Next-Gen Sequencing: A Review

I recently wrote up a review with Dr. Elizabeth Grice of next-generation sequencing technologies. We specifically focused on their applications for microbiome research. While it was published it in a wound care journal, the techniques outlined are broadly applicable to any system with a diversity of microbes. Please have a look at the manuscript below!

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Reference

Hodkinson, B. P., and E. A. Grice. 2015. Next-generation sequencing: a review of technologies and tools for wound microbiome research. Advances in Wound Care 4(1): 50-58.
Download manuscript (PDF file)

Mole Day

This year I am teaching several sections of High School Chemistry, and this past week we had celebrations for Mole Day!  Here's a rundown of some of the snacks and activities.

Moles May Attend:
Make A Mole - For extra credit students can make stuffed animal moles from a pattern (just Google it). It's great to have some homemade moles in attendance at the party!

Food:
Guacamole - Trader Joe's has a guacamole dip called "Avocado's Number" that actually has a picture of Amadeo Avogadro on it, which is a great prop. I also decided to make fresh guacamole for the students, which provided everyone with a bit of a show.
Molasses cookies - OK, so the only real connection here is that 'mol' is in the word, but who doesn't like cookies?!

Sing-Along:
Rock Me Amadeo - The song 'Rock Me Amadeus' by Falco has an awesome video that highlights the time-period in Europe when Amadeo Avogadro was alive (which overlaps with the time period when Wolfgang Amadeus Mozart was alive, which is the reason that the video is set in this time period). Students can sing 'Amadeo' instead of 'Amadeus' on the chorus and you've got a catchy theme song for Mole Day!

Game Show:
More Than, Less Than, or Equal to a Mole - You can take objects that are basically made of one type of compound and ask students if they are more than a mole, less than a mole, or equal to a mole. Students can be split into two teams, and pairs (with one from each team) can stand on either side of a bell and compete to ring in first. If the first to answer is incorrect, the other person gets a chance to guess. I have to give credit to Mrs. Swieson (also of Delaware County Christian School) for coming up with this one!

Most of all, we just partied and had a great time hanging out as a class!

Electronic Publication of Taxa

Hodkinson, B. P., and J. C. Lendemer. 2014. A clarification of effective electronic publication. Taxon 63(4): 911-913.
Download publication (PDF file)

Lecanoromycetes

Please check out the newest publication that we've put together on the phylogeny and taxonomy of the fungal class Lecanoromycetes (the main group of lichen-forming fungi). This publication is more all-encompassing than any before it, including analyses of molecular sequence data from 1307 different fungi!

Miadlikowska, J., F. Kauff, F. Högnabba, J. C. Oliver, K. Molnár, E. Fraker, E. Gaya, J. Hafellner, V. Hofstetter, C. Gueidan, M. A. G. Otálora, B. Hodkinson, M. Kukwa, R. Lücking, C. Björk, H. J. M. Sipman, A. R. Burgaz, A. Thell, A. Passo, L. Myllys, T. Goward, S. Fernández-Brime, G. Hestmark, J. Lendemer, H. T. Lumbsch, M. Schmull, C. L. Schoch, E. Sérusiaux, D. R. Maddison, A. E. Arnold, S. Stenroos, and F. Lutzoni. 2014. A multigene phylogenetic synthesis for the class Lecanoromycetes (Ascomycota): 1307 fungi representing 1139 infrageneric taxa, 317 genera and 66 families. Molecular Phylogenetics and Evolution 79: 132-168.
Download publication (PDF file)
Download supplementary file 1 (PDF file)
Download supplementary file 2 (PDF file)
Download supplementary file 3 (PDF file)
Download supplementary file 4 (PDF file)
Download supplementary file 5 (docx file)
Download supplementary file 6 (PDF file)
Download supplementary file 7 (docx file)

Spider Bite

[WARNING: Graphic Content]

Over the past month, my wife has been dealing with an ulcer on her shoulder that was most likely caused by a spider bite (I'm guessing that some type of Sac Spider is to blame). At first, it raised up like a volcano with a base diameter of about 2-3 cm and a height of about 1-2 cm. Then after a couple of days, the center became necrotic (see "June 15" below). She had the part that was entirely necrotic taken out by a doctor, leaving a bit of a hole in the center ("June 17"). She then treated it daily with honey and kept it covered for the next few weeks. During this time, there was a period of a few days where the whole area started to become red and inflamed ("July 1"), so she got on an antifungal (Fluconazole) and two antibiotics (Keflex & Minocycline), which quickly cleared up that particular issue. It is now healing up nicely ("July 4") and we hope it will finish up without any additional complications!

