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

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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.
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ARTICLE
Genomic Methods and Microbiological Technologies for Proling Novel and Extreme
Environments for the Extreme Microbiome Project (XMP)
Scott Tighe,
1
,
*
,
Ebrahim Afshinnekoo,
2
,
3
,
4
,
*Tara M. Rock,
5
Ken McGrath,
6
Noah Alexander,
2
,
3
Alexa McIntyre,
2
,
3
Soa Ahsanuddin,
2
,
3
Daniela Bezdan,
2
,
3
Stefan J. Green,
7
Samantha Joye,
8
Sarah Stewart Johnson,
9
Don A. Baldwin,
10
Nathan Bivens,
11
Nadim Ajami,
12
,
13
Joseph R. Carmical,
12
,
13
Ian Charold Herriott,
14
Rita Colwell,
15
Mohamed Donia,
16
Jonathan Foox,
2
,
3
,
17
Nick Greeneld,
18
Tim Hunter,
1
Jessica Hoffman,
1
Joshua Hyman,
19
Ellen Jorgensen,
20
Diana Krawczyk,
21
Jodie Lee,
22
Shawn Levy,
23
Nat`alia Garcia-Reyero,
24
Matthew Settles,
25
Kelley Thomas,
26
Felipe G´omez,
27
Lynn Schriml,
28
,
29
Nikos Kyrpides,
30
Elena Zaikova,
9
Jon Penterman,
31
and Christopher E. Mason
2
,
3
,
32
,
1
Advanced Genomics Lab, University of Vermont Cancer Center, University of Vermont, Burlington, Vermont, USA;
2
Department of Physiology and Biophysics,
3
The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute
for Computational Biomedicine, and
32
The Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, New
York, New York, USA;
4
School of Medicine, New York Medical College, Valhalla, New York, USA;
5
Center for Genomics
and Systems Biology, New York University, New York, New York, USA;
6
Australian Genome Research Facility, Gehrmann
Labs, University of Queensland, St Lucia, QLD, Australia;
7
DNA Services Facility, Research Resources Center, University
of Illinois, Chicago, Illinois, USA;
8
Marine Sciences, The University of Georgia, Athens, Georgia, USA;
9
Department of
Biology, Georgetown University, Washington, DC, USA;
10
Signal Biology Inc., Philadelphia, Pennsylvania, USA;
11
DNA Core
Facility, University of Missouri, Columbia, Missouri, USA;
12
Alkek Center for Metagenomics and Microbiome Research and
13
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, USA;
14
Institute of Arctic
Biology, University of Alaska Fairbanks, Fairbanks, Alaska, USA;
15
Center for Bioinformatics and Computational Biology,
University of Maryland Institute for Advanced Computer Studies, College Park, Maryland, USA;
16
Department of Molecular
Biology, Princeton University, Princeton, New Jersey, USA;
17
Department of Invertebrate Zoology, American Museum of
Natural History, New York, New York, USA;
18
One Codex, San Francisco, California, USA;
19
UW Biotechnology
Center, University of Wisconsin - Madison, Madison, Wisconsin, USA;
20
Genspace NYC, Inc., Brooklyn, New York,
USA;
21
Greenland Institute of Natural Resources, Greenland Climate Research Centre, Nuuk, Greenland;
22
Molecular
Diagnostics, Qiagen, Germantown, Maryland, USA;
23
HudsonAlpha Institute for Biotechnology, Huntsville, Alabama,
USA;
24
Institute for Genomics Biocomputing, and Biotechnology, Mississippi State University, US Army Engineer
Research & Development Center, Vicksburg, Mississippi, USA;
25
Genome Center, UC Davis, Davis, California, USA;
26
Hubbard Center for Genome Studies, University of New Hampshire, Durham, New Hampshire, USA;
27
Department of
Planetology and Habitability, Centro de Astrobiolog´ıa (CSIC-INTA), Carretera de Ajalvir, Km 4. 28850 Torrejon
de Ardoz, Madrid, Spain;
28
Epidemiology and Public Health, and
29
Institute for Genome Sciences, University of Maryland
School of Medicine, Baltimore, Maryland, USA;
30
Department of Energy, Joint Genome Institute, Walnut Creek, California,
USA; and
31
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
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 characteriza-
tion 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
themicrobiomeandmetagenomeofthesecomplexsamples. In particular, we present data of a novel
*These authors contributed equally to this work.
