as these may be inciting factors of disease onset or flare-ups, thus it is important to better understand the role of viruses in gut physiology.

This study published in Cell in January of 2015 examined the composition of viruses in multiple inflammatory bowel disease patient cohorts and also compared IBD patients to their household controls. The authors found a significant expansion of one group of bacteriophages that was reportedly not secondary to IBD-associated decreases in bacterial diversity. Increased richness of caudovirales was associated with both CD and UC. This was often accompanied by a decrease in the other predominant bacteriophage species, microviridae. Other viruses comprised less than 5% of all viral sequences that were detected.

We’ll meet again at West End pub at 3:30 pm. The primary objectives of this week’s discussion will be to discuss current and potential methods for reliably sequencing viral DNA, as well as the importance of doing so.

A few papers for relevant background information:

Bernstein et al, 2000. Viruses and inflammatory bowel disease: is there evidence for a causal association? http://www.ncbi.nlm.nih.gov/pubmed/10701147

Breitbart et al, 2003. Metagenomic analyses of an uncultured viral community from human feces. http://www.ncbi.nlm.nih.gov/pubmed/14526037

Perez-Brocal et al. Study of the viral and microbial communities associated with Crohn’s disease: a metagenomic approach. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696940/

Wagner et al, 2013. Bacteriophages in gut samples from pediatric Crohn’s disease patients: metagenomic analysis using 454 pyrosequencing. http://www.ncbi.nlm.nih.gov/pubmed/23749273

Wang et al, 2015. Metagenomic analysis of microbiome in colon tissue from subjects with inflammatory bowel diseases reveals interplay of viruses and bacteria. http://www.ncbi.nlm.nih.gov/pubmed/25939040

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Response to May 29, 2015 Journal Club: “Modulation of Post-Antibiotic Bacterial Community Reassembly and Host Response by Candida Albicans” [Downward et al, 2013]

Although this is a slightly older paper, the role of fungi in the gut microbiota is becoming an increasingly prevalent topic of discussion among microbiome groups, making this discussion a timely one. This group also previously published two papers on the role of antibiotics and fungal microbiota in allergic airway disease (Noverr et al 2004 & Noverr et al 2005). The primary finding of the paper at hand was that the introduction of C.albicans following antibiotic treatment causes marked changes in the reassembly of the microbiome, even in the absence of inflammation.

As discussed in journal club, there were several positive aspects to the study. First, the authors demonstrated the importance of the role of fungi in the gut despite relatively small numbers compared to that of bacteria. This is an aspect of the microbiome that has largely been neglected in recent years but fungi are large organisms and as illustrated, possess a significant ability to impact community reassembly following a disturbance. The inclusion of a multiplex qPCR array was helpful in examining changes to gene expression in response to various microbiome disturbances, although many aspects of this data were not addressed in the discussion. Finally, the relative abundance graphs were a good way to summarize taxonomic distribution, though we did discuss the challenges of pooling this type of data when samples are vastly different from one another.

There were a few other suggested areas for improvement to the study and paper that were discussed as well. For example, it was difficult to compare the bands from the graphs in Fig. 7 as their relative abundances may have been different (ie. Graph C: band for undisturbed ~10% versus band for disturbed + C.albicans ~1%). As a result some of the graphs were difficult to interpret with accuracy. The practice of using a log scale to demonstrate relative abundance followed by a non-log scale to plot taxa is also misleading. In addition, the authors’ use of rarefaction curves was not ideal. Finally, the title of Fig. 7 should read Day 21, not Day 7. Overall there were no major issues with the study or its findings.

We found this paper helpful in demonstrating the need for addressing fungi in microbiome analyses. However, there are still many obstacles to overcome in this area, such as limited fungal databases.

May 2015 Journal Club Paper:

http://www.nature.com/srep/2013/130712/srep02191/full/srep02191.html

Previous Work:

http://www.ncbi.nlm.nih.gov/pubmed/15321991

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC538952/

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Should we care about fungi? The mycobiome: an emerging field of the human microbiome research

penici2With the advancement of next-generation sequencing in the last decade, our knowledge about the trillions of microbes that the human body harbours has exponentially increased. Microbiome research has mostly been focused on the study of bacteria notwithstanding that the classical definition of the microbiome includes viruses and fungi. Despite the numerous microbiome publications, we know only little about the impact of fungi on health and disease. That said, with the increased evidence demonstrating the importance of host bacterial communities on host physiology and their involvement in health, research groups are interested in looking at the impact of fungi as well.

I have selected this publication because it demonstrates the potential effect of fungal colonization on the bacterial composition in vivo. In this study, Downward and colleagues demonstrated that the introduction of C.albicans in a disturbed microbiota was sufficient to alter the reassembly of the bacterial population.

