, 1753 (2007);
et al. Julie C. Dunning Hotopp,
Bacteria to Multicellular Eukaryotes
Widespread Lateral Gene Transfer from Intracellular
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on October 10, 2007
18. A. Yildiz et al., Science 300, 2061 (2003).
19. M. P. Gordon, T. Ha, P. R. Selvin, Proc. Natl. Acad. Sci.
U.S.A. 101, 6462 (2004).
20. X. Qu, D. Wu, L. Mets, N. F. Scherer, Proc. Natl. Acad.
Sci. U.S.A. 101, 11298 (2004).
21. K. A. Lidke, B. Rieger, T. M. Jovin, R. Heintzmann,
Opt. Exp. 13, 7052 (2005).
22. C. Kural et al., Science 308, 1469 (2005).
23. X. L. Nan, P. A. Sims, P. Chen, X. S. Xie, J. Phys. Chem. B
109, 24220 (2005).
24. E. A. Abbondanzieri, W. J. Greenleaf, J. W. Shaevitz,
R. Landick, S. M. Block, Nature 438, 460 (2005).
25. S. Dumont et al., Nature 439, 105 (2006).
26. See supporting material on Science Online.
27. M. Bates, T. R. Blosser, X. Zhuang, Phys. Rev. Lett. 94,
28. M. Heilemann, E. Margeat, R. Kasper, M. Sauer,
P. Tinnefeld, J. Am. Chem. Soc. 127, 3801 (2005).
29. S. Hohng, C. Joo, T. Ha, Biophys. J. 87, 1328 (2004).
30. K. Weber, P. C. Rathke, M. Osborn, Proc. Natl. Acad. Sci.
U.S.A. 75, 1820 (1978).
31. J. E. Heuser, R. G. W. Anderson, J. Cell Biol. 108, 389
32. I. Chen, A. Y. Ting, Curr. Opin. Biotechnol. 16, 35
33. We thank M. Rust for initial discussions of this work,
W. Wang for help with analysis on dye-labeled antibodies,
and S. Liu for providing some DNA constructs. This work
was supported in part by the National
Institutes of Health (grant GM 068518) and a Packard
Science and Engineering Fellowship (to X.Z.). X.Z. is
a Howard Hughes Medical Institute Investigator.
Supporting Online Material
Materials and Methods
Figs. S1 to S7
18 June 2007; accepted 3 August 2007
Published online 16 August 2007;
Include this information when citing this paper.
Widespread Lateral Gene Transfer
from Intracellular Bacteria to
Julie C. Dunning Hotopp,1*†‡ Michael E. Clark,2* Deodoro C. S. G. Oliveira,2Jeremy M. Foster,3
Peter Fischer,4Mónica C. Muñoz Torres,5Jonathan D. Giebel,2Nikhil Kumar,1‡
Nadeeza Ishmael,1‡ Shiliang Wang,1Jessica Ingram,3Rahul V. Nene,1§ Jessica Shepard,1∥
Jeffrey Tomkins,5Stephen Richards,6David J. Spiro,1Elodie Ghedin,1,7Barton E. Slatko,3
Hervé Tettelin,1‡¶ John H. Werren2¶
Although common among bacteria, lateral gene transfer—the movement of genes between
distantly related organisms—is thought to occur only rarely between bacteria and multicellular
eukaryotes. However, the presence of endosymbionts, such as Wolbachia pipientis, within some
eukaryotic germlines may facilitate bacterial gene transfers to eukaryotic host genomes. We
therefore examined host genomes for evidence of gene transfer events from Wolbachia bacteria to
their hosts. We found and confirmed transfers into the genomes of four insect and four nematode
species that range from nearly the entire Wolbachia genome (>1 megabase) to short (<500 base
pairs) insertions. Potential Wolbachia-to-host transfers were also detected computationally in three
additional sequenced insect genomes. We also show that some of these inserted Wolbachia genes
are transcribed within eukaryotic cells lacking endosymbionts. Therefore, heritable lateral gene
transfer occurs into eukaryotic hosts from their prokaryote symbionts, potentially providing a
mechanism for acquisition of new genes and functions.
