New Biotechnology  Volume 27, Number 3  July 2010 REVIEW

Approaching marine bioprospecting in

hexacorals by RNA deep sequencing

Review
Steinar D. Johansen1,2, Åse Emblem1, Bård Ove Karlsen1,2, Siri Okkenhaug1,
Hilde Hansen3, Truls Moum2, Dag H. Coucheron1,4 and Ole Morten Seternes5
1
RNA and Transcriptomics Group, Department of Medical Biology, Faculty of Heath Sciences, University of Tromsø, Norway
2
Marine Genomics Group, Faculty of Biosciences and Aquaculture, Bodø University College, Norway
3
Department of Chemistry, Faculty of Sciences and Technology, University of Tromsø, Norway
4
Centre for Biosafety, Tromsø, Norway
5
Molecular Pharmacology Group, Department of Pharmacy, Faculty of Health Sciences, University of Tromsø, Norway

RNA deep sequencing represents a new complementary approach in marine bioprospecting. Next-
generation sequencing platforms have recently been developed for de novo whole transcriptome
analysis, small RNA discovery and gene expression profiling. Deep sequencing transcriptomics
(sequencing the complete set of cellular transcripts at a specific stage or condition) leads to sequential
identification of all expressed genes in a sample. When combined to high-throughput bioinformatics
and protein synthesis, RNA deep sequencing represents a new powerful approach in gene product
discovery and bioprospecting. Here we summarize recent progress in the analyses of hexacoral
transcriptomes with the focus on cold-water sea anemones and related organisms.

Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267
Hexacoral genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Nuclear genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Mitogenome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
Transcriptome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269
RNA deep sequencing in cold-adapted sea anemones and related hexacorals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
Key species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270
RNA deep sequencing by the 454 and SOLiD platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Towards gene products with relevance to marine bioprospecting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Next-generation bioinformatics and high-throughput protein synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Product candidates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272
Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274

Introduction
Marine bioprospecting aims to identify, explore and exploit mar-
ine natural products for applications in biomedicine, biotechnol-
Corresponding author: Johansen, S.D. (Steinar.Johansen@uit.no) ogy or environmental surveillance. The traditional approach to