ADDRESS CORRESPONDENCE TO: Scott Tighe, Advanced Genomics Lab, Universityof VermontCancer Center,149 Beaumont Ave., Burlington, VT 05405,
USA (Phone: 001-802-656-2482; Fax: 8026562140; E-mail: scott.tighe@uvm.edu).
ADDRESS CORRESPONDENCE TO: Christopher E. Mason,Dept. of Physiology and Biophysics, Weill Cornell Medicine, 1305 York Ave., New York, NY 10021,
USA (Phone: 203-668-1448; Fax: 646-962-0383; E-mail: chm2042@med.cornell.edu).
doi: 10.7171/jbt.17-2801-004
Journal of Biomolecular Techniques 28:3139 © 2017 ABRF
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 Hellcrater 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.
KEY WORDS:metagenomics, whole genome, shotgun sequencing, extremophile, Polyzyme
INTRODUCTION
Revolutionary advances in sequencing technology have
enabled extensive surveys of microbiomes and have sub-
sequently transformed our understanding of the human-
microbe interface. In the last decade, we have entered
into the microbiome era,marked by a rapid increase of
studies exploring the microbial communities that live in
us, on us, and all around us (Fig. 1).
15
Despite these major
technological advances and groundbreaking studies that
have been undertaken in the past few years, there are
few concerted efforts dedicated to investigating the most
elusive of ecological niches: the extremophiles. Physiologic,
chemical, and biologic adaptations allow extremophiles to
thrive in the most acidic, saline, hot, cold, and barophilic
environments on our planet. The secrets to their survival lie
in the versatility and adaptability of their genomes.
68
Taxonomic classication of extremophiles has been a
pioneering eld of study since the 1950s. Efforts to
characterize extremophiles increased after the discovery of
Thermus aquaticus
9
and Taq polymerase,
10
to such a degree
that the International Society of Extremophiles was
established in the 1990s and publishes the dedicated peer-
reviewed journal, Extremophiles.
11
In recent years, in-
ternational efforts, such as the Earth Microbiome Project
(EMP), have initiated large-scale endeavors to map the
distribution of microorganisms (including extremophiles)
across the globe. However, whereas the EMP is one of the
largest contemporary microbiome projects in the world,
12, 13
the methods are signicantly different from those used by
the XMP. This unique consortium was founded in 2014
with the intention to create a comprehensive molecular
prole of various extreme sites using novel culturing
methods, long-read and short-read whole genome shotgun
sequencing (instead of rRNA amplicon-based methods),
improved RNA and DNA extraction methods, methylation
tracing, and for future studies, metaproteomics. Many of
these techniques have yet to be fully developed, which is why
the ABRF MGRG was established, representing a pioneer-
ing research consortium dedicated to characterizing the
current methods and development of new methods for
ubiquitous metagenomics and microbiomes studies.
The understanding of extremophilestheir genomes,
molecular machinery, and how they interact with their
environmentshas potential health and research benets for
humanity. There are applications in bioremediation of polluted
sites deemed too unbearable for most living organisms or as
sources for novel therapeutics in medicine and potentially,
an alternative process for biofuel or energy production. The
metabolic mechanisms of these organisms are rather specialized
and could inspire innovations in such diverse areas as synthetic
biology and research into human survival in space. Many
extreme environments offer relatively accessible proxies for
the harsh environments found beyond Earth. This is why the
consortium members are both academic and corporate, having
diverse backgrounds in areas such as microbiology, genetics,
oceanography, planetary science, geochemistry, and bioinfor-
matics. Whereas most contributors are academic, several others
are coming from industry including researchers from Illumina
(San Diego, CA, USA), Thermo Fisher Scientic (Waltham,
MA, USA), BioO Scientic (Austin, TX, USA), New England
Biolabs (Ipswich, MA, USA), Omega BioTek (Norcross, GA,
USA), and One Codex (San Francisco, CA, USA); see www.
extrememicrobiome.org for details.
14
SAMPLE COLLECTION AND PROCESSING
Since the inception of the XMP, the consortium has begun
collecting and analyzing data from 12 sites across the
world, with more sites under consideration (Fig. 2). This
FIGURE 1
Microbiome and metagenomics publication statistics. PubMed
searches for the keywords metagenomicsand microbiomein
the title of publications by year.
TIGHE ET AL. / GENOMIC METHODS OF THE EXTREME MICROBIOME PROJECT
32 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 1, APRIL 2017
effort differs from other microbiome projects, as each site is
sampled and analyzed as a complete stand-alone project.
