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Introducing PAPRICA

Chasing Microbes in Antarctica - Tue, 08/18/2015 - 14:00

I’m very excited to report that our latest paper – Microbial communities can be described by metabolic structure: A general framework and application to a seasonally variable, depth-stratified microbial community from the coastal West Antarctic Peninsula was just published in the journal PLoS one.  The paper builds on two very distinct bodies of work; a growing literature on microbial community structure and function along the climatically sensitive West Antarctic Peninsula, and a family of new techniques to predict community metabolic function from 16S rRNA gene libraries, which we are calling metabolic inference.

The motivation for metabolic inference is in the large amount of time that it takes to manually curate a likely set of functions for even a small collection of 16S rRNA genes.  In today’s world, where most analyses of microbial community structure consist of many thousand of reads representing hundreds of taxa, it is simply impossible to dig through the literature on each strain to see what metabolic role each is likely to be playing.  Ideally a researcher would use metagenomics or metatranscriptomics to get at this information directly, but it is not advisable or desirable in most cases to sequence hundreds of metagenomes or metatranscriptomes (necessary for the kind of temporal or spatial resolution many of us want these days).  Metabolic inference provides a convenient alternative.

A quick Google Scholar survey of the number of studies since 2005 that have used high throughput 16S rRNA gene sequencing.

A quick Google Scholar survey of the number of studies since 2005 that have used high throughput 16S rRNA gene sequencing.  Over the last ten years we’ve collected an astonishing amount of sequence data from a diverse array of environments, however, much of this data has been from taxonomic marker genes like the 16S rRNA gene, leaving microbial community function largely unknown.  PAPRICA and other methods that try to infer microbial functional potential from 16S rRNA gene data can help bridge this gap.

The basic concept behind all metabolic inference techniques (e.g. PICRUSt, tax4fun, PAPRICA) is hidden state prediction (HSP) (you can find a nice paper on HSP here).  In 16S rRNA gene analysis metabolic potential is a hidden state.  The metabolic inference techniques propose different ways to predict this hidden state based on the information available.

Our small contribution to this effort was to develop a method (PAPRICA – PAthway PRediction by phylogenetIC plAcement) that uses phylogenetic placement to conduct the metabolic inference instead of an OTU (operational taxonomic unit) based approach.  Our approach provides a more intuitive connection between the 16S rRNA analysis and the HSP (or at least it does in my mind) and can increase the accuracy of the inference for taxa that have a lot of sequenced genomes.

Most analysis of large 16S rRNA datasets rely on an OTU based approach.  In a typical OTU analysis an investigator aligns 16S rRNA reads, constructs a distance matrix of the alignments, and clusters the reads at some predetermined distance.  By tradition the default distance has become a dissimilarity of 0.03.  This approach has some advantages.  By clustering reads into discrete units it is easy to quantify the presence or absence of different OTUs, and it allows microbial ecologists to avoid problems with defining prokaryotic species (which defy most of the criteria used to define species in more complex organisms).  To conduct a metabolic inference on an OTU based analyses it is possible to simply reconstruct the likely metabolism for a predefined set of OTUs based on the OTU assignments of published genomes.  This works great, but it limits the resolution of the inference to the selected OTU definition (i.e. 0.03).  For some taxa, such as Escherichia coli (and plenty of more interesting environmental bugs), there are many sequenced genomes that have very similar 16S rRNA gene sequences.  PAPRICA provides a way to improve the resolution of the metabolic inference for these taxa.

Our approach was to build a phylogenetic tree of the 16S rRNA genes from each completed genome.  For each internal node on the reference tree we determine a “consensus genome”, defined as all genomes shared by all members of the clade originating from the node, and predict the metabolic pathways present in the consensus and complete genomes using Pathway-Tools.  To conduct the actual analysis we use pplacer to place our query reads on the reference tree and assign the metabolic pathways for each point of placement to the query reads.  One advantage to this approach is that the resolution changes depending on genomes sequence coverage of the reference tree.  For families, genera, and even species for which lots of genomes have been sequenced resolution is high.  For regions of the tree where there are not many sequenced genomes resolution is poor, however, the method will give you the best of what’s available.