functions. Among Eubacteria, LGT is involved
he transfer of DNA between diverse
organisms, lateral gene transfer (LGT),
facilitates the acquisition of novel gene
in the evolution of antibiotic resistance, patho-
genicity, and metabolic pathways (1). Rare LGT
events have also been identified between higher
eukaryotes with segregated germ cells (2),
demonstrating that even these organisms can
acquire novel DNA. Although most described
LGTevents occur withina singledomainof life,
LGT has been described both between Eu-
bacteria and Archaea (3) and between prokar-
yotes and phagotrophic unicellular eukaryotes
(4, 5). However, few interdomain transfers
involving higher multicellular eukaryotes have
Wolbachia pipientis is a maternally inherited
endosymbiont that infects a wide range of
arthropods, including at least 20% of insect
species, as well as filarial nematodes (6). It is
present in developing gametes (6) and so
provides circumstances conducive for heritable
transfer of bacterial genes to the eukaryotic
hosts. Wolbachia-host transfer has been
described in the bean beetle Callosobruchus
chinensis (7) and in the filarial nematode
Onchocerca spp. (8).
We have found Wolbachia inserts in the
genomes of additional diverse invertebrate taxa,
including fruit flies, wasps, and nematodes. A
comparison of the published genome of the
Wolbachia endosymbiont of Drosophila mela-
nogaster (9) and assemblies of Wolbachia clone
mates (10) from fruit flywhole-genomeshotgun
sequencing data revealed a large Wolbachia
insert in the genome of the widespread tropical
fruit fly D. ananassae. Numerous contiguous,
overlapping, clone sequences (contigs) were
ila retrotransposons and Wolbachia genes. The
large number of these junctions and the deep
sequencing coverage across the junctions indi-
cated that these inserts were probably not due to
observations, we amplified five Drosophila-
Wolbachia junctions with polymerase chain
reaction (PCR) and verified the end sequences
for three of them. Fluorescence in situ hybridiza-
tion (FISH) of banded polytene chromosomes
genes (11) revealed the presence of Wolbachia
genes on the 2L chromosome of D. ananassae
We found that nearly the entire Wolbachia
genome was transferred to the fly nuclear
genome, as evidenced by the presence of PCR-
amplified products from 44 of 45 physically
distant Wolbachia genes in cured strains of D.
ananassae Hawaii verified by microscopy to be
lacking the endosymbiont after treatment with
1The Institute for Genomic Research, J. Craig Venter
Institute, 9712 Medical Center Drive, Rockville, MD 20850,
Rochester, NY 14627, USA.
Division, New England Biolabs Incorporated, 240 County
Road, Ipswich, MA 01938, USA.4Department of Internal
Medicine, Infectious Diseases Division, Washington Uni-
versity School of Medicine, St. Louis, MO 63110, USA.
5Clemson University Genomics Institute, 304 BRC, 51 New
Cherry Street, Clemson, SC 29634, USA.6Human Genome
Sequencing Center, Baylor College of Medicine, Houston,
TX 77030, USA.7Division of Infectious Diseases, University
of Pittsburgh School of Medicine, Pittsburgh, PA 15261,
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-mail:
‡Present address: Institute forGenomeSciences, University of
S-445, 20 Penn Street, Baltimore, MD 21201, USA.
§Present address: Brown University, Providence, RI 02912, USA.
∥Present address: Pace University, New York, NY 10038, USA.
¶These authors contributed equally to this work.
2Department of Biology, University of Rochester,
Fig. 1. Fluorescence microscopy evidence supporting Wolbachia/host LGT. DNA in the polytene
chromosomes of D. ananassae were stained with propidium iodide (red), whereas a probe for the
Wolbachia gene WD_0484 bound to a unique location (green, arrow) on chromosome 2L.
