Monthly Archive for July, 2010

Valencia, The Home Of Sorcerer II And Crew Since September 2009

July 5th

Valencia is located about 140 miles (365 kilometers) from Barcelona.  Valencia has a rich history and  a distinct culture from other Spanish cities.  I have only spent a few months here, but I wanted to share some of the highlights with you all before we set sail and start our Mediterranean sampling season.   A must see in Valencia is the City of Arts and Sciences, it is a series of buildings in the heart of the city center, I swear it looks like an alien city!

City of Arts and Sciences

City of Arts and Sciences

City of Arts and Sciences

Another big event here in Valencia is the Formula 1 race  that is held here yearly.  It is amazing the amount of time and effort that goes into setting up the track and grandstands.  The race course went around the marina……….those cars are really really loud!  I didn’t see this happen live, but the footage is amazing! Click here for the Mark Webber crash.

Formula 1 Race

Click here for a Formula 1 Race Video

While we were in Valencia John and I both celebrated our birthdays.  John’s birthday is the day before mine…..Tea the chef took our dinner requests (John’s was bacon cheese burgers and mine was seared yellow fin tuna)…..it was  a great couple days!

John's Birthday

Another fun outing for the crew was when Craig and Marty rented motorcycles and we followed in the chase car.  The tour took us up into the mountains and some beautiful coastal cities.

Marty and Karolina on Motorcycle Tour

So our time in Valencia is coming to an end,  we are getting the boat ready for an intense 3 months of sampling. We all have really enjoyed our time here, made some great friends and are looking forward to returning in September.  If you have the chance you should come visit Valencia (especially during the World Cup, wow it has been crazy!), it is a great town and I forgot to mention, it has one of the best beaches I have seen in the Med yet!

High-performance comparative metagenomics

Are your carrying out large scale metagenomics analyses to identify differences among multiple sample sites? Are you looking for suitable analysis  tools?

If you have not yet found the right analysis tool, you may be interested in  the latest beta version of JCVI Metagenomics Reports (METAREP)  [Test It].

METAREP is a new open source tool developed for high-performance comparative metagenomics .

It provides a suite of web based tools to help scientists view, query, browse, and compare metagenomics annotation data derived from ORFs called on metagenomics reads or assemblies.

Users can either specify fields, or logical combinations of fields, to filter
and refine datasets
. Users can compare multiple datasets at various functional and taxonomic levels, applying statistical tests as well as hierarchical clustering, multidimensional scaling, and heatmaps (see image gallery).

For each of these features, tab delimited files can be exported for downstream analysis. The web site is optimized to be user friendly and fast.

Feature Summary [download Flyer]:

  • Handle extremely large datasets. Uses scalable high-performance Solr/Lucene search engine (we have indexed 300 million annotation entries, but much larger volumes can be handled as shown by Hathi Trust).
  • Compare 20+ datasets at the same time. Use various compare
    options including statistical tests and plot options to visualize
    dataset difference at various taxonomic and functional levels.
  • Apply statistical tests such as METASTATS (White et al.), a modified
    non-parametric t-test to compare two sample populations (e.g.
    metagenomics samples from healthy and diseased individuals).
  • Export publication-ready graphics. Export heatmaps, hierarchical clustering, and multi-dimensional scaling plots in PDF format.
  • Analyze KEGG metabolic pathways. Summaries include enzyme
    highlights on KEGG maps, pathway enzyme distributions, and
    statistics about pathway coverage at various pathway levels.
  • Search using a SQL-like query syntax. Build your query using 14
    different fields that can be combined logically.
  • Drill down into data using METAREP’s NCBI Taxonomy, Gene
    Ontology, Enzyme Classification or KEGG Pathway browser.
    Install your own METAREP version.
  • Flexible central configuration, METAREP and 3rd party code base is completely open source.
  • Cross-link function with phylogeny. Slice your data at various
    taxonomic and/or functional levels. For example, search for all
    bacteria or exclude eukaryotes or search for a certain (GO/EC
    ID)/taxonomic combination.
  • Generic data format. Data types that can be populated include a
    free text functional description, best BLAST hit information, as well
    as GO ID, EC ID, and HMMs.

