Category: Lab Publications

New lab paper: Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota

New paper out from the lab. This is from a collaboration with Stan Marks in the Vet School at UC Davis. The work was led by Katti Horng with assistance from Holly Ganz.

Citation: Horng KR, Ganz HH, Eisen JA, Marks SL. (2018) Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota. PeerJ 6:e4827https://doi.org/10.7717/peerj.4827

Abstract: Studies involving gut microbiome analysis play an increasing role in the evaluation of health and disease in humans and animals alike. Fecal sampling methods for DNA preservation in laboratory, clinical, and field settings can greatly influence inferences of microbial composition and diversity, but are often inconsistent and under-investigated between studies. Many laboratories have utilized either temperature control or preservation buffers for optimization of DNA preservation, but few studies have evaluated the effects of combining both methods to preserve fecal microbiota. To determine the optimal method for fecal DNA preservation, we collected fecal samples from one canine donor and stored aliquots in RNAlater, 70% ethanol, 50:50 glycerol:PBS, or without buffer at 25 °C, 4 °C, and −80 °C. Fecal DNA was extracted, quantified, and 16S rRNA gene analysis performed on Days 0, 7, 14, and 56 to evaluate changes in DNA concentration, purity, and bacterial diversity and composition over time. We detected overall effects on bacterial community of storage buffer (F-value = 6.87, DF = 3, P < 0.001), storage temperature (F-value=1.77, DF = 3, P = 0.037), and duration of sample storage (F-value = 3.68, DF = 3, P < 0.001). Changes in bacterial composition were observed in samples stored in −80 °C without buffer, a commonly used method for fecal DNA storage, suggesting that simply freezing samples may be suboptimal for bacterial analysis. Fecal preservation with 70% ethanol and RNAlater closely resembled that of fresh samples, though RNAlater yielded significantly lower DNA concentrations (DF = 8.57, P < 0.001). Although bacterial composition varied with temperature and buffer storage, 70% ethanol was the best method for preserving bacterial DNA in canine feces, yielding the highest DNA concentration and minimal changes in bacterial diversity and composition. The differences observed between samples highlight the need to consider optimized post-collection methods in microbiome research.

Source: Effects of preservation method on canine (Canis lupus familiaris) fecal microbiota

New paper out: The Green Berry Consortia of the Sippewissett Salt Marsh: Millimeter-Sized Aggregates of Diazotrophic Unicellular Cyanobacteria 

New paper out from Lizzy Wilbanks et al. including me as one of the co-authors The Green Berry Consortia of the Sippewissett Salt Marsh: Millimeter-Sized Aggregates of Diazotrophic Unicellular Cyanobacteria | Microbiology.

Two Eisen lab papers selected for the PeerJ 2015 Collection

Cool.  Two paper from my lab were selected as highlights of 2014 papers in the Peer J: PeerJ Collection: PeerJ Picks 2015 Collection

The papers were

The microbes we eat: abundance and taxonomy of microbes consumed in a day’s worth of meals for three diet types

Jenna M. Lang,  Jonathan A. Eisen,  Angela M. Zivkovic

and 
PhyloSift: phylogenetic analysis of genomes and metagenomes

Aaron E. Darling,  Guillaume Jospin,  Eric Lowe,  Frederick A. Matsen,  Holly M. Bik,  Jonathan A. Eisen

Thanks PeerJ and all the co-authors for their great work.  I love open science and I particularly think we need continuing experiments on the best ways to do open science.  Thus I like the experiment that is PeerJ in regard to how to publish and how to pay for open access fees.

Full disclosure: I am an Academic Editor at PeerJ.


——–
This is from the “Tree of Life Blog”
of Jonathan Eisen, an evolutionary biologist and Open Access advocate
at the University of California, Davis. For short updates, follow me on Twitter.

