ARTICLES
Genome of the marsupial Monodelphis
domestica reveals innovation in
non-coding sequences
Tarjei S. Mikkelsen1,2, Matthew J.Wakefield3, Bronwen Aken4, Chris T. Amemiya5, Jean L. Chang1, Shannon Duke6,
Manuel Garber1, Andrew J. Gentles7,8, Leo Goodstadt9, Andreas Heger9, Jerzy Jurka8, Michael Kamal1,
Evan Mauceli1, Stephen M. J. Searle4, Ted Sharpe1, Michelle L. Baker10, Mark A. Batzer11, Panayiotis V. Benos12,
Katherine Belov13, Michele Clamp1, April Cook1, James Cuff1, Radhika Das14, Lance Davidow15, Janine E. Deakin16,
Melissa J. Fazzari17, Jacob L. Glass17, Manfred Grabherr1, John M. Greally17, Wanjun Gu18, Timothy A. Hore16,
Gavin A. Huttley19, Michael Kleber1, Randy L. Jirtle14, Edda Koina16, Jeannie T. Lee15, Shaun Mahony12,
Marco A. Marra20, Robert D. Miller10, Robert D. Nicholls21, Mayumi Oda17, Anthony T. Papenfuss3, Zuly E. Parra10,
David D. Pollock18, David A. Ray22, Jacqueline E. Schein20, Terence P. Speed3, Katherine Thompson16,
John L. VandeBerg23, Claire M. Wade1,24, Jerilyn A. Walker11, Paul D. Waters16, Caleb Webber9,
Jennifer R. Weidman14, Xiaohui Xie1, Michael C. Zody1, Broad Institute Genome Sequencing Platform*,
Broad Institute Whole Genome Assembly Team*, Jennifer A. Marshall Graves16, Chris P. Ponting9,
Matthew Breen6,25, Paul B. Samollow26, Eric S. Lander1,27 & Kerstin Lindblad-Toh1
We report a high-quality draft of the genome sequence of the grey, short-tailed opossum (Monodelphis domestica). As the
first metatherian (‘marsupial’) species to be sequenced, the opossum provides a unique perspective on the organization and
evolution of mammalian genomes. Distinctive features of the opossum chromosomes provide support for recent theories
about genome evolution and function, including a strong influence of biased gene conversion on nucleotide sequence
composition, and a relationship between chromosomal characteristics and X chromosome inactivation. Comparison of
opossum and eutherian genomes also reveals a sharp difference in evolutionary innovation between protein-coding and
non-coding functional elements. True innovation in protein-coding genes seems to be relatively rare, with lineage-specific
differences being largely due to diversification and rapid turnover in gene families involved in environmental interactions. In
contrast, about 20%of eutherian conserved non-coding elements (CNEs) are recent inventions that postdate the divergence
of Eutheria and Metatheria. A substantial proportion of these eutherian-specific CNEs arose from sequence inserted by
transposable elements, pointing to transposons as a major creative force in the evolution of mammalian gene regulation.
Metatherians (‘marsupials’) comprise one of the three major groups
ofmodernmammals and represent the closest outgroup to the euthe-
rian (‘placental’) mammals (Supplementary Fig. 1). Metatherians
and eutherians diverged ,180million years (Myr) ago, long before
the radiation of the extant eutherian clades ,100Myr ago1,2.
Although the metatherian lineage originally radiated from North
*Lists of participants and affiliations appear at the end of the paper.
1Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, Massachusetts 02142, USA. 2Division of Health Sciences and Technology, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139, USA. 3Bioinformatics Division, The Walter & Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville Victoria 3050,
Australia. 4TheWellcome Trust Sanger Institute,Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK. 5Molecular Genetics Program, Benaroya Research Institute at
VirginiaMason, 1201 NinthAvenue, Seattle,Washington 98101, USA. 6Department ofMolecular Biomedical Sciences, College of VeterinaryMedicine, North Carolina State University,
4700 Hillsborough Street, Raleigh, North Carolina 27606, USA. 7Stanford University School of Medicine, P060 Lucas Center, Stanford, California 94305, USA. 8Genetic Information
Research Institute, 1925 Landings Drive, Mountain View, California 94043, USA. 9MRC Functional Genetics Unit, Department of Physiology, Anatomy and Genetics, University of
Oxford, South Parks Road, Oxford OX1 3QX, UK. 10Department of Biology, Center for Evolutionary and Theoretical Immunology, University of NewMexico, Albuquerque, NewMexico
87131, USA. 11Department of Biological Sciences, Biological Computation and Visualization Center, Center for Bio-Modular Multi-Scale Systems, Louisiana State University, 202 Life
Sciences Building, Baton Rouge, Louisiana 70803, USA. 12Department of Computational Biology, University of Pittsburgh, 3501 Fifth Avenue, Suite 3064, BST3, Pittsburgh,
Pennsylvania 15260, USA. 13Faculty of Veterinary Science, University of Sydney, New South Wales 2006, Australia. 14Department of Radiation Oncology, Duke University Medical
Center, Box 3433, Durham, North Carolina 27710, USA. 15Department ofMolecular Biology, HughesMedical Institute, Massachusetts General Hospital, and Department of Genetics,
Harvard Medical School, Boston, Massachusetts 02114, USA. 16ARC Centre for Kangaroo Genomics, Research School of Biological Sciences, The Australian National University,
Canberra, ACT 2601, Australia. 17Department of Medicine (Hematology) andMolecular Genetics, Albert Einstein College of Medicine, Ullmann 911, 1300Morris Park Avenue, Bronx,
New York 10461, USA. 18Department of Biochemistry andMolecular Genetics, University of Colorado Health Sciences Center, MS 8101, 12801 17th Avenue, Aurora, Colorado 80045,
USA. 19John Curtin School ofMedical Research, TheAustralianNational University, Canberra, ACT0200, Australia. 20Genome Sciences Centre, British Columbia Cancer Agency, 570
West 7th Avenue, Vancouver, British Columbia V5Z 4S6, Canada. 21Department of Pediatrics, Research Center Children’s Hospital of Pittsburgh, 3460 Fifth Avenue, Room 2109,
Rangos, Pittsburgh, Pennsylvania 15213, USA. 22Department of Biology, West Virginia University, Morgantown,West Virginia 26505, USA. 23Department of Genetics and Southwest
National Primate Research Center, Southwest Foundation for Biomedical Research, San Antonio, Texas 78245, USA. 24Center for Human Genetic Research, Massachusetts General
Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, USA. 25Center for Comparative Medicine and Translational Research, North Carolina State University, 4700
Hillsborough Street, Raleigh, North Carolina 27606, USA. 26Department of Veterinary Integrative Biosciences, Texas A&M University, 4458 TAMU, College Station, Texas 77843,
USA. 27Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA.
Vol 447 | 10 May 2007 |doi:10.1038/nature05805
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America, only one extant species can be found there (the Virginia
opossum), whereas all other species are found in South America
(including more than 65 species of opossums and shrew opossums)
and Australasia (,200 species, including possums, kangaroos, koalas
and many small insectivores and carnivores)3.
All sequenced mammalian genomes until now have come from
eutherian species. Although metatherians and eutherians (together,
‘therians’) share many ancient mammalian characteristics, they have
each evolved distinctive morphological and physiological traits.
Metatherians are particularly noted for the birth of young at a very
early stage of development, followed by a lengthy and complex lacta-
tional period. Genomic analysis will help reveal the genetic innova-
tions that underlie the distinctive traits of each lineage4–6.
Equally important, metatherian genomes can shed light on the
human genome. Comparative analysis of eutherians has greatly
improved our understanding of the architecture and functional
organization of mammalian genomes7–10. Identification of sequence
elements thought to be under purifying selection, on the basis of
cross-species sequence conservation, has led to increasingly refined
inventories of protein-coding genes11,12, proximal and distal regula-
tory elements13,14 and putative RNA genes15. Yet, we still know rela-
tively little about the evolutionary dynamics of these and other
functional elements: how stable is the complement of protein-coding
genes? How rapidly do regulatory sequences appear and disappear?
From what substrate do they evolve?
Comparison of the human genome with genomes from distant
outgroups such as birds (divergence ,310Myr ago) or fish
(,450Myr ago) has provided valuable information.When similarity
between sequences from such distantly related genomes can be
detected, it surely signals functional importance; but the high spe-
cificity of these signals16 is offset by dramatically reduced sensitiv-
ity10,17,18. Simulations have shown that the feasibility of aligning
orthologous genomic sequences declines rapidly once their mean
genetic distance exceeds 1 substitution per site19. The genome of
chicken, the most closely related non-mammalian amniote genome
available, is separated from the human genome by approximately 1.7
substitutions per site in orthologous, neutrally evolving sequences20.
Even moderately constrained functional elements may therefore be
difficult to detect. In contrast, metatherian mammals are well posi-
tioned to address this issue: because unconstrained regions of their
genomes are separated from that of human by only ,1 substitution
per site (see below), most orthologous, constrained sequence should
be readily aligned.
Herewe report the first high-quality draft of ametatherian genome
sequence, which was derived from a female, grey, short-tailed opos-
sum—Monodelphis domestica. The species was chosen chiefly on
the availability and utility of the organism for research purposes.
M. domestica is a small rapidly breeding South American species
that has been raised in pedigreed colonies for more than 25 years
and developed as one of only two laboratory bred metatherians21,22.
M. domestica is being actively used as a model system for investi-
gations in mechanisms of imprinting23–25, immunogenetics26–28, neu-
robiology, neoplasia and developmental biology (reviewed in ref. 6).