VOL 31721 SEPTEMBER 2007
on October 10, 2007
antibiotics (fig. S1) (11). In contrast, only
spurious, incorrectly sized, and weak amplifica-
these inserts (Townsville). The 45 genes assayed
(table S1) are spaced throughout the Wolbachia
genome. Thus, the high proportion of amplified
genes suggests gene transfer of nearly the entire
Wolbachia genome to the insect genome.
A 14-kb region containing four Wolbachia
genes with two retrotransposon insertions was
sequenced (11) from a single bacteria artificial
chromosome (BAC), constituting an indepen-
dent source of DNA as compared with the
largely plasmid-derived whole-genome sequence
of D. ananassae. The two retroelements each
contained 5–base pair (bp) target site duplica-
tions (9/10 bp identical), long terminal repeats,
and gag-pol genes (Fig. 2A) indicating that the
Wolbachia insert is accumulating retroelements.
Insertion of this region appears to be recent, as
shown by the nearly identical target site duplica-
tions and the >90% nucleotide identity between
corresponding endosymbiont genes and sequenced
homologs in the D. ananassae chromosome.
Crosses between Wolbachia-free Hawaii
males (with the insert) and Wolbachia-free
Mexico females (without the insert) revealed
that the insert is paternally inherited by offspring
of both sexes, confirming that Wolbachia genes
infections are maternally inherited, this also con-
firms that PCR amplification in the antibiotic-
Furthermore, the Hawaii and Mexico crosses
revealed Mendelian, autosomal inheritance of
Wolbachia inserts [paternal N = 57, proportion
of offspring with Wolbachia genes (k) = 0.49;
maternalN = 40,k= 0.58].Six physically distant,
inserted Wolbachia genes perfectly cosegregated
they also are closely linked.
Wolbachia loci in 14 D. ananassae lines from
widely dispersed geographic locations revealed
large Wolbachia inserts in lines from Hawaii,
Malaysia, Indonesia, and India (table S2).
Sequence comparisons of the amplicons from
these four lines revealed that all open reading
frames (ORFs) remained intact with >99.9%
identity between inserts. This is compared to an
average of 97.7% identity for the inserts
compared with wMel, the Wolbachia endo-
symbiont of D. melanogaster. These results
indicate the widespread prevalence of D.
ananassae strains with similar inserts of the
Wolbachia genome, probably because of a
single insertion from a common ancestor.
In addition, reverse transcription PCR (RT-
PCR) followed by sequencing (11) demonstra-
ted that ~2% of Wolbachia genes (28 of 1206
genes assayed; table S3) are transcribed in cured
adult males and females of D. ananassae
Hawaii. The complete 5′ sequence of one of
the transcripts, WD_0336, was obtained with
uninfected flies (11), suggesting that this
transcript has a 5′ mRNA cap, a form of
eukaryotic posttranscriptional modification.
Analysis of the transcript quantities of inserted
Wolbachia genes with quantitative RT-PCR
(qRT-PCR) (11) revealed that they are 104times
to 107times less abundant than the fly’s highly
transcribed actin gene (act5C; table S3). There is
no cutoff that defines a biologically relevant
amount of transcription, and assessment of
transcription in whole insects can obscure
it is unclear whether these transcripts are
biologically meaningful, and further work is
needed to determine their importance.
Screening of public shotgun sequencing data
sets has identified several additional cases of LGT
in different invertebrate species. In Wolbachia-
cured strains of the wasp Nasonia, six small
Wolbachia inserts (<500 bp) were verified by
PCR and sequencing (11) that have >96% nucle-
otide identity to native Wolbachia sequences, in
some cases with short insertion site duplications.