——–

Haloferax volcanii, model archaea, and me

When I was a graduate student I was looking around for an extremophile – especially an evolutionarily novel one.  And I settled on this species Haloferax volcanii – a model halophilic archaeon largely because Ford Doolittle and colleagues had started to turn it into a genetic model organism (and because Patrick Keeling, from Ford’s lab convinced me it was a good thing to do).  So I started work on this species – doing DNA repair studies in the lab.  See my PhD thesis for some of the work I did which I never published outside of the thesis for multiple reasons.  But I continued to be interested in this species.  And when I was working at TIGR, an NSF Program Officer approached me asking me to help get the genome sequencing done for this species.  So, well, I did: The Complete Genome Sequence of Haloferax volcanii DS2, a Model Archaeon.  And I became interested in other haloarchaea and eventually started working with Marc Facciotti, in the lab next to mine, in sequencing from across the diversity of the haloarchaea: Sequencing of Seven Haloarchaeal Genomes Reveals Patterns of Genomic Flux and Phylogenetically Driven Sequencing of Extremely Halophilic Archaea Reveals Strategies for Static and Dynamic Osmo-response.

Anyway – enough about me.  The whole point here is to point people to a new paper:  BMC Biology | Abstract | Generation of comprehensive transposon insertion mutant library for the model archaeon, Haloferax volcanii , and its use for gene discovery.  Further evidence for the use of Haloferax volcanii as a model species.  Tools continue to become available for genetic and experimental studies in this species.  So – if you are looking for an unusual and interesting organisms to work on – consider working on this species …

——–
This is from the “Tree of Life Blog”
of Jonathan Eisen, an evolutionary biologist and Open Access advocate
at the University of California, Davis. For short updates, follow me on Twitter.

——–

New lab paper: The microbes we eat: abundance and taxonomy of microbes consumed in a day’s worth of meals for three diet types

A new paper out from my lab (with Jenna Lang as the 1st author and in collaboration with Angela Zivcovic from the UC Davis Food For Health Initiative and the Department of Nutrition):  The microbes we eat: abundance and taxonomy of microbes consumed in a day’s worth of meals for three diet types.  The work in the paper focuses on characterizing the abundance and taxonomy of microbes in food from three model diets.

Basically, Angela prepared meals for these three diets

Food was purchased and prepared in a standard American home kitchen by the same individual using typical kitchen cleaning practices including hand washing with non-antibacterial soap between food preparation steps, washing of dishes and cooking instruments with non-antibacterial dish washing detergent, and kitchen clean-up with a combination of anti-bacterial and non-antibacterial cleaning products. Anti-bacterial products had specific anti-bacterial molecules added to them whereas “non-antibacterial” products were simple surfactant-based formulations. The goal was to simulate a typical home kitchen rather than to artificially introduce sterile practices that would be atypical of how the average American prepares their meals at home. All meals were prepared according to specific recipes (from raw ingredient preparation such as washing and chopping, to cooking and mixing).

And then she blended them and we characterzied the microbial communities in the blended samples:

After food preparation, meals were plated on a clean plate, weighed on a digital scale (model 157W; Escali, Minneapolis, MN), and then transferred to a blender (model 5,200; Vita-Mix Corporation, Cleveland, OH) and processed until completely blended (approximately 1–3 min). Prepared, ready to eat foods that were purchased outside the home were simply weighed in their original packaging and then transferred to the blender. 4 mL aliquots of the blended meal composite were extracted from the blender, transported on dry ice and then stored at −80 °C until analysis. The following analyses were completed using these meal composite samples: (1) total aerobic bacterial plate counts, (2) total anaerobic bacterial plate counts, (3) yeast plate counts, (4) fungal plate counts, and (5) 16S rDNA analysis for microbial ecology.

And Jenna Lang coordainted the sequence analysis and then Angela and Jenna (with some help here and there from me) coordianted the analysis of the different microbial data and the writing of the paper.

Figure 5: Biplot of taxa in sample PCoA space.

Lots of interesting things reported in the paper (read it, I insist).  I note – this is a demonstration project in a way – trying to get the community and others to think about the source pools of microbes that come into our system from our food.  It is by no means comprehensive or conclusive (read the caveats section of the paper).  Congrats to Jenna and Angela for all their hard work. Anyway – the paper is Open Access in PeerJ.  Eat it up.