1871-6784/$ - see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2010.02.019 www.elsevier.com/locate/nbt 267
 REVIEW
marine bioprospecting includes systematic screening of marine
extracts for bioactive products and compounds. Promising
candidates may lead to drug discovery after lengthy and detailed
functional studies, structural characterizations, chemical modifi-
cations and perhaps clinical trials [1,2]. Marine bioprospecting
based on next-generation sequence technologies represents a
revolutionary new approach that complements traditional meth-
ods (Fig. 1). In short, this approach entails high-throughput next-
generation RNA sequencing (RNA deep sequencing) to identify the
complete set of expressed genes and derived gene products. Next,
annotated digital data are stored in large databases and processed
into gene product profiles. The gene products (mainly proteins,
peptides and RNAs) are then produced by high-throughput expres-
Review
FIGURE 1
Schematic view of our analysis strategy using RNA deep sequencing in marine bioprospecting of hexacorals.
268 www.elsevier.com/locate/nbt
New Biotechnology  Volume 27, Number 3  July 2010
sion in cells or cell-free extracts, and subsequently screened for
activities and functions.
The transcriptome represents the full complement of RNA
transcripts expressed in a cell for a specific developmental stage
or physiological condition, and consists of protein-coding RNA
transcripts (mRNAs) and non-protein-coding RNA transcripts
[3,4]. Only a fraction of total cellular RNA is referred to as mRNA,
most of which is at very low abundance and requires extremely
sensitive analytical tools to be identified and characterized. The
key aims of transcriptomics are to obtain an exhaustive catalo-
gue of transcripts, to determine the transcriptional structure of
genes, and to quantify changes in expression levels among
transcriptome samples. Owing to the inherent complexity of
New Biotechnology  Volume 27, Number 3  July 2010
transcriptomics, high-throughput technologies are essential.
The hybridization-based microarray technology has for more
than a decade been the most important approach in large-scale
transcriptome profiling. However, limitations in sensitivities
combined with a narrow dynamic range and complex back-
ground hybridization make microarray less suited for profiling
of non-model organisms [5]. Transcriptome analyses based on
next-generation massively parallel sequencing technologies
have become widely available over the past two to three years.
Platforms represented by the Roche 454 Genome Sequencer, the
ABI SOLiD System and Illumina Genome Analyser are well
established. A recent development in the field is the so-called
third-generation sequencing approach (represented by Helicos)
based on direct single molecule sequencing [6]. In contrast to
other deep sequencing platforms, Helicos appears bias-free
because of lack of PCR amplification steps in library or template
preparations [7]. The power of next-generation sequencing tech-
nologies, including RNA deep sequencing, has recently been
extensively reviewed [7,8].
Cnidarians, sponges and ctenophores share several character-
istic features including lack of bilateral body plan symmetry,
ancient evolutionary origin, and highly unusual life strategies.
These apparently primitive marine animals contain complex gen-
omes with equal or even increased number of genes compared to
the bilateral animals [9–11]. Bioprospecting in tropical and sub-
tropical species has resulted in the discovery of several bioactive
compounds of great benefit to human medicine, including cancer
therapy, vascular diseases and infectious viral diseases such as AIDS
[12–15]. Bioprospecting in species found in Arctic and sub-Arctic
waters has a specific aim in exploiting products adapted to extreme
environmental conditions such as low temperature or seasonal
lighting.
The phylum Cnidaria contains more than 9000 nematocyst-
bearing species. According to recently revised cnidarian taxon-
omy, species are distributed among classes Hydrozoa (hydroids
and fire corals), Scyphozoa (jellyfishes), Cubozoa (box jellyfishes),
Myxozoa (endoparasite worms), Polypodiozoa (endoparasites) and
the largest class Anthozoa (true corals) [16,17]. The anthozoans are
further divided at the subclass level into Hexacorallia and Octo-
corallia possessing six (or divided by six) and eight tentacles per
polyp, respectively. Hexacorals constitute a diverse group of ani-
mals and include reef-building stony corals (order Scleractinia), sea
anemones (order Actiniaria), colonial anemones (order Zoanthi-