Fig_2

Figure from Bowman and Ducklow, 2015.  PAPRICA includes a confidence scoring metric that takes into account the relative plasticity of different genomes.  In this figure each vertical line is a genome (representing a numbered terminal node on our reference tree), with the height and color of the vertical line giving its relative plasticity (which we refer to as the parameter phi).  The genomes identified with Roman numerals are all known to be exceptionally modified, which is a nice validation of the phi parameter.  Many of these are obligate symbionts.  I) Nanoarcheum equitans II) the Mycobacteria III) a butyrate producing bacterium within the Clostridium IV) Candidatus Hodgkinia circadicola V) the Mycoplasma VI) Sulcia muelleri VII) Portiera aleyrodidanum VIII) Buchnera aphidicola, IX) the Oxalobacteraceae.

PAPRICA provides some additional helpful pieces of information.  We built in a confidence scoring metric that takes into account both predicted genomic plasticity and the size of the consensus genome relative to the mean size for the clade (deeper branching clades will have a bigger difference), and predicts the size of the genome and number of 16S rRNA gene copies associated with each 16S rRNA gene, both of which have a strong connection to the ecological role of a bacterium

For our initial application of PAPRICA we selected a previously published 16S rRNA gene sequence dataset from the West Antarctic Peninsula (our primary region of interest).  One thing that we were very interested in looking at was whether we could describe differences between microbial communities organized along ecological gradients (e.g. inshore vs. offshore, or surface vs. deep water) in terms of metabolic structure in place of the more traditional 16S rRNA gene (i.e. taxonomic) structure.  Using PAPRICA to convert the 16S rRNA gene sequences into collections of metabolic pathways we found that we could reconstruct the same inter-sample relationships identified by an analysis of taxonomic structure.  This means that a microbial ecologist can, if they choose, disregard the messy and sometimes uninformative taxonomic structure data and go directly to metabolic structure without losing information.  Applying common multivariate statistical approaches (PCA, MDS, etc.) to metabolic structure data yields information like which pathways are driving the variance between sites, and which are correlated with what environmental parameters.  This information is much more relevant to most research questions than the distribution of different microbial taxa.  It is worth noting that while inter-sample relationships are well preserved in metabolic structure, the absolute distance between samples is much less than for taxonomic structure.  This might have some implications for the functional resilience of microbial communities, which we get into a little bit in the paper.

PAPRICA was an outgrowth of a couple of other papers that I’m working on.  At some point the bioinformatic methods reached a point where separate publication was justified.  As a result, and reflecting the fact that I’m much more an ecologist than a computational biologist, PAPRICA is not nearly as streamlined as PICRUSt (which is even available through an online interface).  I’ve spent quite a bit of time, however, trying to make the scripts user friendly and transportable.  Anyone should be able to get them to work without too much difficulty.  If you decide to give PAPRICA a try and run into an hitches please let me know, either by posting an issue in Github or emailing me directly!  Suggestions for improvement are also welcome.

Glacial Earthquakes May Hold Clues to Future Sea Level Rise - Weather Channel

Featured News - Mon, 08/17/2015 - 12:00
Glacial earthquakes could help us measure how much ice is lost from glaciers around the world, Lamont-Doherty's Meredith Nettles says.

L2-14

Sugar - Sun, 08/16/2015 - 22:33
... so my mother can see I'm wearing a hardhat (Hi Mom).  Galen getting it done, Natalie with commentary, Yogi counting it down ...



Shot L3-01 video

Sugar - Sun, 08/16/2015 - 21:52


HUGE THANKS to all the volunteers who worked so hard to make this project such a great success. It  was a pleasure working with you and getting to know you all.  Also mega thanks to all the landowners who were kind enough, and trusting enough, to let us put a source on their property.  None of this could have happened without your generosity and spirit of curiosity.  Thanks so much.

Dan



What goes bump in the night? We do.