How to analyze your own data: You can install your own METAREP version to analyze your metagenomics annotation data [download source]. We have written a comprehensive manual that describes the installation process step by step [download manual]. Since METAREP only operates on annotated data, raw sequences need to be annotated first. Supported data types that can be loaded for each sequence include functional descriptions, best BLAST hits fields (E-Value, Percent Identity, NCBI Taxon, Percent Sequence Coverage), GO, EC, and HMM assignments. The installation also contains a set of example annotations that can be imported.

Contact Us:

We would like to hear from you. If you have questions or feedback or if you wish to contribute to the METAREP open source project please send an email to metarep-support@jcvi.org

Links:

JCVI’s METAREP Instance

METAREP Flyer

METAREP Manual

METAREP Source Code

Media Day Circus On Sorcerer II

June 23nd

On Monday June 21st  we announced the official start of the Mediterranean leg  of  the Sorcerer II  Global Ocean Sampling Expedition.   Dr. Venter took  time from his busy schedule to fly into Valencia and attend the event as well as representatives from The Life Technology Foundation.  The first part of the day  was dedicated to media (newspapers, radio and T.V.), which included local Valencia  and national outlets from Madrid.  For more on press information please click here, on the linked page you can find press release info and fact sheets.

Dr. Venter With The Media!

Me Being Interviewed, Not Really A Media Circus For Me!

Dr. Venter Being Interviewed

The second half of the afternoon was spent with Spanish collaborators and high ranking  government officials. 

John, Karolina, Tea, Sarah, Charlie, Craig, Heather, Erling, Me, Jeremy and Bea

Sorcerer II All Dressed Up For Media Day!

The next day Craig was invited by his good friend and colleague Dr. Santiago Grisolia to give a talk to a small group of local scientists and students.  The talk was about the  first self-replicating, synthetic bacterial cell, which was recently published in Science.  The announcement made news worldwide and as you could imagine the audience was very intrigued!

Dr. Venter At His talk

Sorcerer II crew, Brett Shipe, Craig, Heather, Dr. Santiago Grisolia and his wife Frances

Genomics of the Indoor Air Environment

Microbial air concentrators in the air mixing room of a large office building. These concentrators work by pulling air through the water-filled chamber (the glowing boxes) and entraining bacteria and particles in liquid. The concentrator on the right is sampling from the building's internal air vent, while the machine on the left is sampling the fresh air entering the mixing room at a 90° angle.

Most of our life is spent in indoors, well-buffered from the constant changes in temperature, humidity, wind and light which shape life outside our homes and offices.  It seems intuitive that the types of microorganisms which inhabit our indoor environment must be different from those on the outside; after all, by removing environmental stresses such as UV, dessication and wind, we eliminate selective pressures on populations.  We spend 23 hours a day indoors, but we know very little about the types of  aerosolized microorganisms  we encounter – and inhale – with every breath.  It has been postulated that ‘everything is everywhere’: microbes are widely distributed around the world, with particular environments selecting for population subsets.  If that is the case, then are the organisms inhabiting a house different from those in a hospital, school or a downtown high rise?  Are cosmopolitan microorganisms simply taking advantage of our climate controlled environment, or are they interested in us in particular, bearing genes for virulence and pathogenicity?  And how are indoor microorganisms adapting to our widespread use of antibiotics, antimicrobials and other bacterial control agents?

Kitchen appliances and workspaces are potential sources of aerosolized microorganisms. We deployed several air samplers in this home, including "Dr. Hibbert" in the kitchen.

Through a grant from the Alfred P. Sloan Foundation, the J. Craig Venter Institute is developing new tools and techniques to examine the composition of microbial populations in the indoor air environment.  As many of these organisms are resistant to cultivation, our approach is modeled on the techniques developed during the Global Ocean Sampling (GOS) Expedition, namely the shotgun sequencing of bulk DNA to generate a genomic snapshot, or metagenome, of the indoor air environment.   A fundamental difference with the GOS expedition is that of scale: a milliliter of open ocean seawater may contain 10,000 microorganisms or more, so filtering a few hundred liters of seawater will often capture enough DNA to construct a high quality random shotgun library for sequencing.  Microbial density in the air is quite different: aerosolized bacteria counts for outdoor air are closer to 10,000 per cubic meter, meaning that a given volume of air contains a million times less organisms than an equivalent volume of seawater.