These include four in Nasonia vitripennis, one in
N. giraulti, and one in N. longicornis (table S4
and Fig. 2B). Amplification and sequencing of
14 to 18 geographically diverse strains of each
species indicated that the inserts are species-
specific. For example, three Wolbachia inserts in
N. vitripennis are not found in the closely related
wMel Mel Mel
wBm Bm Bm
Fig. 2. Schematics of Wolbachia inserts in host chromosomes. (A) Contigs containing Wolbachia sequences
generated from the D. ananassae Hawaii shotgun sequencing project are segregated into sequences
coming from the endosymbiont (wAna) or from the D. ananassae chromosome (Dana) on the basis of
presence or absence of eukaryotic genes in the contigs. These are compared to those from the reference
D. melanogaster Wolbachia genome (wMel) and a D. ananassae BAC. NAD, nicotinamide adenine di-
nucleotide. (B) Fragments of the Wolbachia gene WD_0024 gene have inserted into different positions in
the N. giraulti (NG) and N. vitripennis (NV) genomes with unique insertions in each lineage, including
N. longicornis (NL). (C) A region in the D. immitis genome (Dg2) that is transcribed has introns similar to
sequences from the Wolbachia infecting B. malayi (wBm). All matches in (A) and (B) have >90% nucleotide
identity; those in (C) have >75% nucleotide identity. TPR, tetratrico peptide repeat; CDS, coding sequence.
21 SEPTEMBER 2007VOL 317
on October 10, 2007
species N. giraulti or N. longicornis, which
diversified ~1 million years ago (12). These data
suggest that the Wolbachia gene inserts are of
relatively recent origin, similar to the inserts in
Nematode genomes also contain inserted
Wolbachia sequences. Because Wolbachia infec-
worm Brugia malayi, the genomes of both
organisms were sequenced simultaneously, com-
identity over 90% of the read length on the basis
of the independent BAC-based genome sequence
of wBm, the Wolbachia endosymbiont of B.
malayi (13)]. Despite this, the genome of B.
malayi contains 249 contigs with Wolbachia se-
firmed by long-range PCR and end-sequencing
(11). These include eight large scaffolds con-
taining >1-kb Wolbachia fragments within 8 kb
of a B. malayi gene (table S5). Comparisons of
wBm homologs to these regions suggested that
genome are degenerate. In addition, a single re-
gion <1 kb was examined that contains a de-
generate fragment of the Wolbachia aspartate
aminotransferase gene (Wbm0002). Its location
was confirmed by PCR and sequencing in B.
Of the remaining 21 arthropod and nematode
genomes in the trace repositories (11), we found
six containing Wolbachia sequences. Potential
Wolbachia-host LGT was detected in three: D.
as revealed by the presence of reads containing
homology to both endosymbiont and host ge-
The sequencing of wBm also facilitated the
discovery of a Wolbachia insertion in Dirofilaria
immitis (dog heartworm). The D. immitis Dg2
chromosomal region encoding the D34 immu-
DNAwithin its introns and in the 5′ untranslated
region (5′-UTR) (Fig. 2C). These Wolbachia
genomic fragments have maintained synteny
with the wBm genome (13), suggesting they
may have inserted as a single unit and regions
were replaced by exons of Dg2. A second gene
(DgK) has been identified in other D. immitis
sequences but contains differing number, posi-
tion, size, and sequence of introns (16) and has
no homology to known Wolbachia sequences.
Whole eukaryote genome sequencing proj-
ects routinely exclude bacterial sequences on the
assumption that these represent contamination.
For example, the publicly available assembly of
D. ananassae does not include any of the
Wolbachia sequences described here. Therefore,
the argument that the lack of bacterial genes in
these assembled genomes indicates that bacterial
LGT does not occur is circular and invalid.
continue to be difficult to detect if bacterial se-
quences are routinely excluded from assemblies
events will remain understudied despite their po-
tential to provide novel gene functions and af-
fect arthropod and nematode genome evolution.
intracellular bacteria (17, 18) and its hosts are
that prokaryote-to-eukaryote transfers are un-
References and Notes
1. Y. Boucher et al., Annu. Rev. Genet. 37, 283 (2003).
2. S. B. Daniels, K. R. Peterson, L. D. Strausbaugh, M. G.
Kidwell, A. Chovnick, Genetics 124, 339 (1990).
