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© 2006 Nature Publishing Group
DNA sequence and analysis of human
chromosome 8
Chad Nusbaum
1
, Tarjei S. Mikkelsen
1
, Michael C. Zody
1
, Shuichi Asakawa
2
, Stefan Taudien
3
, Manuel Garber
1
,
Chinnappa D. Kodira
1
, Mary G. Schueler
4
, Atsushi Shimizu
2
, Charles A. Whittaker
1
†, Jean L. Chang
1
,
Christina A. Cuomo
1
, Ken Dewar
1
†, Michael G. FitzGerald
1
, Xiaoping Yang
1
, Nicole R. Allen
1
, Scott Anderson
1
,
Teruyo Asakawa
2
, Karin Blechschmidt
3
, Toby Bloom
1
, Mark L. Borowsky
1
, Jonathan Butler
1
, April Cook
1
,
Benjamin Corum
1
, Kurt DeArellano
1
, David DeCaprio
1
, Kathleen T. Dooley
1
, Lester Dorris III
1
, Reinhard Engels
1
,
Gernot Glo
¨ckner
3
, Nabil Hafez
1
, Daniel S. Hagopian
1
, Jennifer L. Hall
1
, Sabine K. Ishikawa
2
, David B. Jaffe
1
,
Asha Kamat
1
, Jun Kudoh
2
,Ru
¨diger Lehmann
3
, Tashi Lokitsang
1
, Pendexter Macdonald
1
, John E. Major
1
,
Charles D. Matthews
1
, Evan Mauceli
1
, Uwe Menzel
3
†, Atanas H. Mihalev
1
, Shinsei Minoshima
2
†,
Yuji Murayama
2
, Jerome W. Naylor
1
, Robert Nicol
1
, Cindy Nguyen
1
, Sine
´ad B. O’Leary
1
, Keith O’Neill
1
,
Stephen C. J. Parker
1
†, Andreas Polley
3
†, Christina K. Raymond
1
, Kathrin Reichwald
3
†, Joseph Rodriguez
1
,
Takashi Sasaki
2
, Markus Schilhabel
3
, Roman Siddiqui
3
, Cherylyn L Smith
1
, Tam P. Sneddon
5
, Jessica A. Talamas
1
,
Pema Tenzin
1
, Kerri Topham
1
, Vijay Venkataraman
1
, Gaiping Wen
3
†, Satoru Yamazaki
2
, Sarah K. Young
1
,
Qiandong Zeng
1
, Andrew R. Zimmer
1
, Andre Rosenthal
3
†, Bruce W. Birren
1
, Matthias Platzer
3
,
Nobuyoshi Shimizu
2
& Eric S. Lander
1
The International Human Genome Sequencing Consortium
(IHGSC) recently completed a sequence of the human genome
1
.
As part of this project, we have focused on chromosome 8.
Although some chromosomes exhibit extreme characteristics in
terms of length, gene content, repeat content and fraction seg-
mentally duplicated, chromosome 8 is distinctly typical in char-
acter, being very close to the genome median in each of these
aspects. This work describes a finished sequence and gene catalo-
gue for the chromosome, which represents just over 5% of the
euchromatic human genome. A unique feature of the chromosome
is a vast region of ,15 megabases on distal 8p that appears to have
a strikingly high mutation rate, which has accelerated in the
hominids relative to other sequenced mammals. This fast-evolving
region contains a number of genes related to innate immunity and
the nervous system, including loci that appear to be under positive
selection
2
—
these include the major defensin (DEF) gene cluster
3,4
and MCPH1
5,6
, a gene that may have contributed to the evolution
of expanded brain size in the great apes. The data from chromo-
some 8 should allow a better understanding of both normal and
disease biology and genome evolution.
The finished sequence of chromosome 8 contains 145,556,489
bases and is interrupted by only four euchromatic gaps, one gap at
the 8p telomere and one gap containing the centromeric hetero-
chromatin (Fig. 1 and Supplementary Table S1). These gaps are
refractory to current cloning and mapping technology. The esti-
mated total size of the euchromatic gaps is 427 kilobases (kb), based
on direct sizing of three gaps and estimation of the remaining two
gaps at the genome-wide average of ,100 kb each. This corresponds
to ,0.3% of the euchromatic length of the chromosome, similar to
the genome average
1,7–11
. In all, 182.3 megabases (Mb) of finished
sequence were generated by the Broad Institute of MIT and Harvard
(formerly Whitehead Institute/MIT Center for Genome Research
(WICGR)), 27.9 Mb by Keio University School of Medicine, 8.4 Mb
by the Institute of Molecular Biotechnology in Jena, and 5.8 Mb by 10
other groups (Supplementary Tables S2 and S3). These sequences
(which include overlap) were combined to yield the finished path
(see Methods).