dea), tube anemones (order Ceriantharia), mushroom corals (order
Corallimorpharia) and black corals (order Antipatharia) [18]. Here
we summarize current strategies and views on applying RNA deep
sequencing by Roche 454 FLX Genome sequencer and ABI SOLiD
sequencer in marine bioprospecting of cold-water sea anemones
and other hexacorals.
Hexacoral genomics
Nuclear genome
The only sequenced hexacoral draft-grade genome to date is that
of the starlet sea anemone Nematostella vectensis [10], representing
a cnidarian model system for developmental and genomic studies
[19]. Sequencing and analyses of 0.36 gigabases (Gb) of the 0.45-
Gb genome identified more than 18,000 protein-coding genes in
the 15 haploid chromosomes [10]. Nematostella genes appear to be
REVIEW
organized in a vertebrate-like manner with eight of ten spliceo-
somal introns located at the exact same site as in humans [10,20].
Almost half of the Nematostella protein-coding genes are also
present as homologs in bilaterian genomes, including several
genes apparently lost in flies and nematodes [10,21,22]. Hexacor-
als are of special interest in gene discovery and bioprospecting
since several human disease causing genes are represented in the
sea anemone genome [23].
Mitogenome
Hexacorals are difficult to be classified by traditional methods,
but a reliable genetic approach at high resolution is mitochon-
drial genome (mitogenome) analysis. Mitogenomics is the study
Review
of complete mitogenomes, and hexacoral relationships can be
derived from concatameric protein sequence alignments in
phylogenetic analysis (our unpublished results). To date, 27
complete hexacoral mitogenomes have been determined and
reported in the NCBI database [24]. These entries represent five
of the six known hexacoral orders, but additional mitogenomes
from cold-water and tropical species have recently been deter-
mined in our research group and will soon be publicly available
(Table 1).
Typical hexacoral mitogenomes are circular 16–18 kb DNA
molecules encoding at least 13 proteins and 4 structural RNAs
(two ribosomal RNAs and two transfer RNAs) [25,26]. The gene
synteny appears to be conserved within, but not between, different
classes. Interestingly, one or two genes are interrupted by a group I
intron (Table 1). The obligatory ND5 intron is probably the most
complex group I intron known harboring 2–16 mitochondrial
genes inserted within its structure (Table 1, Fig. 2) [25,27]. A subset
of mitogenomes contains a second optional group I intron in the
COI gene that encodes a homing endonuclease probably involved
in intron mobility [28]. In addition, open reading frames encoding
unknown proteins and sequence duplication events are also
observed (our unpublished results).
Hexacoral mitogenomes possess a very low substitution rate
compared to most other metazoans resulting in almost no
sequence variations between individuals and populations of a
species [29,30]. This is exemplified in the sea anemone Metridium
senile when the mitogenome (17.4 kb) of two individuals is com-
pared. Only 12 variant nucleotide positions (0.07% of the mito-
genome) were detected in the comparison of Norwegian and US
specimens (our unpublished results).
Transcriptome
Transcriptome analysis of hexacorals is in its beginning and only a
few tropical and subtropical species of sea anemones and stony
corals have been analysed in depth using expressed sequence tags
(EST) libraries combined with traditional Sanger sequencing [31–
35], microarray analysis [36–39], and more recently by 454 pyr-
osequencing [40].
The transcriptome of the two symbiotic sea anemones,
Aiptasia pallida and Anemonie viridis, and the non-symbiont
sea anemone N. vectensis have been analysed using ESTs com-
bined with Sanger sequencing [10,34,35,41]. More than 10,500
ESTs are available from Nematostella [41] and analysis of the A.
pallida library consisting of 10,000 ESTs from a mixed host/
symbiont cDNA population identified a surprisingly low level of
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TABLE 1
Key features of completely sequenced hexacoral mitogenomes
Species Family kb I-ND5 I-COI Acc No
Sea anemone (Order Actiniaria)
Bolocera tuedia Actiniidae 19.1 + + Unpublished
Urticina eques Actiniidae 22.5 + + Unpublished
Aiptasia pulchella Aiptasiidae 19.8 + + Unpublished
Nematostella sp. Edwardsiidae 16.4 + DQ643835
Metridium senile N1 Metridiidae 17.4 + + Unpublished
Metridium senile Metridiidae 17.4 + + AF000023
Colonial anemone (Order Zoantharia)
Savalia savaglia Parazoanthidae 20.8 + + DQ825686
Stony coral (Order Scleractinia)
Acropora tenuis Acroporidae 18.3 + AF338425
Review