UPDATE: Some press and blog coverage

——–
This is from the “Tree of Life Blog”
of Jonathan Eisen, an evolutionary biologist and Open Access advocate
at the University of California, Davis. For short updates, follow me on Twitter.

——–

New paper from Holly Ganz: Interactions between Bacillus anthracis and Plants May Promote Anthrax Transmission

Holly Ganz is first author on a new paper from her work she did before joining the lab:

PLOS Neglected Tropical Diseases: Interactions between Bacillus anthracis and Plants May Promote Anthrax Transmission.

A little bit about PhyloSift: phylogenetic analysis of genomes and metagenomes

New paper from people in the Eisen lab: PhyloSift: phylogenetic analysis of genomes and metagenomes [PeerJ].

Basically, the concept behind Phylosift is to provide for high quality, automated, high throughput phylogeny-driven analysis of metagenomic sequence data.  The software was developed openly on github and has been available in some form for more than a year.  Aaron, Holly, Erick and I have discussed it extensively in various talks around the world and thus we assume some are already familiar with it.

This project was coordinated by Aaron Darling, who was a Project Scientist in my lab and is now a Professor at the University of Technology Sydney.  Also involved were Holly Bik (post doc in the lab), Guillaume Jospin (Bioinformatics Engineer in the lab), Eric Lowe (was a UC Davis undergrad working in the lab) and Erick Matsen (from the FHCRC).

Abstract:

Like all organisms on the planet, environmental microbes are subject to the forces of molecular evolution. Metagenomic sequencing provides a means to access the DNA sequence of uncultured microbes. By combining DNA sequencing of microbial communities with evolutionary modeling and phylogenetic analysis we might obtain new insights into microbiology and also provide a basis for practical tools such as forensic pathogen detection.

In this work we present an approach to leverage phylogenetic analysis of metagenomic sequence data to conduct several types of analysis. First, we present a method to conduct phylogeny-driven Bayesian hypothesis tests for the presence of an organism in a sample. Second, we present a means to compare community structure across a collection of many samples and develop direct associations between the abundance of certain organisms and sample metadata. Third, we apply new tools to analyze the phylogenetic diversity of microbial communities and again demonstrate how this can be associated to sample metadata.

These analyses are implemented in an open source software pipeline called PhyloSift. As a pipeline, PhyloSift incorporates several other programs including LAST, HMMER, and pplacer to automate phylogenetic analysis of protein coding and RNA sequences in metagenomic datasets generated by modern sequencing platforms (e.g., Illumina, 454).

Figure 1 shows the general outline of the workflow.
Figure 1 showing the Phylosift workflow.

The workflow follows a series of steps including

  • Sequence identity search 
  • Alignment to reference multiple alignment 
  • Placement on a phylogenetic reference tree 
  • Visual presentation of taxonomic summary 
  • Comparison among samples (e.g., using Edge PCA)
In addition, there is a workflow for updating the database behind Phylosift which includes

  • Acquiring new genome data 
  • Gene family search and alignment workflow on each genome 
  • Phylogenetic inference and pruning 
  • Selection of representatives for similarity search 
  • Taxonomic reconciliation 

The paper shows some of the things you can do with Phylosift and some comparison of Phylosift and other methods.

Figure 2. Comparison of QIIME PCA and edge PCA analysis of human fecal samples.

Figure 3: Lineages contributing variation in human fecal sample community structure. (Analyzed using EDGE PCA)

It also provides Krona based output visualization of the taxonomic composition of a sample.

Anyway, more on Phylosift later.  Just thought I would get some out here on the blog.  Thanks to Aaron Darling, Holly Bik, Guillaume Jospin, Eric Lowe and Erick Matsen for all their hard work on this.  And thanks to the Department of Homeland Security for supporting the work.

For more about Phylosift see

——–
This is from the “Tree of Life Blog”
of Jonathan Eisen, an evolutionary biologist and Open Access advocate
at the University of California, Davis. For short updates, follow me on Twitter.

