Genomic Methods and Microbiological Technologies for Profiling Novel and Extreme Environments for the Extreme Microbiome Project (XMP)

Abstract

The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Resource Facilities Metagenomics Research Group (ABRF MGRG) that focuses on whole genome shotgun sequencing of extreme and unique environments using a wide variety of biomolecular techniques. The goals are multifaceted, including development and refinement of new techniques for the following: 1) the detection and characterization of novel microbes, 2) the evaluation of nucleic acid techniques for extremophilic samples, and 3) the identification and implementation of the appropriate bioinformatics pipelines. Here, we highlight the different ongoing projects that we have been working on, as well as details on the various methods we use to characterize the microbiome and metagenome of these complex samples. In particular, we present data of a novel multienzyme extraction protocol that we developed, called Polyzyme or MetaPolyZyme. Presently, the XMP is characterizing sample sites around the world with the intent of discovering new species, genes, and gene clusters. Once a project site is complete, the resulting data will be publically available. Sites include Lake Hillier in Western Australia, the "Door to Hell" crater in Turkmenistan, deep ocean brine lakes of the Gulf of Mexico, deep ocean sediments from Greenland, permafrost tunnels in Alaska, ancient microbial biofilms from Antarctica, Blue Lagoon Iceland, Ethiopian toxic hot springs, and the acidic hypersaline ponds in Western Australia.

Keywords: Polyzyme; extremophile; metagenomics; shotgun sequencing; whole genome.

Figure 1.

Microbiome and metagenomics publication statistics. PubMed searches for the keywords “metagenomics” and “microbiome” in the title of publications by year.

Figure 2.

Current sites of the XMP. The XMP sites span the world with a diversity of samples that test the salinity, temperature, pressure, moisture, and pH limits of life. GUL, Gulf of Mexico; TKM, Turkmenistan; AUS, Australia; ETH, Ethiopia; and ANT, Antarctica. For the most updated list of sites, seewww.extrememicrobiome.org.

Figure 3.

Relationship among different methods of detection. Different methods of detection used in microbiology, including more traditional methods of microscopy and culturing, as well as the novel molecular approaches in metagenomics analysis. Areas of overlap between these methods are also highlighted.

Figure 4.

A 2012 ABRF NARG study on extraction methods. The ABRF NARG developed a mock community standard made up of 1 different organisms (A), showing the breakdown of these organisms’ relative abundances. (B) The extraction yields across different standard commercial extraction kits. The total expected yield (in nanograms of DNA) and cells in the mix were calculated as 450 ng for 1.1 × 10⁸ cells.

Figure 5.

MGRG Polyzyme mixture workflow and extraction results. (A) Omega Bio-tek DNA extraction method to produce longer fragments of DNA suitable for NGS techniques, such as Pacific Biosciences and Oxford Nanopore Technologies. (B) DNA extraction results for samples treated with the lytic enzyme mix, called Polyzyme, and compared with a no-enzyme control or lysozyme. FU, fluorescent units; Soil MB, Soil MoBio kit.

Figure 6.

XMP workflow. Each site is sampled in triplicate at multiple locations for both culture and nucleic acid (DNA and RNA) extraction and sequencing. All data are then run through bioinformatics analysis for the following: 1) taxa classification, 2) functional analysis, and 3) novel molecule discovery. JGI, Joint Genome Institute (Walnut Creek, CA, USA).