Anacropora matthai Acroporidae 17.9 + AY903295

Montipora cactus Acroporidae 17.9 + AY903296
Agaricia humilis Agariciidae 18.7 + DQ643831
Pavona clavus Agariciidae 18.3 + DQ643836
Colpophyllia natans Flaviidae 16.9 + DQ643833
Montastrea annularis Flaviidae 16.1 + AP008973
Montastrea faveolata Flaviidae 16.1 + AP008977
Montastrea franksi Flaviidae 16.1 + AP008976
Mussa angulosa Mussidae 17.2 + DQ643834
Madracis mirabilis Astrocoeniidae 17.0 + EU400212
Pocillopora damicornis Pocilloporidae 17.4 + EF526302
Pocillopora eydouxi Pocilloporidae 17.4 + EF526303
Seriatopora caliendrum Pocilloporidae 17.0 + EF633601
Seriatopora hystrix Pocilloporidae 17.1 + EF633600
Stylophora pistillata Pocilloporidae 17.2 + EU400214
Porites porites Poritidae 18.6 + + DQ643837
Astrangia sp. Rhizangiidae 14.9 + DQ643832
Siderastrea radians Siderastreidae 19.4 + + DQ643838
Mushroom coral (Order Corallimorpharia)
Rhodactis sp. Actinodiscidae 20.1 + + DQ640647
Discosoma sp. CASIZ 168915 Discosomatidae 20.9 + + DQ643965
Discosoma sp. CASIZ 168916 Discosomatidae 20.9 + + DQ643966
Ricordea florida Ricordeidae 21.4 + + DQ640648
Black coral (Order Antipatharia)
Chrysopathes formosa Cladopathidae 18.4 + DQ304771
Note: Presence (+) or absence ( ) of group I introns in the ND5 gene (I-ND5) and COI gene (I-COI) is indicated. Unpublished mitochondrial sequences have been generated in our research
group.

symbiont transcripts [35]. In the A. viridis study 40,000 ESTs
were analysed, revealing novel genes with no homologs in
Nematostella. Homologs to these are present in other symbiotic
cnidarians though, and may thus represent genes involved in
host–symbiont relationships [34].
Transcriptome analyses of the hard corals Acropora millipora,
Acropora palmate and Montastrea faveolata have been performed
using all three methods mentioned above. A library consisting
of about 18,500 ESTs generated from five life stages of both A.
palmata and Montastrea has been established to identify candi-
date symbiosis-related genes as well as candidate genes for
understanding the life history of scleractinian corals [33]. A
subset of these ESTs has been further exploited in a microarray
approach containing 2055 features from A. palmate and 1314
features from Montastrea. In this study differential expression
following exposure to symbiont algae was analysed [39]. Micro-
array has also been used to analyse A. millipora developmental
stages as well as environmental effects on its transcriptome
[36,38]. Finally, 454 deep sequencing of the A. millipora larva
transcriptome has been performed [40] generating more than
600,000 reads assembled into 40,000 contigs, representing some
11,000 different genes.
RNA deep sequencing in cold-adapted sea anemones
and related hexacorals
Key species
Several cold-water hexacoral species are present in the North
European coastal areas, including a variety of sea anemones [42]
and also a few reef-building corals, tube anemones and colonial
anemones. In contrast to their tropical and subtropical relatives,
most cold-water species lack photosynthetic symbiotic
algae. Representative cold-water hexacorals included in our
mitogenome and transcriptome studies are shown in Fig. 3.
We also investigate the reef-building cold-water stony coral
Lophelia pertusa and some tropic and subtropic reference species
raised in our reef-tank facility. Sampling of low-temperature
species is performed at various locations at the coast of
Northern Norway.

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New Biotechnology  Volume 27, Number 3  July 2010 REVIEW

Review
FIGURE 2
Organization of the Metridium senile N1 mitogenome sampled from a cold-water area in Northern Norway. The circular genome (17.4 kb) contains two rRNA genes
and two tRNA genes (red boxes). Two of the 14 protein genes are interrupted by group I introns (schematically presented by their secondary RNA structures).
Three protein genes have intronic locations (ND1 and ND3 in Mse.ND5-717, and HEG in Mse.COI-884). W and M, tRNA for tryptophan and methionin; SSU and LSU,
mitochondrial SSU and LSU rRNA genes; ND1-6, NADH dehydrogenase subunit 1–6; COI-III, cytochrome c oxidase subunit I–III; A6 and A8, ATPase subunit 6 and 8;
Cyt B, cytochrome b; HEG, homing endonuclease gene. P1–P9, conserved helices present in most group I intron structures [27].

