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Seagrass Microbiome Sampling

Recently the Seagrass Microbiome group has been wrapped up in sending (and receiving!) microbiome sampling kits. These kits are part of a larger collaborative project focused on re-sequencing of Zostera marina samples in conjunction with sequencing of additional marine and freshwater Alismatid species and their microbiomes. JGI recently sequenced and released the Zostera marina genome, and we are hoping to build on their efforts and explore population level variation within Zostera marina, as well as differences in genome content and structure between Zostera and other Alismatids, in conjunction with microbiome sequencing.

The sampling kits sent by the seagrass microbiome group have focused on the microbial aspect of this project. We have asked members of the Zostera Experimental Network (ZEN) as well as additional collaborators to sample both plant tissue for sequencing (coordinated through Jay Stachowicz and Jeanine Olsen) and microbiome samples. We are extremely excited about this sample set, as it covers populations of Zostera marina across many different environments, for which we already have extensive metadata through the ZEN group! We are requesting root, sediment (within the rhizosphere), and leaf tissue, as detailed in the diagram below (courtesy of Jeanine Olsen).
microbiome

Collaborators are also sampling at two depths per site (deep and shallow), so that we can examine microbiome differences that may correlate with population depth. We are sampling 24 individuals per site, 12 per depth.

The kits are relatively straightforward and simple to both make and use, even if you’re not an experienced field microbiologist. We followed the kit and samplingl details we previously used (https://seagrassmicrobiome.org/protocols/microbial-sampling-kit/), with a few updates.

The kits now contain:
– 1 5cc syringe (for sediment collections)
– Tubes filled with Zymo buffer (DNA/RNA Shield)
– Plastic forceps
– Plastic spatula
– Parafilm
– Ethanol wipes

Here are a few photos of kit production:

Vann and Firl putting together kits in lab, photo from Katie Dahlhausen (@PhDKD)
Alana Firl and Laura Vann putting together kits in lab, photo from Katie Dahlhausen (@PhDKD)

 

Tubes all ready to go!
Tubes all ready to go!

 

Close up of kit
Close up of kit

 

Completed kits, ready to go!
Completed kits, ready to go!

We have sent out all of the kits, and have already started receiving some completed samples in the mail. Here is a close up of some of the samples from Kotzebue, AK.

Samples for one individual from Kotzebue, AK. From left to right: root, sediment, and leaf.
Samples for one individual from Kotzebue, AK. From left to right: root, sediment, and leaf.

A huge thanks to our collaborators for sampling, and to everyone from the Eisen lab who has helped make and send kits. Stay tuned for updates on sample processing and data !

Marine Algal and Plant Microbiomes Workshop – soliciting comments

So – am participating in a workshop, supported by CIFAR and the Gordon and Betty Moore Foundation over the next few days on “Marine Algal and Plant Microbiomes”.  The workshop is basically trying to come up with a white paper / position paper on the future of such studies and to continue the conversation about this topic afterwards.  We are asking questions like

  • What are the challenges and opportunities in this area?
  •  What are the major scientific questions?
  • How are such systems different from fresh water or terrestrial systems?
  • How are they different?
  • How are marine systems involving other hosts (e.g., coral, sponges, dolphins) comparable (i.e., is there something about marine systems that links them together in any way).
  • What tools and resources could help advance work in this area?

And more

So I am posting here asking for a few bits of information from any readers.

  • Are you interested in participating in follow up discussions on this topic?
  • Do you know of any people we should try to bring into the conversation even if they are not, well, you?
  • Are there any major projects in this area that would be worth engaging?

Any thoughts (on the topic that is) would be welcome.