Timeline (all treatments follow doctors' recommendations):
June 12 - noticed painful swelling
June 15 - Keflex treatment begun (for potential general infection)
June 16 - Keflex treatment terminated, Bactrim treatment begun (for potential MRSA infection)
June 17 - necrotic center removed/biopsied, treatment with honey begun
June 19 - Bactrim treatment terminated (no signs of infection from lab tests)
July 1 - Keflex, Minocycline and Fluconozole treatment begun (swelling and redness seen around the area)

- Brendan

Sticta sylvatica

I recently had a paper published that talks about the status of the rare species Sticta sylvatica in my part of the world (Hodkinson et al. 2014). The article was actually featured on the cover! Here's the abstract:

"The presence of the foliose cyanolichen Sticta sylvatica in eastern North America has been called into question due to the absence of high-quality, verifiable material and the common misuse of its name. Recently, specimens collected in the Great Smoky Mountains have been verified as having the typical S. sylvatica morphology. Although molecular data remain inconclusive regarding the entity’s genetic distinctiveness from the phenotypically dissimilar S. limbata, we argue that the decline in the abundance of this morphological entity worldwide along with the need for further genetic study make continued conservation efforts imperative."

Sticta sylvatica in the field.

- Brendan

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Reference

Hodkinson, B. P., J. C. Lendemer, T. McDonald, and R. C. Harris. 2014. The status of Sticta sylvatica, an ‘exceedingly rare’ lichen species, in eastern North America. Evansia 31(1): 17-24.
Download publication (PDF file)
Download journal issue cover (PDF file)

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[This work was supported by the National Science Foundation under awards EF-1115086 and DEB-1145511.]

Molecular Ecology

A few years back, I began working on a collaborative project led by Greg Bonito to examine fungal and bacterial communities associated with plant roots.  We used next-generation sequencing on the 454 GS-FLX platform for profiling using four different loci (one bacterial and three fungal).  The fungal ITS and LSU primers were identical to those used in my recent Mycosphere paper (Hodkinson & Lendemer 2013), and the 16S primers are the ones I designed for my Environmental Microbiology paper from a couple of years back (Hodkinson et al. 2012).  The paper is finally out in Molecular Ecology; check it out!

- Brendan

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References

Bonito, G., H. Reynolds, M. S. Robeson, J. Nelson, B. P. Hodkinson, G. Tuskan, C. W. Schadt, and R. Vilgalys. 2014. Plant host and soil origin influence fungal and bacterial assemblages in the roots of woody plants. Molecular Ecology 23(13): 3356-3370.
Download publication (PDF file)

Hodkinson, B. P., and J. C. Lendemer. 2013. Next-generation sequencing reveals sterile crustose lichen phylogeny. Mycosphere 4(6): 1028-1039.
Download publication (PDF file)
Download data and sequence-processing scripts (ZIP archive)
Download Ascomycota LSU alignment and analysis files (ZIP archive)
Download Arthoniales LSU alignment and analysis files (ZIP archive)

Hodkinson, B. P., N. R. Gottel, C. W. Schadt, and F. Lutzoni. 2012. Photoautotrophic symbiont and geography are major factors affecting highly structured and diverse bacterial communities in the lichen microbiome. Environmental Microbiology 14(1): 147-161.
Download publication (PDF file)
View publication (publisher's website)Download supplementary phylogeny (PDF file)
Download data and analysis file archive (ZIP file)

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[Support for my work on this project was provided in part by the National Science Foundation under awards EF-0832858, DEB-1011504, and DEB-1145511.]

Lichens As Food

Here's an article about how lichens are a trendy new food item!
http://www.independent.co.uk/life-style/food-and-drink/features/lichen-is-the-surprise-new-ingredient-on-finedining-menus-thanks-to-our-love-of-scandinavian-and-indian-cuisines-9268419.html

Dynamic Periodic Table

Here's a really cool dynamic periodic table that nicely lays out all sorts of information about the various elements (with all sorts of tabs and sliders):

http://www.ptable.com/

I wish there had been something like this when I was in high school and college!

- Brendan

Open Fracture Microbiome

I recently co-authored a paper with a team of dermatologists (those who study skin) and orthopaedists (those who study bone) in which we present the results of microbiome analyses on open fracture wounds. These are the types of wounds where bones actually break through the surface of the skin. We found that the wound-associated microbial communities change over time, becoming significantly more similar to the adjacent skin communities as healing progresses. There are also a number of bacterial groups and aspects of the microbiome that tend to be associated different clinical factors. Please have a look at the publication at the links below!