RNA deep sequencing by the 454 and SOLiD platforms
The analysis strategy presented here combines Sanger (mtDNA
sequencing), 454 (whole transcriptome) and SOLiD (expression
profiling) technologies (summarized in Fig. 1). SOLiD ligation-
based sequencing is conducted at our in-house laboratory facility
[43]. The 454 pyrosequencing is based on sequencing-by-synthesis
generating base-space reads [44]. In short, for each incorporated
dNMP in the newly synthesized DNA a pyrophosphate (PPi) is
released. The PPi is then indirectly detected by luminescence
production recorded as luciferin is converted to oxyluciferin by
luciferase. The 454 GS FLX Titanium version gives 1.2 million
reads per run with an average read length of 400 bp, resulting in
single run yields of at least 0.5 Gb. Thus, 454 pyrosequencing is the
preferred technology in de novo whole transcriptome sequence
analysis.
SOLiD (Sequencing by Oligo Ligation and Detection) sequen-
cing is a high-throughput short read (35, 50, or 75 nt) sequencing
system [45,46]. One single run generates approximately 100 Gb of
sequence from more than a billion reads, and with an order of
magnitude lower cost per base compared to the 454 technology.
SOLiD uses a unique ligation-based color-space sequencing strat-
egy with semi-degenerated short oligonucleotides and a two-base
encoding scheme. Here, each base is independently read twice,
providing an internal proofreading system with more accurate
recording than other next-generation platforms [7,47]. SOLiD
sequencing is highly preferred in small RNA and gene expression
profiling, as well as in whole transcriptome re-sequencing.
Towards gene products with relevance to marine
bioprospecting
Next-generation bioinformatics and high-throughput protein
synthesis
The enormous amounts of data generated by RNA deep sequen-
cing require extended biocomputing resources. Data storage, eva-
luation of sequence quality and retrieval of sequences are usually
performed by software packages available with the sequencing
platforms (454 and SOLiD) [48–51]. In addition, various open-
source and commercial software packages are available for further
analyses [52–58].
Expression and subsequent functional testing of several
proteins simultaneously have recently been reported on
cell-free protein synthesis and high-density protein arrays
[59]. Cell-free protein synthesis offers several advantages over
cell-based methods in large-scale protein production [59–62].
Eukaryotic-based lysates may direct a wide range of protein
modifications and express much protein simultaneously at

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 REVIEW New Biotechnology  Volume 27, Number 3  July 2010
Review

FIGURE 3
Key species representing five distinct orders of the subclass Hexacorallia included in our study. (a) Bolocera tuediae, Order Actiniaria (sea anemones), Family
Actiniidae; (b) Hormathia digitata, Order Actiniaria, Family Hormathiidae; (c) Metridium senile, Order Actiniaria, Family Metridiidae; (d) Urticina eques, Order
Actiniaria, Family Actiniidae; (e) Urticina felina, Order Actiniaria, Family Actiniidae; (f) Protopalythoa sp., Order Zoantharia (colonial anemones), Family
Zoanthidae; (g) Aiptasia pulchella, Order Actiniaria, Family Aiptasiidae; (h) Rhodactis indosinensis, Order Corallimorpharia (mushroom corals), Family
Actinodiscidae; (i) Cerianthus lloydii, Order Ceriantharia (tube anemones), Family Cerianthidae; (j) Caulastrea furcata, Order Scleractinia (stony corals), Family
Flaviidae. Hexacoral species (a)–(e) and (i) are captured from cold-water environments (Northern Norway), and species (f )–(h) and (j) are from our tropical reef-
tank facility. Photos by SDJ.