The selected sites are dened as extreme or novel,based on
such metadata as salinity, temperature, pressure, moisture,
pH, or remoteness, with many sites falling into more
than one category. Table 1 provides details of the samples
collected, including location, suspected types of organisms,
and the sample-processing methods applied (e.g., culturing,
DNA sequencing, and RNA sequencing).
The ABRF MGRG works as a collaborative team to
study these environments, using both traditional and novel
methods as outlined in Fig. 3. This includes a modied,
nucleic acid-free sample collection; extraction of the DNA/
RNA using methods to preserve nucleic acid length;
culturing; microscopy; and multiple types of nucleic acid
sequencing. Culturing methods are included to address
the questions of viability and codependency, as well as the
relationship to the detection of species using next-generation
sequencing (NGS) and bioinformatics analyses. It is well
known that whereas most organisms are unculturable, there
are still gaps in our knowledge about some of the culturable
organisms (or ones observed by microscopy) that are not
amenable to characterization by NGS technologies due
to experimental limitations or challenges with nucleic acid
extraction. The scenario is outlined in Fig. 3.
Not surprisingly, there are major challenges involved with
the XMP as a stand-alone, all-encompassing project, from
sample collection to bioinformatics. These unique samples are
often difcult to collect because of their remote site location
and sometimes, even worse to extract their DNA and RNA as a
result of reticent cells. Samples from harsh environments tend
to have robust cell walls requiring special lysis procedures.
Consequently, one of the major goals of the MGRG was
development of a novel extraction method tailored to difcult
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.
TIGHE ET AL. / GENOMIC METHODS OF THE EXTREME MICROBIOME PROJECT
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 1, APRIL 2017 33
samples that avoided beater beads whenever possible to
minimize unnecessary shearing of DNA. These protocols
included substituting a novel multienzyme blend, called
Polyzyme,in place of lysozyme and further extraction of
DNA to recover long fragment length DNA compatible with
long sequencing strategies. Whereas culturing is not the
primary focus of the projects, it does provide minimum truth
in a sample and also requires multiple techniques, such as the
following: 1) use of a multitude of microbial growth media and
broths (including sample site enrichment media), 2) culturing
of anaerobically and aerobically, 3) incubation at different
times and temperatures, and 4) identication using full-length,
16S DNA sequencing and/or the Microbial Identication
system (Biolog, Hayward, CA, USA).
Samples collected for the XMP are sequenced using the
standard commercial sequencing platforms, including HiSeq
FIGURE 3
Relationship among different methods of de-
tection. Different methods of detection used in
microbiology, including more traditional meth-
ods of microscopy and culturing, as well as the
novel molecular approaches in metagenomics
analysis. Areas of overlap between these meth-
ods are also highlighted.
TAB LE 1
Collection sites and characteristics: phase I of the XMP
Site name Site type Location Types of organisms
Deep-sea brine lakes Hypersaline, methyl hydrate, salt
derived, and halite derived
Gulf of Mexico Barophiles, halophiles, chemotrophs
Door to Hell gas crater High and moderate temperatures,
molten areas of rock and sand
Karakum Desert, Turkmenistan Soil thermophiles, chemotrophs
Lake Hillier Hypersaline, pH neutral,
precipitated salt
Recherche Archipelago,
Western Australia
Halophiles, methylotrophs,
phototrophs, sulfobacteria
Greenland shelf
sediments
Paleoglaciers sediment
(deep-water marine sediments)
Greenland Psychrophiles, halophiles
Acidic hypersaline ponds Hyperacidic ponds Yilgarn Craton, Australia Acidophiles, chemotrophs
Mono Lake Alkaline, hypersaline California, USA Halophiles, alkaphiles, methanogen,
sulfate reducers
Permafrost Deep frozen, high ice pressure of
geologically ancient origin
Alaska, USA; Siberia, Russia Psychrophiles, barophiles
Dry valley lakes Ancient microbial biofilms Victoria Land, Antarctica Heterotrophs, psychrophiles,
chemo/ autotrophs/sulfate reducers
Toxic hot springs Volcanic hot springs acidic, alkaline,
high sulfur, high chlorine,
high temperature
Danakil Depression, Ethiopia Thermophiles, acidophiles,
chemotrophs, sulfate reducers
Great Salt Lake Hypersaline lake Utah, USA Halophiles
Gowanus Canal Industrial toxins, low pH,
black tar sludge
New York, USA Iron bacteria, psychrophiles, chemo/
autotrophs
TIGHE ET AL. / GENOMIC METHODS OF THE EXTREME MICROBIOME PROJECT
34 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 1, APRIL 2017
and MiSeq (Illumina), PacicBiosciences(MenloPark,CA,
USA), Thermo Fisher Scientic, and Oxford Nanopore
Technologies (Oxford, United Kingdom). A mix of these
sequencing technologies allows us to not only assess the
strengths and weaknesses of these different platforms but
also allows us to integrate the data together to generate a
comprehensive molecular prole of each sample and each site.