For example, newborn opossums are remarkable in that they can heal
complete transections of the spinal cord29. Elucidation of themolecu-
lar mechanisms underlying this ability promise important insights
relevant to regenerative medicine concerning spinal cord or peri-
pheral nerve injuries. Other than human, M. domestica is also the
only mammal known in which ultraviolet radiation is a complete
carcinogen for malignant melanoma30, and this has led to its estab-
lishment as a unique neoplasia model. All of these investigations will
directly benefit from the development of genomic resources for this
species.
Below we describe the generation of the draft sequence of the
opossum genome, analyse its large-scale characteristics, and compare
it to previously sequenced amniote genomes. Our key findings
include:
$ The distinctive features of the opossum genome provide an
informative test of current models of genome evolution and support
the hypothesis that biased gene conversion has a key role in deter-
mining overall nucleotide composition.
$ The evolution of random inactivation of the X chromosome in
eutherians correlates with acquisition of X-inactive-specific tran-
script (XIST), elevation in long interspersed element (LINE)/L1 den-
sity and suppression of large-scale rearrangements.
$ The opossum genome seems to contain 18,000–20,000 protein-
coding genes, the vast majority of which have eutherian orthologues.
Lineage-specific genes largely originate from expansion and rapid
turnover in gene families involved in immunity, sensory perception
and detoxification.
$ Identification of orthologues of highly divergent immune genes
and a novel T-cell receptor isotype challenge previous claims that
metatherians possess a ‘primitive’ immune system.
$ Of the non-coding sequences conserved among eutherians,
,20% seem to have evolved after the divergence from metatherians.
Of protein-coding sequences conserved among eutherians, only
,1% seems to be absent in opossum.
$ At least 16% of eutherian-specific conserved non-coding ele-
ments are clearly derived from transposons, implicating these ele-
ments as an important creative force in mammalian evolution.
Extensions to these findings, as well as additional topics, are
reported in a series of companion papers31–41.
Genome assembly and single nucleotide polymorphism discovery
We sequenced the genome of a partially inbred female opossumusing
the whole-genome shotgun (WGS) method7,42. The resulting WGS
assembly has a total length of 3,475megabases (Mb), consistent with
size estimates based on flow cytometry (,3.5–3.6 Gb; Supplementary
Notes 1–2 and Supplementary Fig. 2). Approximately 97% of the
assembled sequence has been anchored to eight large autosomes
and one sex chromosome on the basis of genetic markers mapped
by linkage analysis38 or fluorescence in situ hybridization43 (FISH;
Supplementary Note 3). The draft genome sequence has high con-
tinuity, coverage and accuracy (Table 1; Supplementary Note 4 and
Supplementary Tables 1–7).
To enable genetic mapping studies of opossum, we also created a
large catalogue of candidate single nucleotide polymorphisms
(SNPs). We identified ,775,000 SNPs within the sequenced indi-
vidual by analysing assembled sequence reads.We identified an addi-
tional ,510,000 SNPs by generating and comparing ,300,000
sequence reads from three individuals from distinct, partially
outbred laboratory stocks maintained at the Southwest Foundation
for Biomedical Research (San Antonio, Texas)22,44 (Supplemen-
tary Note 5). The SNP rates between the different stocks range from
Table 1 | Genome assembly characteristics
WGS assembly (monDom5)
Number of sequence reads 38.83 106
Sequence redundancy (Q20 bases) 6.83
Contig length (kb; N50*) 108
Scaffold length (Mb; N50) 59.8
Anchored bases in the assembly (Mb) 3,412
Estimated euchromatic genome size{ (Mb) 3,475
Integration of physical mapping data
Scaffolds anchored on chromosomes 216
Fraction of genome in anchored and oriented scaffolds (%) 91
Fraction of genome in anchored, but unoriented, scaffolds (%) 6
Quality control
Bases with quality score$40 (%) 98
Empirical error rate for bases with quality score$40{ (%) 33 1025
Empirical euchromatic sequence coverage{ (%) 99
Bases in regions with low probability of structural error1 (%) 98
*N50 is the size x such that 50%of the assembly reside in contigs/scaffolds of length at least x.
{ Includes anchored bases and spanned gaps (,2%).
{Based on comparison with 1.66Mb of finished bacterial artificial chromosome (BAC)
sequence.
1Based on ARACHNE assembly certification (see Supplementary Note 4).
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would significantly alter these estimates (Supplementary Note 6 and
Supplementary Table 13).
Transposable elements. Metatherian transposable elements largely
belong to families also found in eutherians, but can be divided into
more than 500 subfamilies, many of which are lineage specific (cata-
logued in Repbase57). At least 52% of the opossum genome can be
recognized as transposable elements and other interspersed repeats
(Table 2)33,35, which is more than in any of the other sequenced
amniotes (34–43%).Notably, theopossumgenome is significantly en-
riched in non-long terminal repeat (LTR) retrotransposons (LINEs,
29%), comprising copies of various LINE subfamilies. Given the low
abundance of segmental duplications, accumulation of transposable
elements seems to be the primary reason for the relatively large opos-
sum genome size. The total euchromatic sequence that is not re-
cognized as transposable elements is rather similar in opossum and
human (1638Mb versus 1568Mb, respectively). The enrichment of
LINEs may be related to the overall low recombination rate in opos-
sum, inasmuch as studies of eutherian genomes have shown that
LINEs occur at elevated densities in regions with low local recombina-
tion rates47.
Conserved synteny
Identification of syntenic segments between related genomes can
facilitate reconstruction of chromosomal evolution and identifica-
tion of orthologous functional elements. Starting from nucleotide-
level, reciprocal-best alignments (‘synteny anchors’), we found that
the opossum and human genomes can be subdivided (at a resolution
of 500 kb) into 510 collinear segments with anN50 length (size x such
that 50% of the assembly is in units of length at least x) of 19.7Mb,
which cover 93% of the opossum genome (Supplementary Fig. 5). If
local rearrangements are disregarded, these segments can be further
grouped into 372 blocks of large-scale, conserved synteny.
Extending this analysis to additional eutherians (mouse, rat and
dog), with chicken as an additional outgroup, we created a high-
resolution synteny map that reveals 616 blocks of conserved synteny
across the five fully sequenced mammals (Supplementary Note 7,
Supplementary Figs 6–7 and Supplementary Table 14). Because the
majority of synteny breakpoints between human, mouse, rat and dog
are clearly lineage specific (see also ref. 10), genomic regions that were
probably contiguous in the last common boreoeutherian ancestor
can be inferred by parsimony (Supplementary Note 8). We found
that themammalian synteny blocks can be used to infer 43 connected
groups in the ancestral boreoeutherian genome (Supplementary Fig.
8). In fact, the largest 30 groups cover 95% of the human genome (see
also ref. 58).
The resulting synteny map can be used to clarify chromosomal
rearrangements during early mammalian evolution. For example,
limited comparative mapping previously revealed that the eutherian
X chromosome contains an ‘X-conserved region’ (XCR) that corre-
sponds to the ancestral therian X chromosome, and an ‘X-added
region’ (XAR), which was translocated from an autosome after the
split from Metatheria59,60. The exact extent of the XCR has been
unclear, however, owing to unclear synteny with non-mammalian
out-groups at its boundary61. Using our high-resolution syntenymap
we can now confidently map the XAR–XCR fusion point to 46.85Mb
on human chromosome band Xp11.3 (Fig. 2).
X chromosome inactivation
In opossum and other metatherian mammals, dosage compensation
for X-linked genes is achieved through inactivity of the paternally
derived X chromosome in females62. In contrast, eutherian dosage
compensation involves inactivation of the paternal X chromosome
at spermatogenesis, reactivation in the early embryo, followed
by random and clonally stable inactivation of one of the two
X chromosomes in each cell of female embryos63. The random in-
activation step is controlled by a complex locus known as the
X inactivation centre (XIC). In the early female embryo, the non-
coding XIST gene is transcribed from the XIC and coats one chro-
mosome, in cis, to initiate silencing of the majority of its genes. It has
been proposed that paternal X chromosome inactivation represents
the ancestral therian dosage compensation system, and that random
X chromosome inactivation is a recent innovation in the eutherian
lineage64,65. The opossum genome sequence provides the first oppor-
tunity to test major hypotheses about the evolution of this system.
No XIST homologue in opossum. We searched all assembled and
unassembled opossum WGS sequence for homology to the human
and mouse XIC non-coding genes but, in agreement with a recent
report66, did not find any significant alignments. (In particular, we
80
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60
50
40
30
20
10
8070605040302010 15014013012011010090
Chr X: 73.2–73.4Chr X: 71.7–72.2Chr 7: 52.4–53.1 Chr X: 55.5Chr Un: 6.0–6.5*
CLCN5PAGE4
PAGE1
GAGE8LMO6
PRAF2
KCND1
HDAC6
WAS
RBM3
SLC38A5
SSX3SSX5
ZNF630ZNF81
ZNF21
CFPRGN UBE1
ZNF157
ZNF41RP2CHST7 UPRTKIAA2022 ABCB7SLC16A2XIST ZCCHC13CHIC1CDX4
Chr X: 26.2–26.3
Human Chr X
O
po
ss
um
p
os
iti
on
(M
b)
(Mb)
Opossum
chromosomes
4
7
X
Un
Figure 2 | Opossum–human synteny for the X chromsome. The dot plot
shows correspondence between the human chromosome (Chr)X and
opossum chromosomes at a resolution of 300 kb. Expanded views, at a
resolution of 50 kb, of the XAR–XCR fusion and the XIC are shown on the
bottom left and right, respectively. In the XIC region, the closest contig on
the distal flank (*) was not anchored in the monDom5 assembly (see
Methods), but has been subsequently mapped near UPRT (opossum
X chromosome ,55Mb) by FISH40.