- Brendan

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Reference

Hannigan, G. D., B. P. Hodkinson, K. McGinnis, A. S. Tyldsley, J. B. Anari, A. D. Horan, E. A. Grice, and S. Mehta. 2014. Culture-independent pilot study of microbiota colonizing open fractures and association with severity, mechanism, location, and complication from presentation to early outpatient follow-up. Journal of Orthopaedic Research 32(4): 597-605.
Download publication (PDF file)
Download supplemental methods (PDF file)
Download sample metadata (QIIME mapping file)
Download OTU table (bzip2-compressed BIOM file)
Download OTU representative sequences (bzip2-compressed FASTA file)

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[This work was funded in part by NIAMS/NIH R00 AR060873.]

Alternative Nitrogen Fixation

During my dissertation research, I found an interesting set of genes in the metatranscriptome of Peltigera praetextata. They were vnfDG and vnfN, two genes in the alternative, vanadium-dependent nitrogen fixation gene cluster. The interesting thing about this was that nitrogen fixation in lichens has always been attributed to the standard molybdenum-dependent system. These gene fragments were very similar to ones from Anabaena, cyanobacteria that are not known to be lichen photobionts, but are closely related to Nostoc, the cyanobiont in Peltigera. After examining metagenomes from other Peltigera species (all associated with Nostoc) and running further analyses, I found that the presence of the vnf gene cluster seems to be widespread (in all five samples checked from five Peltigera species from all over the world). Analyses revealed that the Peltigera-associated sequences all form a group close to, but separate from, Anabaena, which is entirely consistent with them being derived from the main photobiont, Nostoc. Therefore, it seems that lichens with cyanobacteria are actually using both the standard nitrogen-fixation system and an alternative, vanadium-dependent one.

This work could have sweeping implications for studies of biogeochemistry, since certain ecosystems, especially those in the tundra, are dominated by lichens. Ecosystem-wide nitrogen fixation rates are often inferred based on the acetylene-reduction assay (ARA). To interpret the results of this assay, a conversion factor based on the standard molybdenum-dependent system is typically used. However, if a substantial portion of the nitrogen fixation is taking place via alternative means, the standard conversion factor could produce wildly inaccurate interpretations. Even if one is willing to consider that a different conversion factor should be used, determining the proper conversion factor may become especially problematic since the proportion of standard to alternative nitrogen fixation could vary greatly based on the season or micro-environment. Therefore, a greater understanding of vanadium, molybdenum, and nitrogen dynamics may be needed before we can continue to rely blindly upon the commonly-used ARA for ecosystem-wide studies.

Peltigera rufescens, a lichen with a cyanobacterial photobiont from the genus Nostoc, in the Alaskan tundra.

- Brendan

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Reference

Hodkinson, B. P., J. L. Allen, L. L. Forrest, B. Goffinet, E. Sérusiaux, Ó. S. Andrésson, V. Miao, J.-P. Bellenger, and F. Lutzoni. 2014. Lichen-symbiotic cyanobacteria associated with Peltigera have an alternative vanadium-dependent nitrogen fixation system. European Journal of Phycology 49(1): 11-19.
Download publication (PDF file)
Download supplementary table (PDF file)
Download vnfD alignment and analysis files (ZIP archive)
Download vnfN alignment and analysis files (ZIP archive)
Download script for editing GenBank-derived FASTA files (PERL script)

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[This work was supported in part by the National Science Foundation under grants OCI-1053575 and DEB-0919284.]

New Species: Lepidostroma winklerianum

There has recently been amazing burst of discovery in what is now known as the order Lepidostromatales. Until 2007, only one of the species that is now in the order had been described. Then, in a series of recent papers, five new species have been described. The most recent new species is Lepidostroma winklerianum, named after Sieghard Winkler, one of the authors who first described the genus Lepidostroma.

Morphology and anatomy of thallus squamules of Lepidostroma winklerianum. 