3. K. E. Nelson et al., Nature 399, 323 (1999).
4. J. O. Andersson, Cell. Mol. Life Sci. 62, 1182 (2005).
5. W. F. Doolittle, Trends Genet. 14, 307 (1998).
6. R. Stouthamer, J. A. Breeuwer, G. D. Hurst, Annu. Rev.
Microbiol. 53, 71 (1999).
7. N. Kondo, N. Nikoh, N. Ijichi, M. Shimada, T. Fukatsu,
Proc. Natl. Acad. Sci. U.S.A. 99, 14280 (2002).
8. K. Fenn et al., PLoS Pathog. 2, e94 (2006).
9. M. Wu et al., PLoS Biol. 2, e69 (2004).
10. S. L. Salzberg et al., Genome Biol. 6, R23 (2005).
11. Materials and methods are available as supporting
material on Science Online.
12. B. C. Campbell, J. D. Steffen-Campbell, J. H. Werren,
Insect Mol. Biol. 2, 225 (1993).
13. J. Foster et al., PLoS Biol. 3, e121 (2005).
14. S. Sun, K. Sugane, J. Helminthol. 68, 259 (1994).
15. S. H. Sun, T. Matsuura, K. Sugane, J. Helminthol. 65, 149
16. K. Sugane, K. Nakayama, H. Kato, J. Helminthol. 73, 265
17. J. H. Werren, Annu. Rev. Entomol. 42, 587 (1997).
18. J. H. Werren, D. M. Windsor, Proc. Biol. Sci. R. Soc.
London Ser. B 267, 1277 (2000).
19. The D. ananassae BAC 01L18 sequence is deposited in
GenBank (EF426679); the D. ananassae sequence
comparisons from the four lines are deposited in GenBank
(EF611872 to EF611985); the D. ananassae RACE
sequence is deposited in dbEST (46867557) and GenBank
(ES659088); the Nasonia sequences are deposited in
Table 1. Summary of Wolbachia sequences and evidence for LGT in public databases. Junctions were
validated by PCR amplification and sequencing (11), with the number of successful reactions compared
to the number attempted. Species marked with a plus sign are described in the literature as being
infected with Wolbachia. All whole-genome shotgun sequencing reads were downloaded for 26
arthropod and nematode genomes (11). Organisms identified as lacking Wolbachia sequences either
antibiotic-cured insects, they were identified as having a putative LGT event merely on identification of
Wolbachia sequences in a read. All other organisms were considered to have putative LGT events if the
trace repository contained ≥1 read with (i) >80% nucleotide identity over 10% of the read to a
characterized eukaryotic gene, (ii) >80% identity over 10% of the read to a Wolbachia gene, and (iii)
manual review of the BLAST results for 1 to 20 reads to ensure significance (11). NA, not applicable.
Trace repository sequences
Acyrthosiphon pisum (aphid)
Aedes aegypti (mosquito)
Anopheles gambiae (mosquito)
Apis mellifera (honeybee)
Brugia malayi (filarial nematode)
Culex pipiens quinquefasciatus (mosquito)
Daphnia pulex (crustacean)
D. ananassae (fruit fly)
D. erecta (fruit fly)
D. grimshawi (fruit fly)
D. melanogaster (fruit fly)
D. mojavensis (fruit fly)
D. persimilis (fruit fly)
D. pseudoobscura (fruit fly)
D. sechellia (fruit fly)
D. simulans (fruit fly)
D. virilis (fruit fly)
D. willistoni (fruit fly)
D. yakuba (fruit fly)
Ixodes scapularis (tick)
N. giraulti (wasp)
N. longicornis (wasp)
N. vitripennis (wasp)
Pediculus humanus (head louse)
Pristonchus pacificus (nematode)
Tribolium castaneum (beetle)
Dirofilaria immitis (filarial nematode)NA++
*This isolate was previously shown to have Wolbachia reads in its trace repositories that are contaminating reads from the D.
ananassae genome sequencing project (10).
