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Extreme Microbiome Project Aims to Characterize Microbes Living in Harsh Environments

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NEW YORK (GenomeWeb) – Seemingly unlivable environments may be a reservoir of microbial diversity, according to Scott Tighe, research associate at the University of Vermont, who is spearheading a project to characterize microbial communities in harsh conditions.

The Extreme Microbiome Project (XMP) was launched as part of the Association of Biomolecular Resource Facilities' Metagenomics Research Group and is a consortium of microbiologists, geneticists, oceanographers, and bioinformaticists. Tighe was a founder and is the scientific leader of XMP.

Researchers involved in the project are collecting samples from locations such as the Doors of Hell in Turkmenistan — a gas-filled crater that has been burning continuously since 1971 — and Lake Hillier— a high-salinity bright pink lake on an island in Western Australia — and performing metagenomic shotgun sequencing as well as RNA sequencing to catalog and characterize the species that live there.

The goal is to both identify the various species that are able to thrive in such environments, see how they compare to related species and were able to evolve to such habitats, as well as to mine the data for potential new drugs, Tighe said. Because many microbes produce natural products and antibiotics, characterizing biosynthetic pathways, especially of novel microbes, is thought to be a promising method for drug discovery.  

The project is funded through various grants from the individual scientists and academic centers as well as through industry support. Tighe said that the consortium has been working with several vendors to get discounted or free reagents and equipment. For instance, it has received a number of flow cells from Illumina to perform paired-end 2x250 base pair shotgun sequencing on the MiSeq. In return, Tighe said, consortium members upload all their data to Illumina's BaseSpace, enable free access to the data, and cite contributors in published studies.

The vast majority of the sequencing has been performed on Illumina platforms, he said, although the group has also done some sequencing on Oxford Nanopore's MinIon and will begin doing some sequencing on Thermo Fisher's Ion Torrent PGM. Tighe said he is also interested in working with Pacific Biosciences' single-molecule sequencing technology.

For sample and library prep, the researchers have tested technologies from various vendors, including Rubicon Genomics, New England Biolabs, Bioo Scientific, and Illumina.

Thus far, some of the main challenges have been extracting DNA and preparing sequence-ready libraries. Many of the species' genomes have high GC content, which is notoriously difficult for many sequencing technologies.

For instance, Tighe said, it was difficult to extract DNA from the samples from the Doors of Hell site. That project was done in collaboration with Stefan Green, director of DNA services at the University of Illinois at Chicago.

Since the sample was essentially "burning lava sand," Tighe said, it understandably contained very little DNA. To get the DNA out, Tighe had to modify DNA extraction protocols, essentially resuspending the lava sand into a buffer and filtering it through a membrane.

Once they were able to extract the DNA, sequencing it was another challenge, due to its high GC content of at least 65 percent. Ultimately, the researchers were able to sequence the sample using Rubicon's ThruPlex DNA-seq kit and Illumina's MiSeq.

The predominant genus of bacteria they have so far identified from the Doors of Hell location are several varieties of Streptomyces, which are predominantly found in soil and are thermophilic in this location. It made perfect sense that it would be there, Tighe said, given the hot soil-like environment. Streptomyces are high-GC content, filamentous bacteria and are what gives soil its peaty smell. The specific species found in the Doors of Hell lava sand are still being analyzed and will be compared phylogenetically to other Streptomyces species, Tighe added.

From Lake Hillier, which is about 25 percent saline and has a pH of 7.4, the team has identified halophilic or "salt loving" bacteria and Dunaliella — the pink algae that give the lake its color. Dunaliella had always been the suspected culprit for Lake Hillier's fluorescent pink hue, but had never been characterized. "It was nice to nail down once and for all why the lake was pink," Tighe said. For that project, Ken McGrath's team at the Australian Genome Research Facility led the expedition to collect samples, while collaborators at the University of New Hampshire performed the sequencing, he added.

The consortium also plans to study microbes from a variety of other extreme locations. In collaboration with Samantha Joye's marine sciences lab at the University of Georgia, researchers are collecting samples from deep sea brine lakes at a depth of more than 10,000 feet in the Gulf of Mexico and supersaturated saline lakes below the 2-mile deep ice shelf in Antarctica. Joye's lab is currently in the process of collecting samples using a deep sea submersible. The lakes were formed by salt tectonics and are denser than the surrounding seawater.

Also in collaboration with Joye's lab, researchers are studying fecal microbiomes of Adélie penguins, which live in Antarctica. ABRF's Metagenomics Research Group is now analyzing those samples.

Other projects to study iron-rich hyper-saline lakes in Antarctica, Alaskan permafrost, and the deep sea off Greenland are in various stages of planning, and Tighe said the goal is to continue to add interesting extreme sites.

Aside from metagenomic sequencing, Tighe said the researchers are also sequencing RNA when possible and culturing the organisms that they can. There is no set time frame for the project, and Tighe anticipates that the group will sequence samples from about three sites every year.

In addition, he said, the group is in close contact with a sister project, MetaSUB, which is led by Christopher Mason at Weill Cornell Medical College, who is also a co-founder of XMP and head of its computational aspects.

Tighe noted that XMP is separate from and should be complementary to the Earth Microbiome Project, which aims to sequence 200,000 microbial samples from all over the world.

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