Sugar - Sun, 08/16/2015 - 11:30
Steve Harder prepares to detonate a shot.Controlled blasts in deep holes are the source of sound waves for our program.  We set them off in the middle of the night because that is when it is quietest along the county and state roads where our instruments are shallowly buried on profiles across eastern Georgia and listening for sound waves.  During the nights of Aug 7, 8 and 11, our blasting experts Steve Harder, Galen Kaip and Ashley Nauer prepped and detonated 25 blasts along our lines, with some help from other enthusiastic scientists (like me).  Our shots have between 200 and 1600 lbs of explosives – mostly ammonium nitrate emulsion. At each shot, we connect a long wire between the drill hole and a blast box, move back a safe distance from the shot site, wait for the appointed time, and set off the blast. The blast box is used to detonate the shot at a very accurate time. There were two shooting teams, and each has different time windows for blasting to ensure that we only do one blast at a time. If two blasts occurred at the same time, the sound waves could interfere with one another.
Ashley Nauer and Kent Anderson wire up a shot.
When the blast goes off, you feel it more than hear it.  The sound waves radiate out from the shot traveling both within the earth and along the surface. Waves that travel along the surface of the earth (“surface waves”) cause the most ground shaking. If the ground is wet, sometimes a geiser briefly occurs 5-10 seconds are the explosion.  Not surprisingly, plenty of people are interested in experiencing this besides us!  Several of the property owners who very kindly gave us permission to set off these blasts on their land came out in the middle of the night to spectate.
Even putting aside the obvious rush of setting off a bunch of blasts, its fun to be out and about in the Georgia country side at night.  A cacophony of sounds echo around the forests from crickets and frogs.  Immediately after a shot, all of these creatures very briefly go silent – they know that something has happened! And then they ramp up again.  We also see other animals prowling around, including amardillos. A meteor shower occurred during our final night of blasting, which we could see quite well from the rural stretches of Georgia, far from light pollution of population centers.

Donna Shillington, LDEO

Jim Gaherty illuminates a steaming hole that formed over the shot site from the blast.
The shot team filled in this hole the next day.Armadillo patrols one of shot sites.

Earthquake Shakes Parts of New Jersey - Philadelphia Inquirer

Featured News - Fri, 08/14/2015 - 12:00
The quake struck along a branch of the Ramapo Fault, said Lamont's Won-Young Kim. Such small earthquakes happen in the region every few years, he said.

More updates from our field teams...

Sugar - Sun, 08/09/2015 - 12:35

Seventeen teams are rounding up 1953 small seismic stations along our 350-mile-long line across eastern Georgia, and they continue to send texts and pictures with updates on their work…

“21757. Still kickin”
Kevin hunts for missing texans with the metal detector....
“Team 11 is all done and headed home to the mother ship”

“We’re not coming back unless we have all of them!”

“We had a helper at site 20431!”

“Hello Donna Rach and I are crushing it right now”

“Daily check in, we’re making good time so we should see the puppies soon enough”


Making metadata...
“Recovered a Texan at stop 20858. This one doesn’t seem to be working correctly, whenever I press it it just tells me things like “The Cowboys are America’s team” and “Bush was an American hero”. Weird.



“We got to 20170 the one with the ant colony”

Loaded up with Texans and geophones
“Stop 20804. Everything’s fine, except some guy came out of the woods and bit Brent. All he’s saying now is “brains” and is acting super creepy. I’ll keep an eye on it and only use the shovel if necessary”




“Will do! I will let you know if we become stuck… Looks likely”

Unearthing another Texan

“Just beat the downpour and headed for base”

“Stop 20879. Found the Texan disconnected from the geophone on top of where we buried it with pieces of bag around it, looked everywhere for the geophone. Found it about 5 m down the hill near the tree line with bite marks all along it. Either an animal dug it up or a very hungry confused thief”



Picking up litter?
“Found 2 dollars at 21058! Who says geology doesn’t pay well?”

Was not seen on the line...
Was seen on the line... yikes.

Best texts from the field (so far...)

Sugar - Thu, 08/06/2015 - 07:43
Seventeen teams have been out deploying small seismographs and geophones along a 300-mile-long profile across eastern Georgia, and they have been checking in with me regularly by text message. Some highlights from texts and pictures from our groups:



“Team4 is Done! I repeat again, 4 is done! Heading back to the sweet onion city! ☺”

“Still alive”



“Team gruesome twosome on our way back to the hub”

“We are gonna skip installing 21520 because both sides of the streets are well maintained yards and there’s not a great place to put a Texan”

“We’re done! Just kidding haha. We’re on our second!”

“We’re in the zone”

“All geophones buried --- I am beat. Where’s a can of spinach when ya need one, lol”

“It's a long way to the top if you want to study rocks”
"Sunrise at station 21779"
“We’re dirty but doing well!”


“Still digging. Still have not reached China. Will attempt again on next hole”


“On 20186 and we lost our bubble level. We even dug up the last geophone to see if I accidentally buried it”

“We just deployed our last station, 20224. Can we go to Jekyll Island?”