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found no match to the highly conserved 150-bp region overlapping
the critical exon 4 of XIST; this region is so strongly conserved in the
Eutheria that it should be readily detectable if present40.) Analysis of
synteny in the regions surrounding the eutherian XIC also revealed
that it has been disrupted by large-scale rearrangements (Fig. 2)40,41.
In eutherians, the XIC is flanked by the ancient protein-coding genes
CDX4–CHIC1 on one side and SLC16A2–RNF12 on the other side. In
both chicken and frog these four genes are clustered in autosomal
XIC homologous regions (which do not contain homologues of the
XIC non-coding genes66). On the opossumX chromosome, however,
these two pairs of genes are separated by ,29Mb (compared with
,750 Kb in human). Taken together, the evidence strongly suggests
that XIST is specific to eutherians40,41,66.
The Lyon repeat hypothesis. LINE/L1 elements are of particular
interest to the study of X chromosome inactivation. These transpos-
able elements have been proposed to act as ‘boosters’ for the spread of
X chromosome inactivation in cis from the XIC (reviewed in ref. 67).
This hypothesis is supported in part by the observation that in
human, LINE/L1 density is significantly elevated in the XCR
(33%), where nearly all genes are inactivated, but approximates the
autosomal density in the XAR (19%), where many genes escape
inactivation (Fig. 3)61,68. In mouse, we found that the LINE/L1 den-
sity is elevated in both the XCR (35%) and the XAR (32%), which is
consistent with the observation that genes that escape inactivation on
the human XAR are often inactivated in mouse69. As previously
observed in human68, the LINE/L1 elevation in mouse is particularly
dramatic among recent, lineage-specific subfamilies (Supplementary
Fig. 9).
In contrast to human and mouse, the LINE/L1 density on the
opossum X chromosome (22%) is significantly lower than in the
eutherianXCR, and is in fact slightly less than in the autosomal regions
homologous to the eutherian XAR (23%). This difference between
metatherian and eutherianX chromosomes is not readily explainedby
any simple correlation between LINE/L1 density, recombination or
mutation rates. We therefore conclude that LINE/L1 density is
unlikely to be a critical factor for X chromosome inactivation in the
metatherian lineage, and that the approximately twofold increase on
the eutherianX chromosomemaybedirectly related to the acquisition
of XIST and random X chromosome inactivation.
Suppression of large-scale rearrangements. Comparative analyses
have revealed that the structure of the human X chromosome has
remained essentially unchanged since the eutherian radiation10,20,61. A
possible reason is that the requirement for XIST transcripts to spread
across the chromosome from a central location has led to selection
against structural rearrangements. For example, translocation of
LINE/L1-poor XAR segments into the XCR could potentially disrupt
inactivation at more distal loci. Consistent with this hypothesis, our
syntenymap reveals that the XAR andXCRhomologous regions have
experienced several major rearrangements both in the opossum lin-
eage (,15 lineage-specific synteny breakpoints) and in the eutherian
lineage before the eutherian radiation (,9 lineage-specific break-
points; Supplementary Table 15). The low rate of rearrangements
in the human lineage is therefore unlikely to be due to functions or
sequences that were present on the ancestral therian X chromosome,
or in early eutherian evolution.
We note that unlike in human, the mouse X chromosome has
experienced several rearrangements (with 15 lineage-specific synteny
breakpoints), such that the XAR and XCR are no longer two separate
segments. This would be consistent with the more comprehensive
inactivation in themouse imposing weaker constraints on rearrange-
ment. Although little is known about the extent of X chromosome
inactivation in dog or rat, their X chromosomes are also consistent
with this hypothesis. The dogX chromosome is collinear with human
and is enriched for LINE/L1 only in the XCR (33.4% versus 16.8%
for the XAR). The rat X chromosome has accumulated ,4 lineage-
specific synteny breakpoints after the divergence from mouse61, and
is similarly enriched for LINE/L1 in both the XCR (36.7%) and the
XAR (34.5%).
Genes
The gene content of metatherian and eutherian genomes provides
key information about biological functions. We analysed the gene
content of the opossum genome and compared it with that of the
human genome. We focused on instances of rapid divergence and
duplication of protein-coding genes, which have led to lineage-spe-
cific gene complements70.
Gene catalogue. We generated an initial catalogue of 18,648 pre-
dicted protein-coding genes and 946 non-coding genes (primarily
small nuclear RNA, small nucleolar RNA, microRNA and ribosomal
RNA) in opossum34 (Supplementary Note 9 and Supplementary
Data). Regularly updated annotations can be obtained from public
databases (http://www.ensembl.org and http://genome.ucsc.edu).
We next characterized orthology and paralogy relationships
between predicted protein-coding genes in opossum and human11
(Table 3). We could identify unambiguous human orthologues for
15,320 (82%) of the opossum predicted genes, with 12,898 cases
having a single copy in each species (1:1 orthologues). Notably, we
identified orthologues of key T-cell lineage markers such as CD4 and
CD8, which had not been successfully identified by cloning in
metatherian species39. Most (2,704) of the remaining genes are
homologous to human genes, but could not be assigned to ortholo-
gous groups with certainty.
A small number (624) of predicted opossum genes have no clear
homologue among the human gene predictions. Inspection revealed
that most of these are short (median length of 120 amino acids,
compared with 445 for 1:1 orthologues) and probably originate from
pseudogenes or spurious open reading frames. Only eight currently
have strong evidence of representing functional genes without homo-
logues in humans (Supplementary Table 16). These include CPD-
photolyase, which is part of an ancestral photorepair system still
active in opossum71, malate synthase72 and inosine/uridine hydro-
lase. The latter two are ancient genes not previously identified in a
mammalian species.
Conversely, approximately,1,100 current gene predictions from
human have no clear homologue in the initial opossum catalogue
(Supplementary Data). Of these, ,620 can be at least partially
60
LI
N
E/
L1
d
en
si
ty
(%
)
50
40
30
20
10
0
A XAR XCR A XAR XCR A XAR XCR
Opossum Human Mouse
Figure 3 | Enrichment of LINE/L1 correlates with random Xchromosome
inactivation. Box plot of LINE/L1 density in 500-kb intervals across the
autosomes (A), the X-added region (XAR) and its homologous regions in
opossum, and the X conserved region (XCR). Red bar, median; box edges,
25th and 75th percentiles; whiskers, range.
Table 3 | Opossumand human gene predictions and projected gene counts
Protein-coding genes Opossum
Initial predictions 18,648
Orthologues in human* 15,320
1:1 12,898
Many:1 1,016
1:Many 451
Many:Many 582
Homologues in human, but unclear orthology{ 2,704
No predicted homologues in human 624
Projected total{ 18,000–20,000
* Includes some cases where multiple transcripts have inconsistent phylogenies, or where the
predicted orthologue is a putative pseudogene.
{ Includes members of highly duplicated gene families.
{Accounting for missed annotations in opossum and removal of probable pseudogenes.
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aligned to the opossum genome and may not have been annotated as
genes owing to imperfections in the draft assembly or high sequence
divergence. In particular, manual re-annotation identified ortholo-
gues of several rapidly evolving cytokines39. The remaining predic-
tions are dominated by gene families known to have undergone
expansion and rapid evolution in the human lineage, such as
b-defensins and cancer-testis antigens. On the basis of our compar-
ison, we conclude that the opossum genome probably contains
,18,000–20,000 protein-coding genes, with the vast majority having
eutherian orthologues.
Divergence rates among orthologues. We calculated the synonym-
ous substitution rate (KS; substitutions that do not result in amino
acid change because of codon redundancy) of 1:1 opossum–human
orthologues to approximate the unconstrained divergence rate
between the species7,10. The median value of KS is 1.02. Consistent
with expectation, this value is substantially smaller than the chicken–
human KS value (1.7), with the ratio being very close to the ratio of
prior estimates of the divergence times for the two lineages
(,180Myr ago for opossum and ,310Myr ago for chicken).
Notably, the median KS for orthologues located on the XCR is
significantly elevated relative to orthologues located on autosomes
in both species (1.2 versus 1.0; P, 1023; see also refs 34, 35). This is
the opposite to what is observed within Eutheria10, but is consistent
with the expectation that the higher G1C-content and recombina-
tion rate on the opossum X chromosome relative to its autosomes
implies a higher rate of mutation47. A similar elevation can also be
detected in subtelomeric regions34.
Innovation and turnover in gene families. We next studied the
evolution of gene family expansions in the metatherian lineage.
The opossum gene catalogue contains 2,743 (15%) genes that have
probably been involved in one or more duplication or gene conver-
sion event since the last common ancestor with eutherian mammals,
as inferred from low KS between the copies (median5 0.41). The
number of duplications is one-third fewer than the number of
human lineage-specific duplications (4,037; 20%), which may reflect
the lower rate of segmental duplication in the opossum genome.
We found a large number of lineage-specific copies of genes
involved in sensory perception, such as the c-crystallin family of
eye lens proteins73, and taste, odorant74 and pheromone receptors.