During our journal club Friday, May 29th at 3:30pm, Westend Pub I hope to discuss:
(a) The underappreciated impact of fungi in microbiome research
(b) The feasibility and significance of including a mycobiome component in our research
(c) The limitation of the ITS sequencing

Relevant reviews for people interested to read more on the topic include:

The mycobiota: interactions between commensal fungi and the host immune system:

http://www.nature.com/nri/journal/v14/n6/full/nri3684.html

The human mycobiome in health and disease:

http://genomemedicine.com/content/5/7/63

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Going beyond 16S rRNA gene sequencing to examine the functionality of the gut microbiome.

As the technology advances, so does our ability to probe and profile human microbiomes. Next week, I will lead a discussion of the recent paper entitled Functional Dynamics of the Gut Microbiome in Elderly People during Probiotic Consumption.” by Emiley Eloe-Fadrosh et al. This paper combines together many aspects that we’re interested in here at McMaster, including the inclusion of techniques beyond 16S rRNA gene sequencing to gain a more complete understanding of how the elderly gut community is affected by the popular probiotic, Lactobacillus rhamnosus GG (LGG).

During our journal club Friday, May 1st at 3:30pm, Westend Pub I hope to discuss:
(a) the combined use of 16S rRNA gene sequencing and RNAseq
(b) their conclusions regarding the effect of LGG on the gut microbiota
(c) on the more philosophical: discuss their use of data visualizations, and how we can apply their methods to our own datasets

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Introducing: Microbiome Working Group

More and more, we are becoming aware that there are a lot of researchers at McMaster who are dabbling in the microbiome. Some labs are hot-beds of microbial research, whilst others are using microbiome-related datasets as part of a more diverse toolbox to study their phenomenon of interest. The later means that there are often one or two researchers per-lab who are using these tools in semi-isolation.

I am very happy to introduce the Microbiome Working Group, a weekly, drop-in, work/discussion hour with the aim of bringing microbiome researchers together, independent of sample type, laboratory, or skill level. This isn’t a meeting; there are no presentations. This is about getting research and analysis done. Bring your laptop and use the presence of like-minded others as motivation. Test out your new analysis idea on your peers (before you spend months trying it out). Bring any obstacles you might have run into. Come once a month, or come every week.

Microbiome Working Group will occur every Tuesday at 3pm in HSC-3N10A until further notice. Students, postdocs, PIs etc., human microbiome or otherwise, everyone is welcome!

(Note: if this kind of thing interests you, you most likely would get a kick out of our monthly Journal Club as well!).

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Summing up: Bassis et al.’s take on the influence of the URT on other human-associated biogeographies.

Last week, Pat led us in the examination of a new paper from the Huffnagle group at the University of Michigan. This was the first meeting of McMaster’s HMBJC in a few months which meant a very lively discussion since we were bursting to talk of all things microbe!

As a group, we really enjoyed and appreciated this study. It was great to see researchers going back to their ecological roots with discussions of ecosystems, gradients, niches, and how different anatomical sites can affect one another. We really enjoyed how easy this paper was to follow, even for someone outside of the microbiome research field, using simple-to-read graphs and visuals that any scientist could interpret. This paper also tackled some areas which, historically, have been hard to sample or where sampling has been shrouded in doubt (e.g. BALs). Additionally, for our group, the Θyc statistic was interesting to see in use, and this publication led us down a citation-trail to learn of its advantages over metrics that we more commonly employ such as Bray Curtis and Jaccard.

We did comment on how the nasal swab might have captured a slightly different microbial community compared to a nasal wash, which would me more consistent with the other sample types used in this study. A greater proportion of mucosally-associated species obtained from the “scraping” motion of a swab might account for some of the differences between the nasal communities when compared to other anatomical sites which were sampled by “washing”. Additionally, we were sad to see an opportunity to make more intra- and inter-patient comparisons across all sites not taken, but perhaps that will be the group’s next publication.. we look forward to it!

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Bacterial communities in the lung and stomach – how did they get there?

The human microbiota has become more diverse than what scientists believed when the Human Microbiome Project started over a year ago.  Initially, the lung and stomach, two unique mucosal surfaces, were not even initially included in the sample collections.  As culture-independent methods of studying the microbiome has expanded, it revealed that there are diverse and unique communities that exist within these organs, but there is little knowledge about what influences these communities.  A new paper out of Gary Huffnagle’s group at the University of Michigan decided to look at just that.