Field decontamination of an air sampler - this particular picture was taken in the air handling room of a medical center. Each machine was completely disassembled and all of the internal plumbing was replaced to prevent contamination. Those belts on either side of me let out a shrieking 110 dB of sound, hence the earplugs!

An additional issue is in collection efficiency.  The collection efficiency of most cyclone-style air samplers is usually less than 100% – this is especially true as the particle size drops below 1 micron in size.  As aerosolized bacteria are often small and dessicated, this efficiency becomes a problem, and air samplers have to be run for days at a time to collect sufficient DNA for sequencing.  These long collection times lead to  problems with growth and contamination.  Our air samplers are wet-cyclone Spin-Con concentrators, and to prevent bacteria from growing inside the collection chamber, we add a number of bacteriostatic compounds to keep the organisms from multiplying in the collection liquid and skewing our population data.  We have also programmed our collectors to dispense the collected sample into a refrigerated vessel containing additional growth inhibitors, and this is done every 2 hours.  Lastly, we completely clean or replace almost of the tubing and parts on a daily basis – we do this to reduce the chance of biofilms forming inside any of the tubing or collection chambers.   Air sampling is a labor intensive process, but the results have been relatively clean and diverse samples reflecting the actual microbial composition of the air environment.

An array of microbial air samplers at the end of the Scripps Research Pier. This 300 m long pier intercepts air before it reaches land, so is an ideal place to determine the marine microbial component to regional air quality.

Our original dataset was from a high-rise in mid-town Manhattan, where we collected air from an air mixing room over 20 floors up.  Both indoor and outdoor air samples were collected, and these samples form a baseline of data for much of the sampling we are currently conducting in California.  The air in New York City generally arrives from the west, so in addition to its urban signature, it also contains soil, dust and pollen from an entire continent.  In San Diego, the predominant winds are from the Pacific, and we suspect that there will be a strong marine component to populations of microorganisms in both indoor and outdoor environments.  To determine this baseline, we set up an array of samplers at the end of the 1,000 foot long research pier at Scripps Institution of Oceanography.  These samplers ran for five days, and aside from an osprey menacing the water bags, we were able to collect relatively clean marine air prior to being influenced by the terrestrial environment.

Doug Fadrosh checks on our de-aggregation and filtration set up. After the microorganisms are dissociated from the particulate matter and each other, they are filtered through 3.0 and 0.1 micron filters, and the balance - the viral fraction - is collected in the flask.

As can be seen in the pictures accompanying this blog, we have samples in home and a medical center, and we plan to sample in a school and an office building.  Each of these indoor environments is unique, and some of the sites are ten miles inland, and we are interested to see how the marine microbial component in the air attenuates with distance.  We run multiple machines in parallel for several days, and produce two liters of collected liquid, which we then process and concentrate before we attempt to isolate the DNA.  We have noticed that many of the organisms are associated with particles, so we use surfactants and mild physical techniques to de-aggregate the microbes prior to filtration, and we have found that this increases our yield of DNA substantially.

A 0.1 micron filter following de-aggregation and processing. The filtrate had already passed through the 3.0 micron filter, and is composed of bacteria and small particles. This filter contains particles from 5.4 million liters of air!

After the sample has been deaggregated, we pass the liquid through sequential 3.0 and 0.1 micron filters in order to fractionate the sample.  Larger material, particulalry fungal spores, pollen and eukaryotic cells, tend to get trapped on the 3.0 filter, leaving more bacteria on the 0.1 micron filter.  Any material which passes through the 0.1 micron filter is generally viruses and very small particulates – these too will be sequenced.  An example of the quantity of material on a 0.1 micron filter can be seen in the picture on the left – it is surprising just how ‘clean’ 5 million liters of air is!.  In posts to come we will describe more on how we get from filters to DNA and on to libraries, as well as share some of our preliminary results  —  so stay tuned!