Other major lineage-specific duplications were found in the rapidly
evolving KRAB zinc-finger family, and in genes related to toxin
degradation and dietary adaptations, including cytochrome P450
and various gastric enzymes (see also ref. 34).
Innovation in the innate and adaptive immune systems is visible
through substantial duplication or gene conversion involving the
leukocyte receptor and natural killer complexes, immunoglobulins,
type I interferons and defensins32,39. The opossum genome also con-
tains a newT-cell receptor isotype that is expressed early in ontogeny,
before conventional T-cell receptors, andmay provide early immune
function in the altricial young37.
The opossum also shows some surprising gene family expansions
that are without precedent in other vertebrates. Notable among these
are multiple duplications of the nonsense-mediated decay factors
SMG5 and SMG6, and the pre-mRNA splicing factors, KIAA1604
and PRP18. The opossum genome also harbours two adjacent para-
logous copies of DNA (cytosine-5)-methyltransferase 1 (DNMT1),
which catalyses methylation of CpG dinucleotides. It will be inter-
esting to discover if specialized functions have been adopted by these
paralogous genes.
The patterns of evolution among duplicated genes largely mirror
those observed in eutherians34,70. The set of opossum paralogues
is strongly biased towards recent duplications (KS, 0.1) and in gen-
eral have accumulated a disproportionately high number of non-
synonymous mutations (Fig. 4). The median intraspecies ratio of
nonsynonymous to synonymous substitution rates (KA/KS) between
paralogues is 0.51, which is sixfold higher than the interspecies ratio
seen for 1:1 orthologues (0.086). This is consistent with the rapid
gene birth and death model75, which predicts that duplicated genes
either undergo functional divergence in response to positive selection
or rapidly degenerate owing to lack of evolutionary benefit.
Conserved sequence elements
The most surprising discovery to emerge from comparative analyses
of eutherian genomes is the finding that the majority of evolution-
arily conserved sequence does not represent protein-coding genes,
but rather are conserved non-coding elements (CNEs)7,10. The opos-
sum genome provides a well-positioned outgroup to study the origin
and evolution of these elements.
For simplicity, we will refer to sequence elements as ‘amniote
conserved elements’ if they are conserved between chicken and at
least one of opossum or human; ‘eutherian conserved elements’ if
they are conserved between human and at least one of mouse, rat or
dog; and ‘eutherian-specific elements’ if they are eutherian conserved
sequence absent from both opossum and chicken. (‘Metatherian-
specific elements’ surely also exist, but cannot be identified without
additional metatherian genomes.)
Loss of amniote conserved elements in mammals. We first studied
the extent to which amniote conserved elements have been lost in
the human lineage. We focused on ,133,000 conserved intervals
between opossum and chicken (68Mb), ,50% of which overlaps
protein-coding regions (Supplementary Data).
Nearly all (97.5%) of these amniote conserved elements can be
aligned to the human genome (Fig. 5a). We reasoned that some of
the remainder might be orthologous to sequence that lies within gaps
in the current human assembly, or which had been missed by the
initial genome-wide alignment. We therefore repeated the analysis,
focusing only on amniote elements present in opossum and occur-
ring in ‘ungapped intervals’ (that is, syntenic intervals between
human and opossum that have no sequence gaps); the ungapped
intervals contain 63% of all conserved elements.
We found that 99.0% of amniote elements in ungapped intervals
could be unambiguously aligned to the human genome. The remain-
ing 1.0% of amniote elements could not be found even by a more
sensitive alignment algorithm (Fig. 5b), and thus seem to have been
lost in the human lineage.
P
ro
po
rt
io
n
of
g
en
es
(%
)
0.00 0.50 1.00 1.50
KA/KS
Immunity
KRAB/ZnF
Detoxification
Reproduction
Olfaction
All opossum in-paralogues
1:1 orthologues
100
75
50
25
0
Figure 4 | Cumulative distribution of KA/KS values for duplicated genes.
Estimates are shown for pairs of genes duplicated in opossum
(in-paralogues) in the most common functional categories: immunity,
KRAB zinc finger (ZnF) transcription factors, detoxification (including
cytochrome P450, sulphotransferases), reproduction (including
vomeronasal receptors, lipocalins and b-seminoproteins) and olfaction. The
total distributions for opossum in-paralogues and opossum–human 1:1
orthologues are shown for comparison.
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Wenext considered a genome-wide set ofDNase hypersensitive sites
from human lymphocytes, which represent a variety of putative reg-
ulatory elements80. Of the 290 sites that overlap amniote CNEs present
in human, none overlaps instances that are lost in opossum. Of the
2,041 sites that overlap eutherian CNEs in ungapped syntenic regions,
407 (20%) exclusively overlap eutherian-specific elements (Supplem-
entary Data). An example is a 269-bp eutherian-specific CNE in
intron 2 of the apoptosis regulator BCL2, which overlaps a DNase
hypersensitive site, suggesting it has a cis-regulatory function (Fig. 5f).
The fraction of eutherian CNEs overlapping DNase hypersensitive
sites that are eutherian specific is strikingly similar to the fraction of
all conserved non-coding sequence that is eutherian specific (20.5%).
The fraction of miRNAs that correspond to eutherian-specific CNEs
is slightly lower (15%), which is consistent with their higher average
conservation scores. In particular, the results provide strong evidence
that the majority of eutherian-specific CNEs are likely to be genuine
functional elements.
Lineage-specific CNEs associated with key developmental genes.
Wenext explored the distribution of lineage-specific CNEs across the
human genome. Overall, there is a strong regional correlation
between the density of eutherian CNEs shared with opossum and
the density of eutherian-specific CNEs (Spearman’s r5 0.82 for
1-Mb windows; Fig. 6). The densities of amniote CNEs present or
lost in opossum are also positively correlated (Spearman’s r5 0.30).
Previous studies have shown that both eutherian and amniote
CNEs are enriched in certain large, gene-poor regions surrounding
genes that have key roles in development, primarily encoding tran-
scription factors, morphogens and axon guidance receptors10,81,82.
For example, 35% of all eutherian CNEs and 49% of all amniote
CNEs (in ungapped syntenic regions) lie within the 204 largest clus-
ters of CNEs in the human genome (described in ref. 10). The,240
key developmental genes in these regions have relatively low rates
of amino acid divergence (median KA/KS5 0.03) and show little
evidence of lineage-specific loss or duplications. In contrast, we
found that the rate of gain and loss of CNEs in the same regions is
only moderately (,30%) lower than elsewhere in the genome.
Indeed, we identified more than 37,000 lineage-specific CNEs in
these developmentally important regions.
Because experimental studies of CNEs in these regions have fre-
quentlyuncovered cis-regulatory functions affecting thenearbydevelop-
mental genes16,82–85, the substantial innovations in these regions are
candidates for genetic changes underlying differential morphological
andneurological evolution inmammalian lineages. This patternwould
be consistent with the notion that modification of regulatory networks
has been a major force in the evolution of animal diversity86–88.
Eutherian-specific CNEs derived from transposable elements. In
general, each eutherian-specific element must have arisen by one of
three mechanisms: (1) divergence of an ancestral functional element
to such an extent that its similarity is no longer detectable; (2)
duplication of an ancestral functional element giving rise to an ele-
ment without a 1:1 orthologue in other clades; or (3) evolution of a
novel functional element from sequence that was absent or non-
functional in the ancestral genome.
The first mechanism is not likely to account formost of the euther-
ian-specific CNE sequence, at least among those with high conser-
vation scores—if an ancient functional element underwent such
rapid divergence at some point in the eutherian lineage that it is no
longer detectable, then there should be concomitant ‘loss’ of an
amniote conserved element. But, lineage-specific loss seems to be
relatively rare for both amniote elements, as shown above, and for
eutherian elements10.The majority of eutherian-specific conserved
elements therefore probably arose after the metatherian divergence,
either by adaptive evolution of new or previously non-functional
sequence, or by duplication of ancestral elements.
One intriguing source for eutherian-specific CNEs is transposable
elements. A number of researchers have argued that transposable
elements offer an obvious and ideal substrate for the evolution of
lineage-specific functions89–93. Transposable elements contain a vari-
ety of functional subunits that can be exapted and modified by the
host genome89,91, and they can mediate duplication of existing CNEs
to distant genomic locations through transduction or chimaerism92.
Individual instances of CNEs derived from transposable elements
have been described previously14,94,95. However, these cases together
comprise only a trivial fraction of the CNEs in the human genome. It
has thus been unclear whether the evolution of CNEs from transpos-
able elements represents a general mechanism or a rare exception.
When we examined the set of eutherian-specific CNEs, we found a
striking overlap with transposable elements. In ungapped syntenic
intervals, at least 16% of eutherian-specific CNEs overlap currently
recognized transposable elements in human. The fraction is similar
(14%) if we focus only on the most highly conserved elements
(phylo-HMM log2-odds score)$ 60, see above). The overlapping
transposable elements originate from most major transposon fam-
ilies found in eutherians (Table 4), and are not clearly differentiated
from other CNEs in terms of distribution across the genome. This
implies that transposable-element-mediated evolution has been a
significant creative force in the emergence of recent CNEs. The fact
that sequences from transposable elements themselves can be iden-
tified within these CNEs also implies that exaptation of at least a
portion of the transposable element, rather than simply incidental
transduction of adjacent sequence, has been a frequent occurrence.