In their analysis, Bassis et al.  began to look at potential sources of microbes, including the mouth and nasal passages. The mouth is exposed to and produces more biomass daily, with saliva production and access to nutrients via food being chief among them.  The nose is known to harbour important bacteria and potential pathogens.  Based on the findings of their work, it appears that the mouth microbes have greater influence in what reaches the stomach and lungs, but that the lungs can control which bacteria take hold, as they looked at the elimination of Prevotella from the mouth microbial communities into the lung.

In our journal club at 3:30pm on Friday, March 27th at West End pub, I look forward to discussing:
(1) Their methodology for sample collection and sequencing
(2) Their microbial diversity analysis, especially their use of θyc distances
(3) Their model utilizing Prevotella for species elimination

Abstract: No studies have examined the relationships between bacterial communities along sites of the upper aerodigestive tract of an individual subject. Our objective was to perform an intrasubject and intersite analysis to determine the contributions of two upper mucosal sites (mouth and nose) as source communities for the bacterial microbiome of lower sites (lungs and stomach). Oral wash, bronchoalveolar lavage (BAL) fluid, nasal swab, and gastric aspirate samples were collected from 28 healthy subjects. Extensive analysis of controls and serial intrasubject BAL fluid samples demonstrated that sampling of the lungs by bronchoscopy was not confounded by oral microbiome contamination. By quantitative PCR, the oral cavity and stomach contained the highest bacterial signal levels and the nasal cavity and lungs contained much lower levels. Pyrosequencing of 16S rRNA gene amplicon libraries generated from these samples showed that the oral and gastric compartments had the greatest species richness, which was significantly greater in both than the richness measured in the lungs and nasal cavity. The bacterial communities of the lungs were significantly different from those of the mouth, nose, and stomach, while the greatest similarity was between the oral and gastric communities. However, the bacterial communities of healthy lungs shared significant membership with the mouth, but not the nose, and marked subject-subject variation was noted. In summary, microbial immigration from the oral cavity appears to be the significant source of the lung microbiome during health, but unlike the stomach, the lungs exhibit evidence of selective elimination ofPrevotella bacteria derived from the upper airways.

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Fork vs. Spoon: How do you consume your science literature?

Today’s journal club discussion was about how to stay on top of the literature for your field. To me there are three parts to this question:

  1. How do you find out about new papers then what do you do store these until you have time to read them?
  2. How do you read papers: digital vs hard copy? Where do you store and how do you organize the notes you take while reading said papers?
  3. How much time do you spend reading papers each week?

Here are our thoughts on each question but we’d love to know what you do?

1. Finding new papers and keeping a “to read” list

There is a clear dichotomy amongst HMjc members: those who rely on search terms and those who don’t. The first group was the biggest so lets talk about this strategy first. Search termers get notifications, usually through email, when papers with their particular pubmed search terms are published. Some members of this group also get alerts from the individual journals themselves when each issue is published in the form of a table of contents (or TOC) email. The pro of this method is not missing the main papers of interest in a field. The cons of this method are missing papers that don’t match the search terms directly and that it’s time consuming to go through all of the alerts each week. The search termers have the emails but they pretty much just read them as they come in.

The non-search termers rely more on other members of the community to alert them to papers of interest by  following them on google+ and twitter. They also make use of the more mysteriou3466154462_8d3b399cf4_os algorithms in pubchase and google scholar that learn what you like based on what you read, what’s in your library and your publications. The cons to this method include needing to keep a “to read” list as well as the obvious problem of missing key papers and having to do more individual searches. As a member of the non-search termers I can say that although I dread sifting through the soul-crushing deluge of alert emails, I find all of the search termers to be very knowledgeable about current research and often they are the ones I’m following on google+.

I think that the answer here is spork.

2. Digital vs. Paper

For me it’s important to be able to find my notes later… so paper is a problem and I mostly use mendeley and evernote but once a week or so I scribble all over a paper copy. One person has a system of writing “see paper copy” in mendeley when they’ve made hardcopy notes, which I think is brilliant. But pretty much it’s a matter of what method keeps each person most focused.

A couple of people are trying out new gadgets and apps for keeping notes. Livescribe combines a pen a paper combo that lets you take digital notes, includes voice recording and seems very slick. The noteshelf tablet app combined with a good quality stylus lets you scribble on a pdf of the paper you’re reading. I’m eager to see how these work for organizing and searching notes, I’ll keep you posted.

3. How much do you read each week?

The answer is some weeks LOTS and some weeks less. The bottom line is that reading the literature is time consuming so you have to dedicate time to it. Some people like to hide at the coffee shop or the library and others hunker down at their desks but either way you just have to sink your teeth in, as often as you need to, until you feel like you have a handle on your field of study.

Considering how much time is needed for reading abstracts and papers I feel like I need more time-saving strategies for finding and organizing the literature and my notes and would love your input on point 1, or the other two if you insist.

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