 

Culturing Bacterial Isolates from the Seagrass Microbiome

My name is Karley Lujan and I am an undergraduate working on culturing bacterial isolates from the Seagrass microbiome. I joined this project because I am interested in learning about what information we can obtain from studying microbiomes. I think it is fascinating that although we can’t see microorganisms they are extremely prevalent and can have crucial roles in biological systems. The focus at the beginning of this project was to take Seagrass samples from Bodega Bay, create culture samples, and use Sanger sequencing of the 16S rRNA to identify what we grew. Seagrass and sediment samples were taken from Bodega Bay, CA. Then, in order to obtain isolates from the seagrass, we focused on the leaves, roots, and sediment. What we were able to successfully extract DNA from were identified as Shewanella, Pseudoalteromonas, Colwellia, Tenacibaculum, Vibrio and Alteromonas.

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Me (Karley Lujan) at the 2016 UC Davis Undergraduate Research Conference.
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Agarolytic Bacteria on Marine Broth. Tentative identification: Shewanella

Methods:

  • Sample preparation: Dilutions of sediment with PBS, PBS rinse of roots and leaves, ground and crushed leaves with PBS
  • Culturing: Plated the PBS sample solutions onto two of each of the following plate types; one plate for 25℃ and the other at 4℃

Agar Plates/Liquid Media

      • Nitrogen Free
      • Seawater Agar
      • Seawater Medium
      • Difco Marine Broth
  • Selected Colonies: After there was significant growth on the plates we selected various interesting colonies and isolated them by dilution streaking. Single colonies were then grown overnight in the appropriate liquid media and at the appropriate temperature
  • DNA Extraction: Genomic DNA extractions were performed and glycerol stocks were made using the successful liquid cultures. Extracted DNA then went through 16S rRNA gene PCR and gel electrophoresis in order to confirm that enough DNA was present for Sanger Sequencing
  • Sanger Sequencing: 16S rRNA sequences for each isolate were ran through BLAST and phylogenetic trees were built in order to obtain tentative identifications for the isolates

Results: After Sanger sequencing, the data was ran through BLAST to obtain a tentative identification and determine whether or not the microbe was a good candidate for sequencing.

Shewanella: Electrogenic- An electron generator that can be used in microbial fuel cells.

Colwellia: Cold-adapted

Vibrio: Some species of Vibrio can go through morphogenetic changes after going from a liquid to a solid surface. This leads them to change from swimmer cells to swarmer cells.

Pseudomonas: Two bacterial isolates were cable of growing on Nitrogen-Free agar plates at 25⁰C. Identified as part of the genera Pseudomonas, there are some species of this genera capable of aerobically fixing nitrogen. These are of particular interest as we will be further investigating which nitrogen-fixing bacteria are essential for seagrass health.

Currently I am beginning to look at the genomes of the bacteria we decided to sequence and I am also working with bacteria that are capable of growing on the nitrogen-free agar plates. At first it was difficult to extract the DNA from these bacteria but now both have been tentatively identified as Pseudomonas through sanger sequencing of the 16S gene. This is interesting because there aren’t many Pseudomonas that can fix nitrogen which is what these two must be doing in order to survive on the nitrogen-free plates. These two bacteria also have different morphologies which means they could be different species in the genus Pseudomonas. Due to their morphological similarities yet ability to grow on nitrogen-free agar, I think these two bacteria are very interesting and we will be finding out more about them by sequencing and analyzing their genomes.

 

CA sampling for project phylogeny

A few of us at Team Seagrass have been doing fieldwork in Northern CA in order to collect seagrass relatives. We have specifically been targeting freshwater species (highlighted in blue in the below phylogeny). We will continue with marine and brackish species when the tides in the San Francisco Bay are lower.

From https://phylogenomics.wordpress.com/people/post-docs/jenna-lang/seagrass/photo-4/: This is a phylogeny of the seagrasses and their aquatic relatives. This tree was built using parsimony and ~1200bp alignment of the rbcL gene. From Les and Cleland, 1997.
From https://phylogenomics.wordpress.com/people/post-docs/jenna-lang/seagrass/photo-4/: This is a phylogeny of the Alismatales, including the seagrasses and their aquatic relatives. This tree was built using parsimony and ~1200bp alignment of the rbcL gene. From Les and Cleland, 1997.