In contrast, the eutherian CNEs that are present in opossum (and
thus are more ancient) only rarely show overlap with recognizable
transposable elements (,0.7%). We speculate that many of these
CNEs also arose from transposable elements, but that they are difficult
to recognize as suchowing to substantial divergence. In fact, three large
Eutherian
Therian
Amniote
182.7 182.8 182.9 183.0 183.1 183.2
SOX2
(Mb)
S
A
TB
1
W
N
T5
A
FE
Z2
FO
XP
1
R
O
B
O
1/
2
G
B
E1
IG
S
F4
D
ZB
TB
20
LS
A
M
P
EP
H
B
1
ZI
C
1/
4
S
H
O
X2
EV
I1
S
O
X2
LP
P
Figure 6 | Lineage-specific CNEs near key developmental genes. The
densities of eutherian CNEs present (blue) or absent (red) in opossum are
plotted in 1-Mb sliding windows across human chromosome 3. Peaks in the
distributions often correspond to key developmental genes. The expanded
view shows positions of amniote CNEs (purple), eutherian CNEs not
overlapping amniote CNEs (blue) and eutherian-specific CNEs (red) across
a 500-kb gene desert surrounding the SOX2 transcription factor gene. One
amniote CNE present in human has been lost in opossum (orange).
ARTICLES NATURE |Vol 447 | 10 May 2007
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families of ancient paralogous CNEs have recently been discovered
that were clearly distributed around the genome as parts of transpos-
able elements96–98. In each case, only aminority of the family members
still retain evidence of transposon-like features. We also previously
described,100 smaller CNE families that pre-date the eutherian radi-
ation, butwhich had nomembers associatedwith known transposable
elements98. For all but two of these families, we can find orthologues in
the opossum genome for the majority of their members (Supplemen-
tary Note 10 and Supplementary Fig. 10). Moreover, closer inspection
reveals previously unrecognized transposon-like features in several of
these and other ancient CNE families33.
Strikingly, the proportion of eutherian-specific CNEs recognizable
as transposable-element-derived (16%) is very similar to the propor-
tion of the total aligned sequence between the human, mouse and dog
genomes recognizable as ancestral transposable elements (,17% of
,812Mb; the vast majority of which is inactive)10. It is widely sus-
pected that the latter proportion is a significantunderestimate owing to
the difficulty of recognizing transposable elements that inserted more
than,100–200Myr ago7,33. In cases where the transposable-element-
related sequence hallmarks are not essential to the subsequent CNE, or
where evolution of a new function did not follow immediately after the
transposable element insertion, exapted sequences would be expected
to have diverged to the point that they can no longer be readily recog-
nized at a rate similar to inactive insertions. Because this seems to have
occurred formost of the families of ancient CNEs described above, it is
likely that the proportion of all eutherian (not just eutherian-specific)
CNEs derived from transposable elements is substantially higher than
the observed proportion of 16%.
Conclusions
The generation of the first complete genome sequence for a marsupial,
Monodelphis domestica, provides an important resource for genetic
analysis in this unique model organism, as well as the first reference
sequence for metatherian mammals. Our initial results demonstrate
the usefulness of this sequence for comparative analyses of the archi-
tecture and functional organization of mammalian genomes.
The relationship of sequence composition, segmental duplications
and transposable element density with the large and stable karyotype
of the opossum genome has provided new support for an emerging,
general model of chromosome evolution in mammals. In addition,
comparison of the opossum and eutherian X chromosomes revealed
that the evolution of random X chromosome inactivation correlates
with acquisition of XIST, elevation in LINE/L1 density and suppres-
sion of large-scale rearrangements.
Comparative analysis of protein-coding genes showed that the
eutherian complement is largely conserved in opossum. Lineage-
specific genes seem to be largely limited to gene families that are
rapidly turning over in all mammals, although improved annotations
that do not rely on homology to distant species will be required to
complete the opossum gene catalogue. Identification of a wide array
of both conserved and lineage-specific immune genes is particularly
notable because limited success in isolating these genes by cloning has
led to claims that the metatherian immune system is relatively ‘prim-
itive’. Availability of the genome sequence now facilitates more sys-
tematic study of the metatherian immune response39.
At timescales longer than the characteristic time of loss for gene
duplications, it is clear that innovation in non-coding elements
has been substantially more common relative to protein-coding
sequences, at least during eutherian evolution. The opossum genome
sequence has provided the first estimate of the genome-wide rate of
CNE innovation in eutherian evolution, as well as identification of
tens of thousands of lineage-specific elements. It has also provided
evidence that exaptation of transposable elements has a much
greater role in the evolution of novel CNEs than has been previously
realized.
Sequencing of additional metatherian genomes would be helpful
for extending our results by allowing detection of metatherian-
specific coding and non-coding elements. In addition, sampling of
both the American and Australasian lineages would allow the recon-
struction of the genome of their common ancestor, which would
complement ongoing efforts for the boreoeutherian ancestral gen-
ome58. The shorter genetic distance between the ancestral metather-
ian and boreoeutherian genomes (,0.6–0.7 substitutions per site)
would facilitate a more comprehensive analysis of short and weakly
conserved functional elements, for which the phylogenetic distri-
bution and evolutionary origins are still difficult to ascertain.
METHODS SUMMARY
WGS sequencing and assembly. Approximately 38.8million high-quality
sequence reads were assembled using an interim version of ARACHNE21
(http://www.broad.mit.edu/wga/).
SNP discovery. The SNP discovery was performed using ARACHNE and
SSAHA-SNP99. Linkage disequilibrium was assessed using Haploview100.
Genome alignment and comparisons. Synteny maps were generated using
standard methods7,10.
Gene prediction and phylogeny.Opossumprotein-coding andnon-codingRNA
genes were predicted using a modified version of the Ensembl genebuild pipe-
line101, followed by several rounds of refinement using Exonerate102 and manual
curation. Orthology and paralogy were inferred using the PhyOP pipeline11,34.
Conserved element prediction. Amniote conserved elements were inferred
from pairwise BLASTZ alignment blocks with more than 75% identity
for$100 bp. Eutherian conserved elements were inferred using phastCons14.
Eutherian elements that did not fall within a 10-kilobase or longer synteny
‘net’103 were ignored.
Phylogeny of conserved elements. For amniote conserved elements, pairwise
best-in-genome BLASTZ alignments of opossum to human and vice versa were
used to infer their phylogenetic distributions. For eutherian conserved elements,
concomitant BLASTZ/MULTIZ alignments to opossum and chicken were used.
A conserved element was called absent from a species if it was not covered by a
single aligned nucleotide in the relevant alignment.
Correction for assembly gaps and initial alignment artefacts. A conserved
element was considered to be in an ungapped syntenic interval if it was flanked
by two synteny anchorswithin 200 kb on the same contigs in both the human and
opossum assemblies. All conserved elements in ungapped syntenic intervals were
realigned using water (http://emboss.sourceforge.net). Putatively eutherian-
specific elements, including XIST, were also searched against all opossum
sequencing reads using MegaBLAST.
Table 4 | Eutherian-specific conserved non-coding elements derived from
transposons
Transposon family All log2-odds score$ 60
Number of
CNEs*
Overlapped
length (kb){
Number of
CNEs*
Overlapped
length (kb){
SINE/MIR 9,617 364 363 49
LINE/L1 6,619 286 194 36
LINE/L2 7,616 303 290 47
LINE/CR1 2,520 136 203 36
LINE/RTE 867 48 56 11
LTR/MaLR 1,995 65 25 3.7
LTR/ERV1 140 5.1 1 0.2
LTR/ERVL 992 36 12 2.8
DNA/Tip100 242 9.3 2 0.6
DNA/MER1_type 2,427 93 54 9
DNA/MER2_type 113 5.3 4 0.9
DNA/Tc2 162 8.5 6 1.4
DNA/Mariner 250 14.6 20 3.3
DNA/AcHobo 151 5.1 3 0.3
Unknown (MER121) 49 4 10 1.6
Total 33,760 1,383 1,243 203
Fraction of overlapped CNEs 16% 14%
*Number of eutherian-specific CNEs in ungapped syntenic regions overlapping annotated
transposable elements.
{Total length of annotated transposable element sequence overlapping the CNEs (this is less
than the total length of CNEs overlapping transposable element sequence).
NATURE |Vol 447 | 10 May 2007 ARTICLES
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Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 5 December 2006; accepted 3 April 2007.
1. Kumar, S. & Hedges, S. B. A molecular timescale for vertebrate evolution. Nature
392, 917–920 (1998).
2. Woodburne, M. O., Rich, T. H. & Springer, M. S. The evolution of tribospheny and
the antiquity of mammalian clades. Mol. Phylogenet. Evol. 28, 360–385 (2003).
3. Tyndale-Biscoe, C. H. Life of Marsupials (CSIRO Publishing, Collingwood, Victoria,
2005).
4. Wakefield, M. J. & Graves, J. A. M. Marsupials and monotremes sort genome
treasures from junk. Genome Biol. 6, 218 (2005).
5. Graves, J. A. M. & Westerman, M. Marsupial genetics and genomics. Trends
Genet. 18, 517–521 (2002).
6. Samollow, P. B. Status and applications of genomic resources for the gray, short-
tailed opossum, Monodelphis domestica, an American marsupial model for
comparative biology. Aust. J. Zool. 54, 173–196 (2006).
7. Mouse Genome Sequencing Consortium. Initial sequencing and comparative
analysis of the mouse genome. Nature 420, 520–562 (2002).
8. Rat Genome Sequencing Project Consortium. Genome sequence of the Brown
Norway rat yields insights intomammalian evolution.Nature428,493–521 (2004).