Finally, RNA extraction is accomplished using a standard
TRIzol LS procedure
15
combined with a bead-beater step
using Matrix A lysing matrix (MP Biomedicals, Santa Ana,
CA, USA). RNA sequencing, coupled with functional and
phylogenetic bioinformatics, provides results on the dynam-
ics of the metatranscriptome at these sites, notably, the
metabolic systems of extremophiles in situ.
DEVELOPMENT OF A POLYZYME MPZ MIXTURE FOR
ENHANCED DNA AND RNA EXTRACTION
For a metagenomics study to be as comprehensive as pos-
sible, high-efciency, high-yield, and unbiased nucleic acid
extraction methods are required. Whereas it is currently
possible to extract nucleic acids, it is not possible to extract
100% of all organisms, especially from different domains
of life (i.e., bacteria, fungi, viruses, archaea, and other
eukaryotes) or even species to species, in some cases.
Moreover, the question of viability remains a challenge in
the eld of metagenomics, as it is not possible to determine
whether the nucleic acids recovered belong to living
organisms. These challenges create major obstacles for the
applications of metagenomics research described earlier. A
key question that must be addressed is which organisms are
present in a sample, and in what phase of growth ordormancy
are they? As a result of these concerns, a small ABRF study was
conducted in 2012 that focused on addressing 2 areas: rst,
comparing DNA extraction efciencies on a known bacterial
mix using different methods
16
and second, investigating
methods to increase extraction efciencies using Polyzyme.
Figure 4A shows the species breakdown of a bacterial mix
standard developed by the ABRF Nucleic Acids Research
Group (NARG). Figure 4B depicts the different DNA yields
of this mock community using different DNA extraction kits.
With the recognition of the limitations of nucleic acid
extraction efciency, the MGRG investigated alternative
methods to increase DNA yields before downstream
processing and analyses. These new methods take into
account previous ABRF data, data from scientic corporate
partners, and current ongoing studies by members of the
ABRF MGRG, Metagenomics and Metadesign of the
Subways and Urban Biomes International Consortium,
17
and International Metagenomics and Microbiome Stan-
dards Alliance.
18
Standardization of these methods have
included the use of Polyzyme, a novel enzyme blend of
microbial lytic enzymes that digest cell-wall components
and allow for more efcient lysis of the resulting
sphearoplasts or protoplasts. Polyzyme was originally
designed by S.T. in 2006 and further rened by
MilliporeSigma (Billerica, MA, USA) and includes 6
enzymes that specically target the cell wall of bacteria,
yeast, and fungi [see MilliporeSigma MetaPolyZyme]. This,
when used in combination with the XMP multifaceted
DNA extraction method that uses hexadecyltrimethylam-
monium bromide, SDS, phenol chloroform, and magnetic
beads, proves increased recovery of high MW DNA on
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 organismsrelative
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 1.1 310
8
cells.
TIGHE ET AL. / GENOMIC METHODS OF THE EXTREME MICROBIOME PROJECT
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 1, APRIL 2017 35
many sample types (Fig. 5) needed for third-generation
sequencing platforms. This research is still underway, and
efciency data will be published in a future manuscript.
DEVELOPMENT OF MICROBIAL
REFERENCE STANDARDS
Another major challenge for metagenomics and micro-
biome research is the lack of microbial reference standards
and controls for determination of protocol efciencies for
DNA extraction, DNA sequencing, and bioinformatics.
The ABRF MGRG is working with the National Institute
of Standards and Technology and Genomics Standard
Consortium (GSC) to design reference standards for this
application. Standardized mock communitiescan serve
as positive controls for microbiome and metagenomics
studies, allowing researchers to evaluate the reliability and
limitations of their results and interpretations. The MGRG
has developed 3 Class I microbial reference standards that
are distributed by the American Type Culture Collection
FIGURE 5
MGRG Polyzyme mixture workflow and extrac-
tion 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 Technolo-
gies. (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.