VOL 317 21 SEPTEMBER 2007
on October 10, 2007
GenBank (EF588824 to EF588901); the Brugia malayi Download full-text
contigs are available in GenBank (DS237653, DS238272,
DS238705, DS239028, DS239057, DS239291,
DS239377, and DS239315); the Brugia malayi scaffolds
are available in GenBank (AAQA01000958,
AAQA01000097, AAQA01001500, AAQA01000425,
AAQA01001819, AAQA01001498, AAQA01000384,
AAQA01001952, AAQA01000736, AAQA01000571, and
AAQA01000369); and the microarray primers sequences
that were used for RT-PCR are deposited in ArrayExpress
(A-TIGR-28). This work was supported by an NSF grant to
J.H.W. and H.T., a National Institute of Allergy and
Infectious Diseases grant to E.G., and New England
Biolabs Incorporated support to B.E.S.
Supporting Online Material
Materials and Methods
Tables S1 to S5
13 March 2007; accepted 2 July 2007
Published online 30 August 2007;
Include this information when citing this paper.
Draft Genome of the Filarial Nematode
Parasite Brugia malayi
Elodie Ghedin,1,2# Shiliang Wang,2David Spiro,2Elisabet Caler,2Qi Zhao,2
Jonathan Crabtree,2Jonathan E. Allen,2* Arthur L. Delcher,2† David B. Guiliano,3
Diego Miranda-Saavedra,4‡ Samuel V. Angiuoli,2Todd Creasy,2Paolo Amedeo,2
Brian Haas,2Najib M. El-Sayed,2§ Jennifer R. Wortman,2Tamara Feldblyum,2Luke Tallon,2
Michael Schatz,2† Martin Shumway,2Hean Koo,2Steven L. Salzberg,2† Seth Schobel,2
Mihaela Pertea,2† Mihai Pop,2† Owen White,2Geoffrey J. Barton,4Clotilde K. S. Carlow,5
Michael J. Crawford,6Jennifer Daub,7|| Matthew W. Dimmic,6Chris F. Estes,8Jeremy M. Foster,5
Mehul Ganatra,5William F. Gregory,7Nicholas M. Johnson,9Jinming Jin,10
Richard Komuniecki,11Ian Korf,12Sanjay Kumar,5Sandra Laney,13Ben-Wen Li,14Wen Li,13
Tim H. Lindblom,8Sara Lustigman,15Dong Ma,5Claude V. Maina,5David M. A. Martin,4
James P. McCarter,6,16Larry McReynolds,10Makedonka Mitreva,16Thomas B. Nutman,17
John Parkinson,18José M. Peregrín-Alvarez,1Catherine Poole,5Qinghu Ren,2Lori Saunders,13
Ann E. Sluder,19Katherine Smith,11Mario Stanke,20Thomas R. Unnasch,21Jenna Ware,5
Aguan D. Wei,22Gary Weil,14Deryck J. Williams,7Yinhua Zhang,5Steven A. Williams,13
Claire Fraser-Liggett,2¶ Barton Slatko,5Mark L. Blaxter,7Alan L. Scott23
Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of
people in the developing world. We have sequenced the ~90 megabase (Mb) genome of the human
filarial parasite Brugia malayi and predict ~11,500 protein coding genes in 71 Mb of robustly
assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis
elegans revealed that, despite these genes having maintained little conservation of local synteny
during ~350 million years of evolution, they largely remain in linkage on chromosomal units.
More than 100 conserved operons were identified. Analysis of the predicted proteome provides
evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the
molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer
a foundation for rational drug design.
third of all humans, mainly in the developing
world, carry a nematode infection. Parasitic
worms typically cause chronic, debilitating in-
fections that are often difficult to treat and that,
despite the high cost to human health, have been
neglected in biomedical research. Current
knowledge of nematode molecular genetics
and developmental biology is largely based on
extensive studies of the free-living, bacterio-
vorous species Caenorhabditis elegans. Here, we
present the initial analysis of the genome of the
human filarial parasite Brugia malayi.