high yields [59,63]. Automated platforms performing high-
throughput protein production and functional screening are
now available including the DAPA procedure (DNA array to
protein array) [64].
Product candidates
Comparative analysis of our current 1.2 million 454 sea anemone
transcriptome reads (500,000 Bolocera tuedia, 500,000 Hormathia
digitata and 200,000 Urticina eques) (Fig. 3a,b,d) against annotated
sequences in public databases identified thousands of different
derived proteins, some with relevance to biomedicine and bio-
technology. Among these are several enzymes, fluorescent pro-
teins and peptide toxins.
The key color determinants in hexacorals are fluorescent and
non-fluorescent chromo proteins [65]. The green fluorescent pro-
tein (GFP) was first described in the jellyfish Aequorea [66], and is
now widely applied as fluorescent labels in molecular cell biology
and molecular genetic research. Various chromoproteins have
been discovered in tropical and subtropical hexacorals and include
the fluorescence colors green, cyan, red, yellow and chromo-red
proteins [65,67–69]. The presence of homologous genes in cold-
water sea anemones provides new properties and applications of
fluorescent labels.
The sea anemones are rich sources of two classes of peptide
toxins, the cytolysins and neurotoxins [70,71], with important
biological roles in predation and defense. Cytolysins are pep-
tides or polypeptides belonging to four defined groups, each
with distinct structural and functional characteristics. Arctic
and sub-Arctic genera of sea anemones are known to express
all the cytolysin groups [70]. The neurotoxin peptides (usually
30–60 amino acids in size) are related to peptide toxins found in
venomous snakes, spiders, bees or scorpions. The neurotoxins
are produced in specialized stinging cells (nematocysts) located
at the tip of tentacles, and some appear differentially expressed
because of intron retention at specific developmental stages
[72]. Both the sodium and potassium voltage-gated channel
toxin transcripts are identified in our 1.2 mill 454 reads.
Figure 4 presents an amino acid sequence alignment of sodium
channel toxins found in representative sea anemones. Channel
neurotoxins are important molecular tools in cell biology

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New Biotechnology  Volume 27, Number 3  July 2010 REVIEW

Review
FIGURE 4
Amino acid sequence alignment of sodium channel neurotoxins from some sea anemones. The putative Urticina eques toxin (Ue-1) and Hormathia digitata toxin
(Hd-1) generated from our RNA deep sequencing analysis represent the Type 1 and Calitoxins, respectively. The leader peptide is cleaved off at the cleavage
tandem sequence is indicated. Additional sequences are from [72–74] and their references.

and neurobiology research, but it remains to be seen if the
cold-water sea anemone toxins possess unique functional
properties.
Perspectives
Sequence analyses of complete genomes and whole transcrip-
tomes will revolutionize our understanding of early diverging
animals such as the hexacorals. Hopefully the complete genome
sequencing of the Nematostella sea anemone [10] soon will
be followed by more species representing both model
and non-model organisms (e.g. cold-adaptive species). The tran-
scriptome, which has a deeper complexity than the correspond-
ing genome, reflects the gene expression at a given
developmental stage or tissue type. In fact, the composition
of the transcriptome varies by changes in physiological states
and external factors such as temperature or various pollutants.
This gives new possibilities and applications for these benthic
animals in environmental surveillance. Transcriptome profiling
and analysis of hexacoral model systems, represented by the sea
anemones Nematostella and Aiptasia, and the stony coral Acro-
pora [11], will bring new valuable information as references for
gene composition and expression patterns in comparative stu-
dies. Next-generation transcriptome sequence data from non-
model genera adapted to low-temperature environments (e.g.
Lophelia, Urticina, Bolocera, Hormathia and Ceriantus) will raise
many new and interesting questions. Are there unique sets of
genes in low-temperature adapted species? Is the lack of photo-
synthetic symbionts reflected in gene expression profiles? How
do homologous proteins in related hexacoral species from tro-
pical and low-temperature environments maintain their func-
tions? These and many other relevant questions will be
approached by the next-generation massively parallel sequen-
cing technologies and experimental functional studies in the
next few years.
Acknowledgements
We thank members in our research groups at University of Tromsø
and Bodø University College for interesting discussion and

www.elsevier.com/locate/nbt 273
 REVIEW
continued enthusiasm. We also acknowledge colleges at Eurofins
MWG Operon for technical discussions and 454 sequencing
services, and colleges at Applied Biosystems for discussion and
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