TAB LE 2
Bacterial species used in the 3 genomic DNA microbial reference standards developed by the ABRF MGRG and XMP
Organism Designation Gram reaction Genome size, Mb
Staphylococcus epidermidis PCI 1200 ATCC 12228 + 3.50
Chromobacter violaceum NCTC 9757 ATCC 12472 22.75
Micrococcus luteus NCTC 2665 ATCC 4698 + 3.17
Pseudoalteromonas haloplanktis TAC125 ATCC 35231 24.04
Haloferax volcanii DS2 ATCC 29605 + 4.11
Bacillus subitilis subsp. Subtilis str. 168 ATCC 23857 + 5.81
Halobacillus halophilus DSM 2266 ATCC 35676 + 5.55
Escherichia coli K-12 substr. MG1655 ATCC 700926 25.58
Entercoccus faecalis OG1RF ATCC 47077 + 3.57
Pseudomonas fluorescens F113 ATCC 13525 28.68
TIGHE ET AL. / GENOMIC METHODS OF THE EXTREME MICROBIOME PROJECT
36 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 1, APRIL 2017
(ATCC; Manassas, VA, USA; Table 2). These standards are
composed of Bio-safety level 1 organisms of differing Gram
reaction and guanine-cytosine content. Class I genomes are
the simplest to sequence and assemble and can be applied to
all sequencing platforms.
19
COMPUTATIONAL ANALYSES
Figure 6 summaries an example of the workow of one
XMP site: Lake Hillier, Australia. After the wet lab
component and sequencing are complete, the sequence
reads were analyzed using a specially developed XMP
bioinformatics pipeline, which includes 3 major com-
ponents: 1) taxa classication, 2) functional analysis, and
3) novel molecule discovery. Not surprisingly, pipeline
challenges exist, as some of the organisms have not
been described, sequenced, or detected previously using
molecular techniques and therefore, lack reference
genomic data. To address this challenge, the bioinfor-
matics team is working on a pipeline strategy that
includes an ensemble approach of current taxa classi-
cation and quantication analytics to ensure a thorough
and comprehensive examination of the samples. More-
over, a database is being constructed of all unmapped,
uncharacterized sequences, which can be used for future
queries. Novel assemblies of synthetic metagenomes and
related metadata, known as genomes from metagenomes
(GFM), as well as minimum information about extracted
GFM, will be considered for deposition into the National
Center for Biotechnology Information databank where
possible.
2022
The concept of a complete molecular prole of each
site is an important goal of the XMP and the primary
reason for using shotgun whole-genome sequencing. To
that end, the functional analysis component will include
searching for abundant functional biomolecular path-
ways,aswellasscreeningforantimicrobialresistance
genes and markers. Moreover, one of the more exciting
aspects of the XMP bioinformatics analysis is the search
for novel gene clusters and molecules that can be used for drug
development, such as new antibiotics. MetaBGC is an
algorithm developed by the M.D. lab for the discovery of
biosynthetic gene clusters (BGCs), which will be used on
unassembled or minimally assembled complex metagenomic
data and cultured and uncultured microbes.
23
The applica-
tion of the MetaBGC pipeline previously has been demon-
strated successfully on the New York City PathoMap datasets
5
by detecting novel and predicted thiopeptide BGCs on steel
subway railings after Hurricane Sandy.
17
CONCLUSION
The XMP is helping to make unique contributions to the
eld of microbiome and metagenomics research, speci-
cally in new methods and product development. As
extremophilic samples are extraordinarily difcult to work
with, they require new approaches that can be applied to
other microbiome projects: both small contributions, such
as development of nucleic acid-free reagents, standards, and
protocol, or complex questions, such as discovering clues to
synthetic gene clusters or new antibiotics. Nonetheless,
discoveries made in the eld of microbiome will have a
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).
TIGHE ET AL. / GENOMIC METHODS OF THE EXTREME MICROBIOME PROJECT
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 1, APRIL 2017 37
major impact on understanding health; the way we handle
food; the way we build buildings, subways, boats, and
airplanes; or disease transmission, by comprehending the
metagenomics of biolms, for example.
17
Regardless of the
area of research, the eld of microbiome research remains
one of the fastest growing and most exciting areas of biologic
research today, with possibly the most signicant discoveries
yet to come.