9. Chimpanzee Sequencing and Analysis Consortium. Initial sequence of the
chimpanzee genome and comparison with the human genome. Nature 437,
69–87 (2005).
10. Lindblad-Toh, K. et al. Genome sequence, comparative analysis and haplotype
structure of the domestic dog. Nature 438, 803–819 (2005).
11. Goodstadt, L. & Ponting, C. P. Phylogenetic reconstruction of orthology, paralogy,
and conserved synteny for dog and human. PLoS Comput. Biol. 2, e133 (2006).
12. Clamp, M. et al. Gene content of the human genome. Nature (submitted).
13. Xie, X. et al. Systematic discovery of regulatory motifs in human promoters and 39
UTRs by comparison of several mammals. Nature 434, 338–345 (2005).
14. Siepel, A. et al. Evolutionarily conserved elements in vertebrate, insect, worm, and
yeast genomes. Genome Res. 15, 1034–1050 (2005).
15. Pedersen, J. S. et al. Identification and classification of conserved RNA secondary
structures in the human genome. PLoS Comput. Biol. 2, e33 (2006).
16. Nobrega, M. A., Ovcharenko, I., Afzal, V. & Rubin, E. M. Scanning human gene
deserts for long-range enhancers. Science 302, 413 (2003).
17. Ovcharenko, I., Stubbs, L. & Loots, G. G. Interpreting mammalian evolution using
Fugu genome comparisons. Genomics 84, 890–895 (2004).
18. Prabhakar, S. et al. Close sequence comparisons are sufficient to identify human
cis-regulatory elements. Genome Res. 16, 855–863 (2006).
19. Margulies, E. H. et al. An initial strategy for the systematic identification of
functional elements in the human genome by low-redundancy comparative
sequencing. Proc. Natl Acad. Sci. USA 102, 4795–4800 (2005).
20. Hillier, L. W. et al. Sequence and comparative analysis of the chicken genome
provide unique perspectives on vertebrate evolution.Nature432, 695–716 (2004).
21. VandeBerg, J. L. The gray short-tailed opossum (Monodelphis domestica) as a
model didelphid species for genetic research. Aust. J. Zool. 37, 235–247 (1990).
22. VandeBerg, J. L. in UFAW Handbook on the Management of Laboratory Animals.
Vol. 1 Terrestrial Vertebrates (eds Poole, T. & English, P.) 193–209 (Blackwell
Science, Oxford, 1999).
23. Murphy, S. K. & Jirtle, R. L. Imprinting evolution and the price of silence. Bioessays
25, 577–588 (2003).
24. Rapkins, R.W. et al. Recent assembly of an imprinted domain from non-imprinted
components. PLoS Genet. 2, e182 (2006).
25. Weidman, J. R. et al. Phylogenetic footprint analysis of IGF2 in extant mammals.
Genome Res. 14, 1726–1732 (2004).
26. Deakin, J. E. et al. Evolution and comparative analysis of the MHC Class III
inflammatory region. BMC Genomics 7, 281 (2006).
27. Deakin, J. E., Olp, J. J., Graves, J. A. & Miller, R. D. Physical mapping of
immunoglobulin loci IGH@, IGK@, and IGL@ in the opossum (Monodelphis
domestica). Cytogenet. Genome Res. 114, 94H (2006).
28. Belov, K. et al. Reconstructing an ancestral mammalian immune supercomplex
from a marsupial major histocompatibility complex. PLoS Biol. 4, e46 (2006).
29. Wintzer, M. et al. Strategies for identifying genes that play a role in spinal cord
regeneration. J. Anat. 204, 3–11 (2004).
30. VandeBerg, J. L. et al. Genetic analysis of ultraviolet radiation-induced skin
hyperplasia and neoplasia in a laboratory marsupial model (Monodelphis
domestica). Arch. Dermatol. Res. 286, 12–17 (1994).
31. Baker, M. L. et al. Analysis of a set of Australian northern brown bandicoot
expressed sequence tags with comparison to the genome sequence of the south
American grey short-tailed opossum. BMC Genom. 8, 50 (2007).
32. Belov, K. et al.Characterization of the opossum immune genome provides insights
into the evolution of the mammalian immune system. Genome Res. doi:10.1101/
gr.6121807 (2007).
33. Gentles,A.J.etal.Evolutionarydynamicsof transposableelements intheshort-tailed
opossum Monodelphis domestica. Genome Res. doi:10.1101/gr.6070707 (2007).
34. Goodstadt, L., Heger, A., Webber, C. & Ponting, C. P. An analysis of the gene
complement of a marsupial Monodelphis domestica: Evolution of lineage-specific
genes and giant chromosomes. Genome Res. doi:10.1101/gr.6093907 (2007).
35. Gu, W. et al. Phylogenetic detection, population genetics, and distribution of
active SINEs in the genome of Monodelphis domestica. Gene
doi:10.1016/j.gene.2007.02.028 (2007).
36. Mahony, S., Corcoran, D. L., Feingold, E. & Benos, P. V. Regulatory conservation of
protein coding and miRNA genes in vertebrates: lessons from the opossum
genome. Genome Biol. (in the press).
37. Parra, Z. E. et al. A new T-cell receptor discovered in marsupials. Proc. Natl Acad.
Sci. USA (submitted).
38. Samollow,P. B. et al.Amicrosatellite-based, physicallyanchored linkagemap for the
gray, short-tailed opossum (Monodelphis domestica). Chromosome Res. advance
online publication, doi:10.1007/s10577-007-1123-4 (25 February 2007).
39. Wong, E. S., Young, L. J., Papenfuss, A. T. & Belov, K. In silico identification of
opossum cytokine genes suggests the complexity of the marsupial immune
system rivals that of eutherian mammals. Immunome Res. 2, 4 (2006).
40. Hore, T., Koina, E., Wakefield, M. J. & Graves, J. A. M. The region homologous to
the X-chromosome inactivation centre has been disrupted in marsupial and
monotreme mammals. Chromosome Res. 15, 147–161 (2007).
41. Davidow, L. S. et al. The search for amarsupial XIC reveals a breakwith vertebrate
synteny. Chromosome Res. 15, 137–146 (2007).
42. Venter, J.C.etal.Thesequenceof thehumangenome.Science291,1304–1351(2001).
43. Duke, S. E. et al. Integrated cytogenetic BACmap of the genome of the gray short-
tailed opossum, Monodelphis domestica. Chromosome Res. advance online
publication, doi:10.1007/s10577-007-1131-4 (6 April 2007).
44. VandeBerg, J. L. The laboratory opossum (Monodelphis domestica) in laboratory
research. ILAR J. 38, 4–12 (1997).
45. Rens,W. et al. Karyotype relationships between distantly relatedmarsupials from
South America and Australia. Chromosome Res. 9, 301–308 (2001).
46. Belle, E. M., Duret, L., Galtier, N. & Eyre-Walker, A. The decline of isochores in
mammals: an assessment of the GC content variation along the mammalian
phylogeny. J. Mol. Evol. 58, 653–660 (2004).
47. Jensen-Seaman, M. I. et al. Comparative recombination rates in the rat, mouse,
and human genomes. Genome Res. 14, 528–538 (2004).
48. Duret, L., Eyre-Walker, A. & Galtier, N. A new perspective on isochore evolution.
Gene 385, 71–74 (2006).
49. Dumas, D. & Britton-Davidian, J. Chromosomal rearrangements and evolution of
recombination: comparison of chiasma distribution patterns in standard and
robertsonian populations of the house mouse. Genetics 162, 1355–1366 (2002).
50. Myers, S. et al. A fine-scale map of recombination rates and hotspots across the
human genome. Science 310, 321–324 (2005).
51. Hope, R.M. Selected features ofmarsupial genetics.Genetica90, 165–180 (1993).
52. Sharp, P. J. &Hayman, D. L. An examination of the role of chiasma frequency in the
genetic system of marsupials. Heredity 60, 77–85 (1988).
53. Holm, P. B. Ultrastructural analysis of meiotic recombination and chiasma
formation. Tokai J. Exp. Clin. Med. 11, 415–436 (1986).
54. Samollow, P. B. et al. First-generation linkage map of the gray, short-tailed
opossum, Monodelphis domestica, reveals genome-wide reduction in female
recombination rates. Genetics 166, 307–329 (2004).
55. Bailey, J. A. et al.Hotspots of mammalian chromosomal evolution. Genome Biol. 5,
R23 (2004).
56. Webber, C. & Ponting, C. P. Hotspots of mutation and breakage in dog and human
chromosomes. Genome Res. 15, 1787–1797 (2005).
57. Jurka, J. et al. Repbase Update, a database of eukaryotic repetitive elements.
Cytogenet. Genome Res. 110, 462–467 (2005).
58. Ma, J. et al. Reconstructing contiguous regions of an ancestral genome. Genome
Res. 16, 1557–1565 (2006).
59. Kohn, M. et al. Wide genome comparisons reveal the origins of the human X
chromosome. Trends Genet. 20, 598–603 (2004).
60. Graves, J. A. Sex chromosome specialization and degeneration in mammals. Cell
124, 901–914 (2006).
61. Ross, M. T. et al. The DNA sequence of the human X chromosome. Nature 434,
325–337 (2005).
62. Cooper, D. W., Johnston, P. G., Graves, J. A. & Watson, J. M. X-inactivation in
marsupials and monotremes. Sem. Dev. Biol. 4, 117–128 (1993).