Donna Shillington, LDEO

Digging Holes and Filling Batteries -- A party in Vidalia, Georgia

Sugar - Tue, 08/04/2015 - 07:52

The SUGAR deployment team arrived en-masse on Saturday bringing the Line 2 personnel total to a whopping 45! The day started off with science and overview lectures by the SUGAR principle investigators Donna Shillington and Dan Lizarralde.  Students diligently rearranged the ten’s of Texan boxes into a makeshift lecture hall, complete with a projector and a Bluetooth sound system. 

With the science lecture complete and stomachs full of pizza, the entire group ventured out to conduct a practice deployment under the watchful eyes of the PASSCAL instrument team.  All 17 teams participated in the activity, standing in a single file line in front of our hotel digging practice holes, connecting the Texans to the geophones, and mindfully orientating them with their handy-dandy bubble levels. 

After a sweat filled hour under the Georgia sun, we caravanned back to the instrument center for a “battery party”. I call it a battery party in honor of the “streamer parties” that students will often participate in on active source seismic research cruises in which kilometers of cable need to be reeled off and rearranged.  In our case a battery party consisted of the 32 students placing 2 D-cell batteries inside each of the 2,000 Texans.  The instrument center quickly transformed from an orderly lecture hall into a mass of empty battery boxes and disassembled Texans though despite the apparent chaos, we got the job complete and the Texans filled in only a few short hours. 

Next up will be flagging the instrument locations and the actual deployment.  We have our fingers and toes crossed for dry weather and safe road conditions as the student teams prepare to set off on their flagging and deployment expeditions. 

Natalie Accardo - Columbia University, LDEO


The SUGAR2 deployment team hails from all across the United States
covering more than 15 states and 21 different universities/institutions.   

The deployment team sits with rapt attention listening to
the science and overview lecture.

Students practice digging holes and deploying Texans
near our hotel in Vidalia, Georgia.
Students and PASSCAL personnel take over the instrument center
filling 2,000 Texans with D-cell batteries.
The "battery party" comes to an end as the last Texans are filled and
the boxes are rearranged for easy late-night programming by the PASSCAL team.  



2000 “Texans” with all the fixin’s….

Sugar - Sun, 08/02/2015 - 09:21
During our project, we plan to record sound waves generated by a series of controlled blasts on two profiles, one with 2000 instruments (“Texans”) deployed along a 350-mile-long profile across Georgia and another with 700 Texans deployed along an 80-mile-long profile.  In total, that’s 2000 instruments and 2700 deployments!! Lot of instruments means lots of stuff.   The basic components of the instruments themselves were shipped in ~160 big plastic boxes arranged into ~18 pallets.  Each of these instruments will be powered by two D-cell batteries. To power the instruments for both lines, we needed 5500 D-cell batteries.  We picked them up from the Lowes in Vidalia as a 2000-lb pallet.  For each station, we also need flags to mark the locations, and bags and tape to protect the data recorder.  We very quickly filled up our 1800-square-foot field center in Lyons, GA with all these goodies…

Donna Shillington,  LDEO

Freshly delivered pallets of boxes holding all the science equipment
The PASSCAL team re-arranged the boxes into a T for their own devious reasons :)The trusty Silverado loaded down with 2000 pounds of batteries! (Dan for scale).



Drill, Baby Drill! Drilling and filling for the SUGAR seismic shots

Sugar - Fri, 07/31/2015 - 12:14
We are using sound waves to image the subsurface of Georgia along two long transects.  It is like creating a huge x-ray of the geology in the region. Thousands of instruments (termed “Texans”) will record sound waves that are generated from a series of controlled seismic sources (“shots”) that we will set off along the line. 

For the last few weeks, the seismic source team, based at the University of Texas – El Paso, and the drillers have been hard at work drilling twenty-six 60- to 100-foot-deep holes that will contain the explosives used to create the sound waves.  Once the holes are drilled (the first stage of which is termed spudding), emulsion explosives with boosters and caps are carefully installed in the base of the hole and the remaining height is filled in with dirt and gravel (“stemming”). 

Now with the 26 shots drilled and patiently waiting for the electronic signal to blow, all we have left to do is deploy the 2,000 instruments that will record the sound waves … An easy feat for the 50+ scientists, students, and engineers descending on Vidalia, GA over the next few days.  Stay tuned for our progress and adventures as we continue on this epic scientific undertaking.

Natalie Accardo - LDEO

The SUGAR seismic source and science team from left to right:
Steve Harder, Dan Lizarralde, Ashley Nauer, and Galen Kaip
The drill rig set up and drilling a shot on SUGAR Line 2.