Sorcerer II back at Sea!

June 13th 2010

After we collected and processed the sample from Blanes on May 26th we dropped off the collaborators on the dock, and  set sail for France.  After a overnight sail we reached our last Spanish sample site, it is in Spanish waters but monitored by French scientist. 

CTD Profile From Last Spanish Sample

Other than collecting our Spanish sample the main reason for the trip to France was to get some specialized boat work done in La Ciotat .  From May 28th-June 8th Sorcerer II was docked at Composite Works Construction and Super Yacht Refit for the rigging work.

Sorcerer II In The Yard, La Ciotat France

Our yard manager was Marion Gorman, some of you might know her better as Frenchie, she has done many passages on Sorcerer II over the years.  Although still in the yachting industry, she is taking a break from boat deliveries  and is focusing on delivering a baby (She is 8 months pregnant!)

Marion, Our Yard Manager

As always the crew took time to enjoy the local scenery, food and culture.

View of La Ciotat

We also celebrated Jeremy’s birthday!

Jeremy's Birthday in La Ciotat

 On June 8th we left France for Valencia Spain, with a stop on the Island of Mallorca  (we actually stopped in the city of Palma) for supplies. While in Spain we will prepare for the upcoming intense sampling season in the  Mediterranean/Black Seas, it will all be kicked off with a big media day event.

Sailing Past Mallorca Spain

The Re-Sampling of Blanes By Karolina Ininbergs

May 26th 2010

After docking in Barcelona and picking up Jeff, who just finished the lake sampling with Chris up in the Pyrenees, we headed north-east towards Blanes Bay. We were also joined by Bea Diez, her PhD student Roy McKenzie, Meri Antó and Vanessa Balague from ICM, Barcelona. It was a beautiful sunny day and just enough wind to set the reacher for some smooth sailing.

After about three hours we reached the sampling site, located just outside the small fishing harbour in Blanes. Vanessa had just been up there the day before for the monthly BBMO sampling, and mentioned that it seemed to be a lot of plankton in the water. We did a quick plankton tow with our net, but when looking in the microscope the sample seemed dominated by unicellular plankton, hard to identify by morphology alone.

Sampling went smoothly and the ICM team was dropped off at the dock in Blanes, where their colleague Clara was waiting with a car to take them and their samples back to Barcelona.

Sorcerer II is now sailing north along the coast for an overnight trip to the next sampling station, which will be the last one in Spanish water until we head back at the end of summer.  

Guests and crew for the Blanes sampling 2010. Up: Vanessa, Bea, Roy, Sarah, Charlie, On deck: Tea, Jeremy, Meri, Clara, me and Jeff.

Bea and Sorcerer II first mate John Henke in Blanes harbour.

BBMO – Blanes Bay Microbial Observatory-By Karolina Ininbergs

May 25th 2010

In 2008 I spent three months working at the Institute of Marine Sciences in Barcelona, hosted by Beatriz Diez in the Marine Microbiology group, headed by Carles Pedrós-Alió. One of the many microbial research projects at ICM is focused on environmental monitoring of marine microbes in Blanes Bay, in the North Western Mediterranean. At the Blanes Bay Microbial Observatory  scientists from ICM/CSIC in Barcelona are exploring the role of planktonic viruses, bacteria, archaea and microbial eukaryotes in the marine environment. Sampling is done monthly and oceanographic as well as microbial data is obtained routinely, including temperature, salinity, chlorophyll, inorganic and organic nutrients, dissolved oxygen, and light penetration. Blanes Bay is an example of an oligotrophic (nutrient-poor) coastal ecosystem, virtually unaffected by human and terrestrial influences. The sampling site has a good historical dataset, with records dating back to the 1950’s.

Our first visit to Blanes with Sorcerer II was in November 2009 when we sampled three stations along a transect, starting with the BBMO site. I remember that the wind was strong when we left Barcelona that day and it was a bumpy ride up to Blanes Bay, we could feel that it was the end of the sampling season in the Med.