(a–b) Fully grown and young squamules. [scale=1 mm] 

(c) Section through squamule.  [scale=100 μm]

(d) Photobiont cells.  [scale=10 μm]

(e) Upper cortex in section view. [scale=10 μm]

(f) Upper cortex in surface view. [scale=10 μm]

Here is a key to the currently-known species in the order Lepidostromatales:

1a Thallus crustose, undifferentiated, lacking distinct cortical structures (Sulzbacheromyces); basidiomata clavarioid; Neotropics (northeastern Brazil) ... Sulzbacheromyces caatingae

1b Thallus microsquamulose to squamulose, with distinct cellular cortex; basidiomata clavarioid or club-shaped ... 2

2a Thallus microsquamulose, composed of contiguous glomerules with cortex formed by distinctly lobate, jigsaw-puzzle-shaped cells (Ertzia), medulla absent; basidiomata clavarioid; tropical Africa (Rwanda) ... Ertzia akagerae

2b Thallus distinctly squamulose, composed of scattered to dense, rounded to reniform squamules with cortex formed by polygonal or jigsaw-puzzle-shaped cells (Lepidostroma), medulla present; basidiomata clavarioid or club-shaped ... 3

3a Basidiomata clavarioid; squamules lacking raised margin and maculae; photobiont layer above medulla, more or less uniform, with scattered cells throughout medulla and in lower portion of squamules; photobiont cells lacking pyrenoids; Neotropics (Costa Rica, Colombia) ... Lepidostroma calocerum

3b Basidiomata club-shaped; squamules with conspicuous, slightly raised white margin, their surface maculate; photobiont layer below medulla, forming pyramidal columns protruding upwards; photobiont cells with pyrenoid(s) ... 4

4a Upper cortex with jigsaw-puzzle-shaped cells in surface view; tropical Africa (Rwanda) ... Lepidostroma rugaramae

4b Upper cortex with polygonal cells in surface view; Neotropics (Mexico) ... 5

5a Squamules 1.5–3 mm diam.; upper cortex multi-layered; basidia 2-sterigmate ... Lepidostroma vilgalysii

5b Squamules 0.5–1.5(–2) mm diam.; upper cortex single-layered; basidia 4-sterigmate ... Lepidostroma winklerianum

- Brendan

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Hodkinson, B. P., B. Moncada, and R. Lücking. 2014. Lepidostromatales, a new order of lichenized fungi (Basidiomycota, Agaricomycetes), with two new genera, Ertzia and Sulzbacheromyces, and one new species, Lepidostroma winklerianum. Fungal Diversity 64(1): 165-179.
Download publication (PDF file)
View publication (website)
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[This work was supported in part by the National Science Foundation under DEB-0715660.]

New Genera: Ertzia & Sulzbacheromyces

For a recent paper establishing the new order Lepidostromatales, we examined the morphological and molecular diversity of the genus Lepidostroma, and it became clear that there were three very different types of species. The core group of four species has rounded to reniform squamules. However, one species is outside of this group and has a microsquamulose thallus that forms contiguous glomerules with a cortex of jig-saw-puzzle-shaped cells. We gave this species its own genus and called it Ertzia akagerae, naming the genus after Damien Ertz, the primary describer of the species. Another species outside of the core group has an entirely crustose thallus, so we gave it a new genus and named it Sulzbacheromyces caatingae. The genus was named for Marcelo Sulzbacher who described the single species with colleagues. Although it's still inconclusive, there also could be photobiont differences associated with the split of these three genera. The samples of Lepidostroma and Sulzbacheromyces from which we were able to obtain algal DNA reads yielded sequences that were very different from one another, but were both from groups not known to be associated with any other types of lichenized fungi.

- Brendan

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Hodkinson, B. P., B. Moncada, and R. Lücking. 2014. Lepidostromatales, a new order of lichenized fungi (Basidiomycota, Agaricomycetes), with two new genera, Ertzia and Sulzbacheromyces, and one new species, Lepidostroma winklerianum. Fungal Diversity 64(1): 165-179.
Download publication (PDF file)
View publication (website)

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[This work was supported in part by the National Science Foundation under DEB-0715660.]

New Order: Lepidostromatales

Some colleagues from the Field Museum and I recently described a new order of fungi, Lepidostromatales (Hodkinson et al. 2014). One thing that makes it unique is that it is the only order of basidiomycete fungi containing only lichenized members. The ordinal placement of the family Lepidostromataceae had previously been uncertain, but our molecular analyses confirmed its isolated position and made it clear that the best treatment would be to give it its own separate order. Please have a look at the new paper in Fungal Diversity for a key to all of the species in the new order!

- Brendan

[Update: I decided to also post the key to the species here.]

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Hodkinson, B. P., B. Moncada, and R. Lücking. 2014. Lepidostromatales, a new order of lichenized fungi (Basidiomycota, Agaricomycetes), with two new genera, Ertzia and Sulzbacheromyces, and one new species, Lepidostroma winklerianum. Fungal Diversity 64(1): 165-179.
Download publication (PDF file)
View publication (website)

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[This work was supported in part by the National Science Foundation under DEB-0715660.]