Galen Kaip prepares the source charges (white tubes) on the truck bed as
the drillers complete a shot hole.
The source team carefully lowers the prepared seismic charges into the complete shot hole.
Ashley Nauer (red hat) stands waiting with shovel in hand to fill the remaining height of
the hole with sand and gravel.   
The drill team monitors the process of spudding, the very first stage of drilling the
shot hole, for SUGAR line 2.
The source team and drill team push on late into the night to ensure the completion of the
final shot for the entire SUGAR experiment.  

Ocean Currents Could Shed Light on Flight 370 Mystery - Business Insider

Featured News - Fri, 07/31/2015 - 12:00
Ocean currents could have carried debris from a missing Malaysia Airlines flight around the Indian Ocean to Reunion Island, says Lamont's Arnold Gordon.

Ramping up for bigger, badder SUGAR Part 2

Sugar - Tue, 07/28/2015 - 23:11
We are in Georgia gearing up for the second phase of field work for the SUGAR project, which will involve collecting seismic refraction data along two profiles spanning eastern Georgia. In the coming weeks, we’ll deploy thousands of small seismometers along county and state roads across the region, which will record sound waves generated by a series of controlled blasts. We can use the sound waves to make pictures of geology beneath the surface. Geological structures beneath Georgia record the most profound events involved in the formation and evolution of the eastern North America continent. In particular, we want to image an ancient suture between Africa and North America that formed when these continents collided to create the supercontinent Pangea, frozen magma bodies from one of the biggest volcanic outpourings in Earth’s history, and continental stretching and thinning that lead to the breakup of Pangea and formation of the Atlantic Ocean.


Map of SUGAR lines, showing two possible locations of the ancient suture (red dotted lines)

We collected similar data in western Georgia last year during the first phase of the SUGAR experiment imaging these same features. During that field program, we deployed 1200 seismometers and set off 11 controlled blasts along a 250-mile-long line, which felt like a big project at the time. But this year, we will go even bigger! In eastern Georgia, we need to span an even larger area to encompass our geological targets. One of the reasons that we need to look at a bigger swath of the earth is that there is a debate about the location of the suture here – it could be as far north as Milledgeville, GA or as far south as Baxley, GA. (In case you are not up on your Georgia geography, those towns are ~100 miles apart). This means longer profiles, more instruments and more blasts! We will deploy a total of 2700 seismometers and detonate 26 blasts along two profiles. The longer profile spans 350 miles from Winder, GA to the Florida-Georgia state line near St Mary’s Georgia. Stay tuned!

Donna Shillington, LDEO 



Interview: Climate Change in Central Asia - The Diplomat

Featured News - Thu, 07/23/2015 - 12:00
Lamont-Doherty's Benjamin Orlove discusses the repercussions of climate change in Central Asia.

Stay Tuned for SUGAR 2!!

Sugar - Wed, 07/22/2015 - 21:57
In just a few short weeks a mass of students and scientists will descend on southern Georgia with work boots and sunscreen in hand to take part in the second portion of the SUGAR active source experiment.  Make sure to stay tuned for regular updates on our progress and to learn more about the exciting science that motivates this amazing field expedition!

What's Inside the Earth? - PBS News Hour

Featured News - Thu, 06/25/2015 - 12:00
Lamont-Doherty's Christine McCarthy discusses some of the ways scientists study what is inside the Earth.

The True Face of Mercury - BBC

Featured News - Thu, 06/11/2015 - 12:00
Almost 20 years ago, NASA researchers began developing the Messenger probe, which would photograph the entire surface of Mercury. Lamont-Doherty Director Sean Solomon discusses the mission he led.

How Does Oil Form? - NBC News

Featured News - Wed, 06/10/2015 - 12:00
Lamont-Doherty's Peter Kelemen breaks down the process of oil formation for NBC News.

California's Vanishing Clouds Could Intensify Drought - KQED

Featured News - Mon, 06/08/2015 - 12:00
Fog and clouds play a vital role in keeping coastal temperatures down. But that benefit could be disappearing. Lamont-Doherty's Park Williams explains.

New Estimates of the Deep Carbon Cycle - Deep Carbon Observatory

Featured News - Fri, 06/05/2015 - 12:00
In a new study, Lamont-Doherty's Peter Kelemen assesses how much carbon is taken up by the Earth’s crust during hydrothermal processes at or near mid-ocean ridges, and how much carbon is released into the atmosphere through volcanic degassing and diffuse venting.

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