63. Heard, E. Recent advances in X-chromosome inactivation. Curr. Opin. Cell Biol. 16,
247–255 (2004).
64. Wakefield, M. J., Keohane, A. M., Turner, B. M. & Graves, J. A. Histone
underacetylation is an ancient component of mammalian X chromosome
inactivation. Proc. Natl Acad. Sci. USA 94, 9665–9668 (1997).
65. Reik,W.& Lewis, A. Co-evolution of X-chromosome inactivation and imprinting in
mammals. Nature Rev. Genet. 6, 403–410 (2005).
66. Duret, L. et al. The Xist RNA gene evolved in eutherians by pseudogenization of a
protein-coding gene. Science 312, 1653–1655 (2006).
67. Lyon, M. F. Do LINEs have a role in X–chromosome inactivation? J. Biomed.
Biotechnol. 2006, 59746 (2006).
68. Bailey, J. A., Carrel, L., Chakravarti, A. & Eichler, E. E. Molecular evidence for a
relationship between LINE-1 elements and X chromosome inactivation: the Lyon
repeat hypothesis. Proc. Natl Acad. Sci. USA 97, 6634–6639 (2000).
69. Disteche, C. M., Filippova, G. N. & Tsuchiya, K. D. Escape from X inactivation.
Cytogenet. Genome Res. 99, 36–43 (2002).
70. Emes, R. D., Goodstadt, L., Winter, E. E. & Ponting, C. P. Comparison of the
genomes of human andmouse lays the foundation of genome zoology. Hum. Mol.
Genet. 12, 701–709 (2003).
ARTICLES NATURE |Vol 447 | 10 May 2007
176
Nature ©2007 Publishing Group

71. Kato, T. Jr et al. Cloning of a marsupial DNA photolyase gene and the lack of
related nucleotide sequences in placental mammals. Nucleic Acids Res. 22,
4119–4124 (1994).
72. Kondrashov, F. A. et al. Evolution of glyoxylate cycle enzymes in Metazoa:
evidence of multiple horizontal transfer events and pseudogene formation. Biol.
Direct 1, 31 (2006).
73. Wistow, G. et al. cN-crystallin and the evolution of the bc-crystallin superfamily in
vertebrates. FEBS J. 272, 2276–2291 (2005).
74. Grus,W. E., Shi, P., Zhang, Y. P. & Zhang, J. Dramatic variation of the vomeronasal
pheromone receptor gene repertoire among five orders of placental and
marsupial mammals. Proc. Natl Acad. Sci. USA 102, 5767–5772 (2005).
75. International HumanGenome Sequencing Consortium. Finishing the euchromatic
sequence of the human genome. Nature 431, 931–945 (2004).
76. Dermitzakis, E. T. & Clark, A. G. Evolution of transcription factor binding sites in
Mammalian gene regulatory regions: conservation and turnover. Mol. Biol. Evol.
19, 1114–1121 (2002).
77. Griffiths-Jones, S. et al. miRBase: microRNA sequences, targets and gene
nomenclature. Nucleic Acids Res. 34 (database issue), D140–D144 (2006).
78. Michael, M. Z. et al. Reduced accumulation of specific microRNAs in colorectal
neoplasia. Mol. Cancer Res. 1, 882–891 (2003).
79. Lagos-Quintana, M., Rauhut, R., Lendeckel, W. & Tuschl, T. Identification of novel
genes coding for small expressed RNAs. Science 294, 853–858 (2001).
80. Crawford, G. E. et al. Genome-wide mapping of DNase hypersensitive sites using
massively parallel signature sequencing (MPSS).Genome Res. 16, 123–131 (2006).
81. Sandelin, A. et al.Arrays of ultraconserved non-coding regions span the loci of key
developmental genes in vertebrate genomes. BMC Genom. 5, 99 (2004).
82. Woolfe, A. et al. Highly conserved non-coding sequences are associated with
vertebrate development. PLoS Biol. 3, e7 (2005).
83. Bailey, P. J. et al. A global genomic transcriptional code associated with CNS-
expressed genes. Exp. Cell Res. 312, 3108–3119 (2006).
84. de la Calle-Mustienes, E. et al. A functional survey of the enhancer activity of
conserved non-coding sequences from vertebrate Iroquois cluster gene deserts.
Genome Res. 15, 1061–1072 (2005).
85. Pennacchio, L. A. et al. In vivo enhancer analysis of human conserved non-coding
sequences. Nature 444, 499–502 (2006).
86. Carroll, S. B. Evolution at two levels: on genes and form. PLoS Biol. 3, e245 (2005).
87. Davidson, E. H. & Erwin, D. H. Gene regulatory networks and the evolution of
animal body plans. Science 311, 796–800 (2006).
88. Stathopoulos, A. & Levine, M. Genomic regulatory networks and animal
development. Dev. Cell 9, 449–462 (2005).
89. Britten, R. J. Mobile elements inserted in the distant past have taken on important
functions. Gene 205, 177–182 (1997).
90. Britten, R. J. & Davidson, E. H. Gene regulation for higher cells: a theory. Science
165, 349–357 (1969).
91. Brosius, J. Genomes were forged by massive bombardments with retroelements
and retrosequences. Genetica 107, 209–238 (1999).
92. Kazazian, H. H. Jr. Mobile elements: drivers of genome evolution. Science 303,
1626–1632 (2004).
93. Marino-Ramirez, L., Lewis, K. C., Landsman, D. & Jordan, I. K. Transposable
elements donate lineage-specific regulatory sequences to host genomes.
Cytogenet. Genome Res. 110, 333–341 (2005).
94. Cooper, G. M. et al. Distribution and intensity of constraint in mammalian
genomic sequence. Genome Res. 15, 901–913 (2005).
95. Silva, J. C. et al. Conserved fragments of transposable elements in intergenic
regions: evidence for widespread recruitment of MIR- and L2-derived sequences
within the mouse and human genomes. Genet. Res. 82, 1–18 (2003).
96. Bejerano, G. et al.Adistal enhancer and an ultraconserved exon are derived from a
novel retroposon. Nature 441, 87–90 (2006).
97. Nishihara, H., Smit, A. F. & Okada, N. Functional noncoding sequences derived
from SINEs in the mammalian genome. Genome Res. 16, 864–874 (2006).
98. Xie, X., Kamal, M. & Lander, E. S. A family of conserved noncoding elements
derived from an ancient transposable element. Proc. Natl Acad. Sci. USA 103,
11659–11664 (2006).
99. Ning, Z., Cox, A. J. & Mullikin, J. C. SSAHA: a fast search method for large DNA
databases. Genome Res. 11, 1725–1729 (2001).
100. Barrett, J. C., Fry, B.,Maller, J. &Daly,M. J. Haploview: analysis and visualization of
LD and haplotype maps. Bioinformatics 21, 263–265 (2005).
101. Birney, E. et al. Ensembl 2006.Nucleic Acids Res. 34 (database issue), D556–D561
(2006).
102. Slater, G. S. & Birney, E. Automated generation of heuristics for biological
sequence comparison. BMC Bioinformatics 6, 31 (2005).
103. Kent,W. J. et al.Evolution’s cauldron:duplication, deletion, and rearrangement in the
mouse and human genomes. Proc. Natl Acad. Sci. USA 100, 11484–11489 (2003).
Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements Generation of the Monodelphis domestica sequence at the
Broad Institute of MIT and Harvard was supported by grants from the National
Human Genome Research Institute (NHGRI). For work from other members of the
Opossum Genome Sequencing Consortium, we acknowledge the support of the
National Institutes of Health (NHGRI, NIAID, NLM), the National Science
Foundation, the Robert J. Kleberg Jr and Helen C. Kleberg Foundation, the State of
Louisiana Board of Regents Support Fund, State of Colorado support funds, the
Pittsburgh Foundation, TATRC/DoD, the UK Medical Research Council and the
Australian Research Council. We thank colleagues at the UCSC genome browser
for providing data (BLASTZ/MULTIZ alignments, synteny nets, and annotations).
We thank L. Gaffney for assistance in preparing the manuscript and figures, and
J. Danke for flow cytometry data.
Author Information All analysed data sets can be obtained from http://
www.broad.mit.edu/mammals/opossum/. This Monodelphis domestica
whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank
under NCBI accession code AAFR00000000. SNPs have been deposited in the
dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP/). Reprints and
permissions information is available at www.nature.com/reprints. The authors
declare no competing financial interests. Correspondence and requests for
materials should be addressed to K.L.-T. (kersli@broad.mit.edu), T.S.M.
(tarjei@broad.mit.edu) and E.S.L. (lander@broad.mit.edu).
Broad Institute Genome Sequencing Platform members Jennifer Baldwin1, Amr
Abdouelleil1, Jamal Abdulkadir1, Adal Abebe1, Brikti Abera1, Justin Abreu1, St
Christophe Acer1, Lynne Aftuck1, Allen Alexander1, Peter An1, Erica Anderson1, Scott
Anderson1, Harindra Arachi1, Marc Azer1, Pasang Bachantsang1, Andrew Barry1, Tashi
Bayul1, Aaron Berlin1, Daniel Bessette1, Toby Bloom1, Jason Blye1, Leonid Boguslavskiy1,
Claude Bonnet1, Boris Boukhgalter1, Imane Bourzgui1, Adam Brown1, Patrick Cahill1,
Sheridon Channer1, Yama Cheshatsang1, Lisa Chuda1, Mieke Citroen1, Alville
Collymore1, Patrick Cooke1, Maura Costello1, Katie D’Aco1, Riza Daza1, Georgius De
Haan1, Stuart DeGray1, Christina DeMaso1, Norbu Dhargay1, Kimberly Dooley1, Erin
Dooley1, Missole Doricent1, Passang Dorje1, Kunsang Dorjee1, Alan Dupes1, Richard
Elong1, Jill Falk1, Abderrahim Farina1, Susan Faro1, Diallo Ferguson1, Sheila Fisher1,
Chelsea D. Foley1, Alicia Franke1, Dennis Friedrich1, Loryn Gadbois1, Gary Gearin1,
Christina R. Gearin1, Georgia Giannoukos1, Tina Goode1, Joseph Graham1, Edward
Grandbois1, Sharleen Grewal1, Kunsang Gyaltsen1, Nabil Hafez1, Birhane Hagos1,
Jennifer Hall1, Charlotte Henson1, Andrew Hollinger1, Tracey Honan1, Monika D.