Sampling stations during the transit from England to Spain, and the Blanes transect sites

Dr Chris Dupont, JCVI San Diego

Chris Dupont had flown in from San Diego for the two week sampling trip, which besides the Blanes transect also included sites around Mallorca and off the south-east coast of Spain. Joining us for the transect sampling was also Elisabet, a lab technician from ICM, who came along to collect some extra samples for our Spanish collaborators.

Eli collecting samples on Sorcerer II

 When we reached Blanes another group of scientists from ICM joined us for the day sampling at BBMO. Dr. Josep Gasol, Dr. Dolors Vaqué and Dr. Bea Diez shared their expertise from sampling at BBMO and helped out during the day.

Since one of the interesting aspects of the time series in Blanes is the seasonal variation in phytoplankton abundance, with different species dominating at different times of the year, our collaborators suggested that if we would have the opportunity it would be interesting to sample BBMO again at the beginning of the sampling season in 2010. Luckily our schedule and permits for this year enables us to start off the season by collecting a spring time sample at BBMO.

Tomorrow we head back to Blanes Bay to collect another set of samples from this long-term microbial monitoring station.

Stay tuned for more details on tomorrows sampling together with our Spanish collaborators.

Sampling team in Blanes 2009. On the boom: Sarah Dyste, Sorcerer II logistics coordinator, Bea Diez, ICM, Elli, ICM, me, on deck: Chris Dupont, JCVI, Dolors Vaqué, ICM, Pep Gasol, ICM, Jeremy Niles, Sorcerer II crew, in the middle: Captain Charlie Howard.

Tourist Time in Barcelona!

May 20th 2010

After two weeks on the road, I am back on Sorcerer II as we prepare for the Mediterranean sampling season.  We are docked in Port Olympic right in the heart of Barcelona.  One aspect of this year’s blogs is to share some of the experiences and places we get to visit.  We are delayed in Barcelona waiting for a boat part, so we had a few days to explore  Barcelona.  Last year I spent a week in Barcelona and did the jump on jump off bus tour, which was good.  The main thing you see on the bus tour involves the architecture works of  Antoni Gaudí , which is great and very impressive to see especially the Sagrada Familia.  The one thing I thought the bus tour didn’t describe in great detail was Barcelona’s long history and interesting culture.  Marc Lliros, a collaborator on the lakes samples we had collected in Spain, was born and raised in the Barcelona area and offered to give me a walking tour of the city.  And walk we did………. for about 8 hours………I think it really is the best way to see a city, it was exhausting but very educational.  As we walked around Marc described the history of the city, historical importance of each building and the culture of the Spanish/Catalan people. 

Roman Tombs in the heart of Barcelona

Fresh seafood at the huge market

Cow tongue for sale at the market, who wants some!?!?

I am not really sure what this is!

One afternoon Jeremy and I took the train out to the base of Montserrat  a mountain about 40 miles outside of Barcelona where the  Santa Maria de Montserrat is loctated. The monastery is Catalonia’s most important religious retreat, there are also miles of hiking trails.

Santa Maria de Montserrat

Jeremy with Santa Maria de Montserrat in the background

The Great Blizzard Sample of Lake Redon!

May15th 2010

We decided to do the  3 lakes in the Banyoles area first because the weather in the Pyrenees was so bad that we wouldn’t have been able to get up the mountain to sample Lake Redon.  Lake Redon is a pristine Alpine lake that is sampled weekly by Spanish researchers.   On Tuesday May 11th the weather prediction for the next few days in the Pyrenees said clear skies, so we loaded up the van and Chris, Emilio, Maria and myself took off on the 341 kilometer (211 miles) drive.

Route to Lake Redon

Driving up the Pyrenees

The drive was amazing……. clear skies, beautiful green mountains and crystal clear lakes.  Our destination was The Center for Mountain Research, a research station of the University of Barcelona. The research station is run by Lluís Camarero and is at the base of the mountain which Lake Redon is located.