——–

New EisenLab paper: PhyloSift: phylogenetic analysis of genomes and metagenomes [PeerJ]

New paper from people in the Eisen lab (and some others): PhyloSift: phylogenetic analysis of genomes and metagenomes [PeerJ].  This project was coordinated by Aaron Darling, who was a Project Scientist in my lab and is now a Professor at the University of Technology Sydney.  Also involved were Holly Bik (post doc in the lab), Guillaume Jospin (Bioinformatics Engineer in the lab), Eric Lowe (was a UC Davis undergrad working in the lab) and Erick Matsen (from the FHCRC).  This work was supported by a grant from the Department of Homeland Security.

Abstract:

Like all organisms on the planet, environmental microbes are subject to the forces of molecular evolution. Metagenomic sequencing provides a means to access the DNA sequence of uncultured microbes. By combining DNA sequencing of microbial communities with evolutionary modeling and phylogenetic analysis we might obtain new insights into microbiology and also provide a basis for practical tools such as forensic pathogen detection.

In this work we present an approach to leverage phylogenetic analysis of metagenomic sequence data to conduct several types of analysis. First, we present a method to conduct phylogeny-driven Bayesian hypothesis tests for the presence of an organism in a sample. Second, we present a means to compare community structure across a collection of many samples and develop direct associations between the abundance of certain organisms and sample metadata. Third, we apply new tools to analyze the phylogenetic diversity of microbial communities and again demonstrate how this can be associated to sample metadata.

These analyses are implemented in an open source software pipeline called PhyloSift. As a pipeline, PhyloSift incorporates several other programs including LAST, HMMER, and pplacer to automate phylogenetic analysis of protein coding and RNA sequences in metagenomic datasets generated by modern sequencing platforms (e.g., Illumina, 454).

For more about Phylosift see

New paper from Eisen lab: Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes KMG-I project

A new paper of possible interest discussing one of the new phases of the GEBA Genomic Encyclopedia of Bacteria and Archaea project. Genomic Encyclopedia of Type Strains, Phase I: the one thousand microbial genomes KMG-I project | Kyrpides | Standards in Genomic Sciences.

 

New paper from the Eisen lab: Sporulation phylogenetic profiling

Quick post here. This paper came out a few months ago but it was not freely available so I did not write about it until now as it just showed up in Pubmed Central. It was published in the Journal of Bacteriology but they do not release material for free onto their website or Pubmed Central for a few months. Alas, as I was kind of a peripheral player in the main work in the paper (I helped them with the phylogenetic profiling part) I did not end up pushing as hard as I should have for paying the open access fee to make it available earlier / openly.

Here is a link to the paper: Gene Conservation among Endospore-Forming Bacteria Reveals Additional Sporulation Genes in Bacillus subtilis.

It is from Richard Losick’s lab at Harvard and it is one I am very very pleased with. Basically, Losick’s lab has been studying sporulation in Bacillus subtilis like forever. And in 2005 we wrote a paper on the genome of another member of the same phylum that also sporulates (Carboxydothermus hydrogenoformans): Life in Hot Carbon Monoxide: The Complete Genome Sequence of Carboxydothermus hydrogenoformans Z-2901.

And in that paper we did a phylogenetic profile based analysis of sporulation genes and found a set of genes that were (on average) in all the sporulating species and not in non sporulating species.  Among this set of genes were quite a few that nobody had ever shown to be involved in sporulation.  We predicted that they were likely involved in sporulation. 
And then I waited, since I did not really work on sporulation.  And in a series of discussions with Losick and people in his lab found out that they had evidence that many of these genes in B. subtilis were involved in sporulation.  And the latest paper is in essence a follow up on some of those discussions (well, really it is a lot of work from Losick’s lab with a little input from those conversations to guide some of the experimental tests). 
——–
This is from the “Tree of Life Blog”
of Jonathan Eisen, an evolutionary biologist and Open Access advocate
at the University of California, Davis. For short updates, follow me on Twitter.

——–