Huard1, Leanne Hughes1, Brian Hurhula1, M. Erii Husby1, Asha Kamat1, Ben Kanga1,
Seva Kashin1, Dmitry Khazanovich1, Peter Kisner1, Krista Lance1, Marcia Lara1, William
Lee1, Niall Lennon1, Frances Letendre1, Rosie LeVine1, Alex Lipovsky1, Xiaohong Liu1,
Jinlei Liu, Shangtao Liu1, Tashi Lokyitsang1, Yeshi Lokyitsang1, Rakela Lubonja1, Annie
Lui1, PenMacDonald1, Vasilia Magnisalis1, KebedeMaru1, Charles Matthews1, William
McCusker1, SusanMcDonough1, TeenaMehta1, JamesMeldrim1, LouisMeneus1, Oana
Mihai1, Atanas Mihalev1, Tanya Mihova1, Rachel Mittelman1, Valentine Mlenga1, Anna
Montmayeur1, LeonidasMulrain1, AdamNavidi1, JeromeNaylor1, Tamrat Negash1, Thu
Nguyen1, Nga Nguyen1, Robert Nicol1, Choe Norbu1, Nyima Norbu1, Nathaniel Novod1,
Barry O’Neill1, Sahal Osman1, Eva Markiewicz1, Otero L. Oyono1, Christopher Patti1,
Pema Phunkhang1, Fritz Pierre1, Margaret Priest1, Sujaa Raghuraman1, Filip Rege1,
Rebecca Reyes1, Cecil Rise1, Peter Rogov1, Keenan Ross1, Elizabeth Ryan1, Sampath
Settipalli1, Terry Shea1, Ngawang Sherpa1, Lu Shi1, Diana Shih1, Todd Sparrow1, Jessica
Spaulding1, John Stalker1, Nicole Stange-Thomann1, Sharon Stavropoulos1, Catherine
Stone1, Christopher Strader1, Senait Tesfaye1, Talene Thomson1, Yama Thoulutsang1,
Dawa Thoulutsang1, Kerri Topham1, Ira Topping1, Tsamla Tsamla1, Helen Vassiliev1,
Andy Vo1, Tsering Wangchuk1, Tsering Wangdi1, Michael Weiand1, Jane Wilkinson1,
Adam Wilson1, Shailendra Yadav1, Geneva Young1, Qing Yu1, Lisa Zembek1, Danni
Zhong1, Andrew Zimmer1 & Zac Zwirko1
Broad Institute Whole Genome Assembly Team members David B. Jaffe1, Pablo
Alvarez1, Will Brockman1, Jonathan Butler1, CheeWhye Chin1, Sante Gnerre1 & Iain
MacCallum
Affiliation for participants: 1Broad Institute of MIT and Harvard, 7 Cambridge Center,
Cambridge, Massachusetts 02142, USA.
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METHODS
WGS sequencing and assembly. Approximately 38.8million high-quality
sequence reads were derived from paired-end reads of 4- and 10-kb plasmids,
fosmid and BAC clones, prepared from primary tissue DNA from a single female
opossum. The reads were assembled using an interim version of ARACHNE21
(http://www.broad.mit.edu/wga/). No comparative data were used in the assem-
bly process. An intermediate assembly (monDom4) was used for the majority of
the analyses reported here. The most recent version (monDom5) has identical
sequence content and scaffold structure, but includes additional FISH data as
described in Supplementary Note 2.
SNP discovery. The SNP discovery was performed using ARACHNE by com-
parison of the two haplotypes derived from the opossum assembly using only
high-quality discrepancies supported by two or more reads each. Sequence reads
from three additional individuals were also aligned to the reference assembly,
and SNPs were discovered using SSAHA-SNP99. Linkage disequilibrium was
assessed using Haploview100.
Genome alignment and comparisons. The assembly versions used in all com-
parative analyseswere hg17 or hg18 (human),mm8 (mouse), rn4 (rat), canFam2
(dog), monDom4 ormonDom5 (opossum) and galGal3 (chicken). The number
of aligned nucleotides was counted directly from unfiltered, pairwise BLASTZ
alignments (obtained from http://genome.ucsc.edu). Synteny maps were gener-
ated using standard methods7,10, starting from 320,000 reciprocal-best syntenic
anchors identified by PatternHunter104 (see SupplementaryNote 7). Reconstruc-
tion of the boreoeutherian ancestral karyotype is described in Supplementary
Note 8.
Gene prediction and phylogeny. Opossum protein-coding and non-coding
RNA genes were predicted using a modified version of the Ensembl genebuild
pipeline101, followed by several rounds of refinement using Exonerate102 and
manual curation. Orthology and paralogy were inferred using the PhyOP pipe-
line with all predicted opossum and human (Ensembl v40) gene transcripts as
input andKS as the distancemetric11,34. Coding regionswere aligned according to
their amino acid sequences using BLASTP. KA and KS were estimated using the
codeml program105, with default settings and the F3X4 codon frequency model.
Functional categories were identified using the Gene Ontology106.
Conserved element prediction. Amniote conserved elements were inferred
directly from pairwise BLASTZ alignments of chicken to opossum or human.
Every alignment blockwithmore than 75% identity for$100 bpwas classified as
an amniote conserved element. Eutherian conserved elements were inferred
using phastCons14 on BLASTZ/MULTIZ107,108 alignments of human to mouse,
rat and dog. The nonconserved model was fitted to fourfold degenerate sites
from 15,900 human RefSeqs projected onto the same alignments, using phyloFit
and REV. A separate model was fitted for the X chromosome. The scaling para-
meter for the conservedmodel was estimated by phastCons. Target coverage and
expected element length were set to 12.5% and 12 bp, respectively. Predicted
eutherian conserved elements that did not fall within a 10-kb or longer synteny
‘net’103 between human, mouse and dog were ignored. The coding status of each
element was inferred from$1 nucleotide overlap with entries in the UCSC
human ‘known genes’ track109. Proportions are reported out of the total length
of the elements considered. Eutherian CNEs were classified as transposable-
element-derived if they showed more than 20% nucleotide overlap (med-
ian5 100% for all elements, 54% for elements with log2-odds score$ 60) with
human RepeatMasker annotations.
Phylogeny of conserved elements. For amniote conserved elements, pairwise
best-in-genome BLASTZ alignments of opossum to human and vice versa were
used to infer their phylogenetic distributions. For eutherian conserved elements,
concomitant BLASTZ/MULTIZ alignments to opossum and chicken were used.
A conserved element was called absent from a species if it was not covered by a
single aligned nucleotide in the relevant BLASTZ alignment.
Correction for assembly gaps and initial alignment artefacts. A conserved
element was considered to be in an ungapped syntenic interval if it was flanked
by two PatternHunter synteny anchors within 200-kb of each other on the same
contigs in both the human and opossum assemblies. All conserved elements
(represented by human or opossum, as appropriate) in ungapped syntenic inter-
vals were realigned to the unmasked genome sequence (in opossum or human)
using the water program (http://emboss.sourceforge.net) with default para-
meters and a gap extension penalty of 4. A randomly permuted version of each
element was also realigned. For amniote conserved elements, only the longest
interval with$75% identity from within the originating alignment block (see
above) was realigned. Amniote elements were called lost, and eutherian elements
were called eutherian-specific if their Smith–Waterman realignment score,
divided by the length of the element, did not exceed the corresponding score
for the permuted element plus one. (Conservatively calling an element found if
its score simply exceeded the score of the permuted element resulted in 15% of
eutherian CNEs in ungapped regions and 8% of those with log2-odds score$ 60
being called eutherian-specific.) Putatively eutherian-specific elements, includ-
ing XIST, were also searched against all opossum sequencing reads using dis-
contiguous MegaBLAST.
104.Ma, B., Tromp, J. & Li, M. PatternHunter: faster and more sensitive homology
search. Bioinformatics 18, 440–445 (2002).
105. Yang, Z. PAML: a program package for phylogenetic analysis by maximum
likelihood. Comput. Appl. Biosci. 13, 555–556 (1997).
106. Ashburner, M. et al. Gene ontology: tool for the unification of biology. Nature
Genet. 25, 25–29 (2000).
107. Blanchette, M. et al. Aligning multiple genomic sequences with the threaded
blockset aligner. Genome Res. 14, 708–715 (2004).
108. Schwartz, S. et al. Human-mouse alignments with BLASTZ. Genome Res. 13,
103–107 (2003).
109. Hsu, F. et al. The UCSC known genes. Bioinformatics 22, 1036–1046
(2006).
doi:10.1038/nature05805
Nature ©2007 Publishing Group