Center for Mountain Research Station

Lake Redon

Lluís showed us around the station, including the well equipped labs, dorms and full kitchen.  After our tour we were told some bad news, we might have enjoyed great weather for our drive up, but that was all about to change. A big snow storm was on the way………our plan was to take a helicopter from the station directly to Lake Redon……but the helicopters can’t fly in snow storms.  Since weather up in the mountains can be unpredictable, we just decided to see how it was in the morning and go from there.

We woke up to this!

As you can see from the above picture, we weren’t flying anywhere!  Instead we took the day to catch up on emails/work and get some rest……it had been a week straight of driving and sampling and a day of down time was greatly needed!  During the day we discussed a few options on how we could collect the water from the lake and bring it back to the lab to filter.  Our time was limited (I had to join Sorcerer II and Chris had to catch a flight back home), so we decided that if the weather was still bad we would assemble a hiking team and hike the 2-3 hours up to the lake and carry water back down.  A couple problems for me were 1. I hate cold weather 2. I didn’t bring any cold weather gear up there, I had jeans and tennis shoes!  Lucky for me I am bigger than most Spanish men, so nobody had gear I could borrow……..Chris wasn’t so lucky!

Chris and Emilio about to set out on the hike for water!

So Thursday morning Emilio, Maria, Chris, Lluís, Jean-Christopher, a few post docs and grad students set out in the blizzard  to collect as much water as they could carry down the mountain…………..me and my big feet stayed inside…….warm and dry!

Hiking team

Lake Redon, yes that is a boat, just incase the ice breaks!

Water collection

About 5 hours later the team returned with 70 liters of water.  I asked Chris how it went and he replied with “That was the worse experience of my life!”

0.1um filter from Lake Redon Sample

The water they collected looked very clear and I was worried 70 liters wouldn’t have enough biomass for DNA extractions.  As you can see from the above filter picture there was plenty of biomass on the filters.

After a long hard day (well not for me, but for everyone else) Emilio and Lluís took us all out for a great local meal.  At the restaurant they had the town paper, which had done a story on us……….all the facts aren’t correct, but not bad for never interviewing Chris or myself!

Newspaper article

Friday May 14th we loaded up the van and drove back to Blanes.  We stored the samples in Emilio’s lab and unloaded the gear.  It has been a very successful 2 weeks of sampling, we collected samples from a variety of lakes.  These are very unique sites and I am sure some great scientific work will come from these samples for the Venter Institute and all our collaborators that we worked with.

Tomorrow we are driving to Barcelona, Chris will fly back to San Diego and I will join Sorcerer II and her crew, which are making their way to Barcelona from Valencia.

I would like to thank all the collaborators and people that helped make this 2 week road sampling trip possible, not only did we collect great samples, you all showed us your country, culture, history and great food!  Thanks again!

Lake Vilar, The Final Lake In Banyoles

May 10th 2010

On Monday May 10th we headed back to sample the last lake in the Banyoles area.  Lake Vilar is another meromictic lake located about 1 kilometer (1/2 mile) from Lake Siso and has a maximum depth of 10 meters (32 feet).   Sulfide is present during the entire year, although restricted to the deeper, high conductivity waters.  A stable chemocline exists at 5 meters (16 feet) between the oxygenated surface and the sulfide-rich bottom.  Here, dense populations of photosynthetic sulfur bacteria can develop when enough light penetrates.

Lake Vilar Information

Like the previous two lake sample we rowed out to the deepest section of Lake Vilar and collected a profile of the water column.  So we could compare all three lake systems we collected in the same region in the water column (above anoxic zone and in the anoxic zone)

Lake Vilar Profile

0.1um Filter from Lake Vilar

The appearance of every filter from each lake site in the Banyoles region was very distinct.  With all the different environmental and chemical factors that vary from all three lake sites it is going to be very interesting to see the microbial diversity in the sample sites and different depths within the same lake system.  The entire team was very happy with the sample trips and what we accomplished the last 3 days, I myself was disappointed with one thing…………I never saw the Banyoles Monster……….I heard all the stories, I called him out, I even mocked him (this is why I sent Chris in the dinghy and stayed on land!), but no sign of him!! For more about the Banyoles Monster click here.