We have been following the methods detailed in https://seagrassmicrobiome.org/sample-collection-and-preservation/ and have managed to collect a few representative species from the following clades: PotamogetonaceaeAlismataceae, Hydrocharitaceae, Najadaceae, and the Lilaceae.

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Example leaf and root from Alisma species, collected at Cosumnes River

For further plant identification and the construction of a ‘host phylogeny’ we will be using chloroplast DNA markers.

In addition to what we have sampled thus far, we have also managed to sample some outgroups (MyriophyllumCeratophyllum, and Camboba). We will hopefully be able to sample additional outgroup species shown on the above tree.

We have been successful in 7 out of the 10 locations that we have tried. The lack of success so far can be attributed to drought conditions in CA and the increase in invasive submerged aquatic vegetation (e.g. Myriophyllum, Water Milfoil; and Eichhornia crassipes, Water Hyacinth). Water Milfoil and Water Hyacinth form dense mats below and above water, respectively, outcompeting native vegetation. Even in sites where we been successful, much of the area we surveyed has been overrun with these two invasive species.

Obviously drought has also been an issue for us, as many of the lakes and tributaries have little to no water in them this year, resulting in a lack of any aquatic vegetation. Water levels in Folsom Lake, for example, are so far reduced that the Park Service has had to build additional parking lots on the water side of the boat ramp in order for people to be able to get close to the new shoreline.

Map of Folsom Lake, with parking instructions drawn on by park official. X marks location of new parking lot, which is still at 15 minute walk to the shoreline.
Map of Folsom Lake, with parking instructions drawn on by park official. X marks location of new parking lot, which is still at 15 minute walk to the shoreline.

In the following weeks we will head up to the foothills in search of remaining freshwater species, as well as explore the Bay Area coastline and salt marsh/Delta area for marine and brackish species. Stay tuned!

Sample preservation experiment

During ZEN DNA extractions we noticed that samples preserved in the Zymo buffer were forming a precipitate with the C1 solution from the MoBio kit. Furthermore, many of the samples also resulted in very low DNA yields, perhaps correlated with the precipitate formation. Any microbiologist will tell you that there are many different ways to preserve samples from the field, but there does not seem to be a universal *best* method.

We decided the best way to approach this problem for the Seagrass Microbiome project was to explore a variety of sample preservation methods and see which approximated the ‘real’ microbiome best (as measured by preservation on dry ice). We chose the following methods to try: Dry ice, Zymo, RNA-later, Drierite, and Ethanol. We also wanted to investigate how different preservation methods performed over time. We chose 4 time points post sampling for our extractions: 24 hours, 1 week, 2 weeks, and 1 month.

We drove to Putah Creek in Winters in search of submerged aquatic plants in the Alismatales (the order that contains the seagrasses). Here is a photo of our study site: 20150611_133052

We found a bed of Elodea canadensis growing near the shore and started sampling.

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We pulled out whole plants and divided them into root and leaf for each of our trial preservation methods, with 3 replicates per method per time point. We extracted DNA at each of the different time points, and did PCR of the 16S SSU rRNA gene, with subsequent library preparation and sequencing on the Illumina Mi-Seq. We used QIIME to analyze the sequence data and compare species diversity between our preservation methods.

PCA
Weighted unifrac PCoA

Our PCoA plot shows that preservation in Zymo best approximates the community captured using dry ice (our control), and that Drierite and Ethanol were both very different from anything else. RNA-later appears close to Zymo and dry ice, but we only have 5 data points for it. For the RNA-later and Ethanol extractions, we extracted directly from the solution without pelleting, which may have affected our end result.

Taxonomy summary plot: Order level
Taxonomy summary plot: Order level

The taxonomy table supports the trend seen in our PCoA plot, with Drierite and Ethanol being very different, and Zymo, dry ice, and our 5 RNA-later time points showing similar communities.