Brugia malayi is endemic in Southeast Asia
and Indonesia. Like other filarial nematodes,
B. malayi develops through four larval stages
into an adult male or female (fig. S1), entirely
within one of two host species—a mosquito
vector (Culex, Aedes, and Anopheles) and
humans, where adult worms can live for more
than a decade. B. malayi was chosen for whole-
genome sequencing (1) because it is the only
he phylum Nematoda is speciose and
abundant and, although most species are
free-living, many are parasitic. Over one-
major human filarial pathogen that can be
maintained in small laboratory animals. Most
filarial nematodes, including B. malayi, carry
three genomes: nuclear, mitochondrial (available
at GenBank, accession no. AF538716), and that
of an alphaproteobacterial endosymbiont, Wol-
bachia. We present here the draft assembly and
annotated genome of the TRS strain of B.
malayi. We provide comparative analyses with
Caenorhabditis and another well-annotated
member of the superphylum Ecdysozoa, Dro-
sophila melanogaster, to further illuminate the
originsof novelty andlossof ancestralcharacters
in the model species and the parasite. Compara-
tive genome analysis reveals key features of
Nematoda that define the scope of molecular
phylum. The analysis also uncovers adaptations
that appear to have evolved in the B. malayi
and to the presence of the parasite’s Wolbachia
The B. malayi nuclear genome is organized
as five chromosomes (2), including an XY sex-
determination pair, and has been estimated to be
80 to 100 megabases (Mb) (3, 4). The sequence
of the B. malayi nuclear genome was obtained
to ~9× coverage with the use of whole-genome
shotgun (WGS) sequencing (1, 5). The sequences
were assembled into scaffolds totaling ~71 Mb
of data with a further ~17.5 Mb of contigs not
integrated into any scaffold (orphan contigs).
The repeat content of the B. malayi genome,
estimated at ~15% (1), may have contributed
significantly to assembly difficulties (5, 6). From
these sequence data, we estimate that the B.
malayi genome is 90 to 95 Mb (Table 1) (5). In
comparison, the C. elegans genome is 100 Mb
and the Caenorhabditis briggsae genome 104
Mb. The overall G + C content (30.5%) is lower
than that of C. elegans (35.4%) or C. briggsae
The complement of protein-coding genes
was derived by automated gene prediction from
the ~71-Mb assembly and by manual annotation
of selected gene families (table S1). The 11,515
robustly predicted gene-coding regions occupy
~32% of the sequence at an average density of
162 genes/Mb (Table 1).
in the unannotated portion of the genomic
14,500 and 17,800 protein-coding genes, agree-
ing with previous estimates (7). Even the higher
estimate is lower than the 19,762 (WormBase
data release WS133) and 19,507 (6) genes re-
ported for C. elegans and C. briggsae, respec-
tively, which suggests that parasitic nematode
genomes have fewer genes than their free-living
counterparts, echoing a pattern observed in bac-
For the six scaffolds longer than 1 Mb,
totaling ~25 Mb of the genome, the arrangement
of B. malayi genes was compared with that of
their C. elegans orthologs (Fig. 1). Linkage is
in general conserved: For large regions of the
B. malayi genome, orthologs map predominantly
to one (or, in the case of scaffold 14972, two)
C. elegans chromosome(s) (Fig. 1, A to C),
which indicates maintenance of linkage of these
genes despite ~350 million years of separation
(8). However, local gene order is not conserved
(Fig. 1D). The largest, 6.5-Mb scaffold contains
interdigitating blocks of genes that map to
chromosomes 4 and X of C. elegans, which
suggests there were ancient breakage and fusion
events between linkage groups. These data
support a model where within-linkage group
rearrangements have been many times more
common than between-linkage group transloca-
21 SEPTEMBER 2007VOL 317
on October 10, 2007