AUTHORSHIP
C.E.M. and S.T. cofounded and led the MGRG and XMP. E.A. helped
coordinate the XMP, especially regarding analysis of samples. T.M.R.
and A.M. manage the XMP website (www.extrememicrobiome.org).
E.A., S.T., and C.E.M. led the writing of the manuscript. R. Colwell,
S.S.J., T.M.R., N.K., D.B., A.M., K.M., and S.A. provided critical
manuscript edits, and S.A. helped form at and nalize the manuscript for
submission. S.J.G., S.J., S.S.J., D.K. I.C.H., K.M., F.G., E.J., and S.T.
are responsible for sample collection, logistics, and technical arrange-
ments at remote sites. E.A. A.M., N. Ajami, J.R.C., R. Colwell, S.J.G.,
M.D., J.F., N.G., M.S., K.T., and C.E.M. provided complex
bioinformatics analysis. D.A.B., D.B., N. Alexander, N.B., J. Hoffman,
J. Hyman, J.L., S.L., N.G-R., K.T., T.M.R., K.M., J.P., S.T., and
E.Z. preformed the lab processing. N. Alexander, N.G-R., S.J.G., S.T.,
K.M., K.T., I.C.H., T.M.R., and C.E.M. provided Oxford Nanopore
Technologies, Illumina, or Pacic Biosciences sequencing services. L.S.
and N.K. led the GSC and provided technical guidance and support.
T.H. is the Executive Board liaison to ABRF. The manuscript was
reviewed and approved by all authors.
ACKNOWLEDGMENTS
The authors thank the ABRF for hosting the XMP through the
MGRG subcommittee. The authors thank Chuan-Yu Hsu at
Mississippi State University; Steve Simpson, Feseha Abebe-Akele,
Jordan Ramsdell, and Krystalynne Morris at University of New
Hampshire; John Lizamore and Don Cater at Department of Parks
and Wildlife (Western Australia); Susannah Tringe from the Joint
Genome Institute for technical input and guidance during the initial
assembly of the consortium; and Robert Prill at IBM for bioinfor-
matics support (MegaBlast). All work related to XMP has been done
solely by in-kind contributions through the research members and
associated core labs. All materials for extraction, sequencing, and
analysis have been contributed by the devoted corporate research
partners of XMP, including Mostafa Ronaghi, Rob Cohen, and
Clotilde Teiling at Illumina; Fiona Stewart at New England Biolabs;
Adam Morris at Bioo Scientic; Mike Farrell and Ken Guo at Omega
Bio-tek; Ryan Kemp at Zymo Research; Sam Minot at One Codex;
Manoj Dadlani and Nur Hasan at CosmosID; Anjali Shah and Abizar
Lakdawalla at Thermo Fisher Scientic; and Thomas Juehne, George
Yeh, and Robert Gates at MilliporeSigma. The authors give special
thanks to George Kourounis for collection of samples from the
Darvaza crater Door to Hell,Turkmenistan. The authors also thank
Michael Micorescu from Oxford Nanopore Technologies for de-
signing special software for the XMP and helping with work in
Antarctica. The authors thank Tarun Khurana at Illumina and Mike
Farrell at Omega Bio-tek for agreeing to help test the Polyzyme. The
authors thank the Epigenomics Core of Weill Cornell Medicine,
funding from Starr Cancer Consortium grants (I7-A765, I9-A9-071),
Irma T. Hirschl and Monique Weill-Caulier Charitable Trusts, Bert L
and N Kuggie Vallee Foundation, WorldQuant Foundation, Pershing
Square Sohn Cancer Research Alliance, NASA (NNX14AH50G,
NNX17AB26G), U.S. National Institutes of Health (R25EB020393,
R01NS076465, R01AI125416, R01ES021006), Bill and Melinda Gates
Foundation (OPP1151054), and Alfred P. Sloan Foundation (G-2015-
13964).The authors thank Europlanet 2020 RI from the European
Unions Horizon 2020 research and innovation programme, under grant
agreement No 654208 funded Danakil work, for the Ethiopian Hot
Springs.
DISCLOSURES
The authors have read and understood the Journal of
Biomolecular Techniquespolicies on declaration of interests
and herein declare the following interests: N.Greeneld is
employed by and retains ownership in Reference Genomics
(One Codex). R. Colwell is the founder of CosmosID.
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