Looking at variation in community structure across time points within Zymo we see little to no change.

Weighted unifrac PCoA showing time point variation within Zymo
Weighted unifrac PCoA showing time point variation within Zymo

However, we see some change in community structure by the month mark with dry ice.

Weighted unifrac PCoA showing time point variation within dry ice
Weighted unifrac PCoA showing time point variation within dry ice

Take home messages from our sample preservation experiment:

  • Using Drierite as a preservation technique does not capture the community assembly well.
  • RNA-later and Ethanol could have had better success with pelleting and removing the supernatant prior to extraction. We may investigate this further in the future.
  • In spite of our initial concerns regarding precipitate forming in the Zymo buffer, Zymo is the clear winner in our trial.
  • There are slight changes in the community assembly by the month mark, in all methods (depending on whether you use weighted or unweighted unifrac PCoA).
  • Due to the current ease of sampling using dry ice, we will continue this method except in situations where samples must be kept at room temperature (we will then use Zymo).

20150902_072734

HAPPY SAMPLING 🙂 Please see https://seagrassmicrobiome.org/sample-collection-and-preservation/ for more details regarding sampling and preservation protocols. 

MBL Microbial Diversity Summer Course

I spent this past summer at the MBL Microbial Diversity summer course and I’ve written a post about it which can be found here: http://microbe.net/2015/09/28/mbl-microbial-diversity-summer-course/

While at the course, I also cultured a handful of microbes from seagrass leafs/roots and several different amoeba from seagrass bed sediments including one really cool “follow the leader” snail slime like amoeba (slime mold?).

Visualizing Microbiome Data: Choropleth Style

A Pipette and an Open Mind

Recently I’ve needed to visualize spatial changes in my microbiome data that are easily interpretable by other people. The best solution I’ve come across is simply projecting the data onto a drawing of my organism. Like this:

Zostera marina high resolution Alpha diversity of samples from pieces of a seagrass plant projected onto a drawing of that plant.

I’ve used SitePainter to produce these in the past, and in some ways it’s great. I got the figure I wanted and I’ve received a lot of positive feedback about it. The only problem is it has a steep learning curve and is the most dysfunctional GUI I’ve ever come across (generating the figure above took several days of my time, the first time). So when it became necessary to produce over forty more images like it I decided to search for a better way.

My first instinct was that there should be an easy way…

View original post 694 more words

ASM Highlights

The Seagrass Team (Hannah, Jenna & I) hit up ASM this year which was in New Orleans. In case you missed ASM, Jonathan took the effort to compile all the tweets together (#ASM2015) and his efforts can be found: here.

However in case you don’t want to wade though thousands of tweets I’ve included some some brief highlights:

NCBI’s Targeted Loci Blast (MOLE-BLAST)

  • Using MOLE-BLAST you can blast specific 16S or ITS sequences against NCBI currated databases for those marker genes. This seems like it could be really useful if you want to identify bacterial or fungal taxonomy using a marker gene approach. Also, MOLE-BLAST appears to use a tree based approach to help you find the nearest neighbor for taxonomy assignment.

Carl Zimmer’s Talk on Microbiomes and the Hyperbolome

The Session that kept Redefining the Tree of Life (aka Unearthing the Dark Matter of Microbial Metabolism and Diversity)

  • Unfortunately, I missed this session, but apparently both Brett Baker (@archaeal) and Laura Hug (@LAHug_) shook things up
  • Brett Baker brought Thorarchaeota to the party, a monophyletic group that looks like it branches between Lokiarchaeota and Eukaryotes
  • Then Laura Hug showed up with Woesearchaeota and Pacearcheoata, not sure where they fit in, but cool

Contributions to “Extreme” Microbiology by Female Scientists Session

  • The whole session was awesome (and extreme), but Emmie de Wit gave a heartfelt (and tear producing) talk on being a first responder to the Ebola outbreak in Liberia
  • Also, I now really want to go to the Bonneville Salt Flats that Betsy Kleba talked about

Honorable mentions: John Zehr (Talk on open ocean Nitrogen fixing symbionts), David Baltrus (Talk on fungal endophytes with bacterial symbionts in their hyphae), Michael Wagner (Talk on syntrophy between nitrite and ammonia oxiders and alternative substrates for nitrification), Tom Marshburn (A real life astronaut!), Tom Sharpton (A Shotgun Metagenome Annotation Pipeline (ShotMAP))

Hannah and I presented posters at the meeting, her poster (left, #1237) and my poster (right, #1236) can be seen below:

20150601_162116 20150601_162124

Of course, Team Seagrass wasn’t the only ones from the Eisen lab presenting at ASM. David Coil (@davidacoil), Srijak Bhatnagar (@srijakbhatnagar) and Megan Krusor (@MKrusor) also presented posters this year! See tweets with photos below.

Some might say that the real highlight of ASM was New Orleans itself; it was my first time there and I really enjoyed being immersed in the culture (the music!), city and food (especially the food). Below is a picture of me touching the Mississippi River (my first time!). Unfortunately, the water was too murky to search for any seagrass or seagrass relatives.

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Potomac Samples: not what we expected, but still some interesting connections

Last summer I went on a sampling expedition to the Chesapeake Bay for some SAV (submerged aquatic vegetation) collection. I came back with leaf and root samples from the Potomac River from a few different SAV species. Ideally, we thought the microbial community would correlate with the salinity gradient across the sites or the host species.

Neither of those patterns are discernible in this data set as far as the beta diversity plots are concerned, but I found some other interesting things while sifting through these plots. For instance, the samples cluster based on the site location (P1-P4). The communities at P1 and P3 look really similar and P4 is within the tail end of their cluster, while P2 is totally different. location

For reference, here’s a map of our sites:

Screen Shot 2015-06-05 at 11.21.11 AM
Whitehead, Andrew et al. “Genomic Mechanisms of Evolved Physiological Plasticity in Killifish Distributed along an Environmental Salinity Gradient.” Proceedings of the National Academy of Sciences of the United States of America 108.15 (2011): 6193–6198. PMC. Web. 5 June 2015.

Although all the sites were visited, I only found SAV species at P1-4. There are some useful patterns in the water and soil chemistry data (courtesy of Greg Mayer from Texas Tech University) that show the same correlation pattern as the site locations (as expected since each location has its own distinct chemistry data). Some of the chemistry data shows different patterns from site location, so I’ll have to sift through those next and see what looks relevant. I also ran a core microbiome script for each site, but haven’t looked at the output yet.

In addition, the leaf and root samples are pretty distinct:

sampletype

The alpha diversity graphs are a whole ‘nother beast that I’m going to explore some other time. That’s all for now, but I feel that there are some interesting lines of investigation to pursue and more scripts to run.

A first look at the first ZEN run

PNAs (blockers for mitochondrial and chloroplast amplification)

  • More organelles were filtered from the leaves than the roots, sediment, or water.
average % loss by sample type
Leaf 26.5518607264
Root 10.6381529905
Sediment 5.1730348426
Water 13.0591363787
  • using PNAs did not result in an overall loss of reads
# filtered reads
PNA 389011
no PNA 411059
  • in progress: do we see more chimeras with PNA use?

Run summary statistics

    • 533 samples, including kit controls and PNA tests
    • reads per sample (after filtration) ranged from 5 – 206,661
    • the kit control with the most reads had 6549
    • 306 (57.4%) of the samples had fewer than 6549 reads, we’ll call these low-abundance samples
    • breakdown of low-abundance samples by type
Leaf Water Roots Sediment
126 (41.1%) 109 (35.6%) 32 (8.8%) 33 (10.8%)
  • in progress: not sure, I published this post by accident