We assessed the local accuracy of the clone path by aligning paired-
end sequences from a human Fosmid library (WIBR2, representing
£10 physical coverage) to the finished sequence
7
. Errors in the clone
path were detected by identifying discrepancies between the pre-
dicted and observed distances between Fosmid ends
7
. This revealed
two deleted clones, which were replaced. Finally, an independent
quality assessment exercise commissioned by NHGRI estimated the
accuracy of the finished sequence at less than 1 error in 100,000
bases
12
(J. Schmutz, personal communication).
Several analyses support the idea that nearly the entire euchro-
matic region of chromosome 8 is present and accurately represented.
From the well-curated RefSeq
13
data set 681 transcripts (from 573
unique genes) mapped to chromosome 8. All but one of these are
present and complete in the finished sequence. The finished sequence
shows excellent co-linearity with the genetic map
14
(Supplementary
LETTERS
1
Broad Institute of MIT and Harvard, 320 Charles St, Cambridge, Massachusetts 02141, USA.
2
Department of Molecular Biology, Keio University School of Medicine, 35
Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
3
Genome Analysis, Institute of Molecular Biotechnology, Beutenbergstrasse 11, Jena 07745, Germany.
4
National Human
Genome Research Institute, National Institutes of Health, 50 South Drive Rm 5529, Bethesda, Maryland 20 982, USA.
5
HUGO Gene Nomenclature Committee (HGNC), The
Galton Laboratory, Department of Biology, University College London, Wolfson House, 4 Stephenson Way, London NW1 2HE, UK. †Present addresses: MIT Center for Cancer
Research, 77 Massachusetts Avenue E18-570, Cambridge, Massachusetts 02139, USA (C.A.W.); McGill University and Genome Quebec Innovation Centre, Montreal, Quebec
H3A 1A4, Canada (K.D.); Department of Genetics and Pathology, Uppsala University, SE-751 85 Uppsala, Sweden (U.M.); Photon Medical Research Center, Hamamatsu
University School of Medicine, Handayama, Hamamatsu, Shizuoka 431-3192, Japan (S.M.); Boston University Bioinformatics and Systems Biology Program, 24 Cummington St,
Boston, Massachusetts 02215, USA (S.C.J.P.); TraitGenetics GmbH, Am Schwabeplan 1b, 06466 Gatersleben, Germany (A.P.); University Clinic for Child and Adolescent
Psychiatry, University of Duisburg-Essen, Virchowstr. 174, 45147 Essen, Germany (K.R.); GSF-Forschungszentrum fu¨r Umwelt und Gesundheit, Ingolsta
¨dter Landstraße 1,
85674 Neuherberg, Germany (G.W.); Signature Diagnostics AG, Voltaireweg 4B, 14469 Potsdam, Germany (A.R.).
Vol 439|19 January 2006|doi:10.1038/nature04406
331
© 2006 Nature Publishing Group
Fig. S1). Among 247 sequence-based genetic markers (Supplemen-
tary Table S4) there are six discrepancies. One discrepancy consists of
eight markers and spans a region in 8p23 known to be the site of a
polymorphic inversion in the human population
15,16
(see below).
Five discrepancies each consist of single markers out of order by one
position; all occur in small regions where the genetic map shows no
recombination in one of the two sexes (Supplementary Table S4). The
sequence also shows good agreement with the radiation hybrid (RH)
map
17
(Supplementary Table S5).
We produced a manually curated gene catalogue, containing 793
gene loci and 301 pseudogene loci (see Methods). The catalogue
includes all previously known genes on chromosome 8 (Table 1).
According to the Hawk2 categorization scheme
18
, there are 614
‘known’ genes, 109 ‘novel CDS’, 43 ‘novel transcripts’, 14 ‘putatives’
and 13 ‘gene fragments’. The small set of novel and putative categories
were annotated by spliced expressed sequence tag (EST) evidence
only; some ‘putative novel’ loci may prove to be pseudogenes.
Comparison of manual annotation performed at the Broad Institute
of MIT and Harvard to manual annotation for specific regions done
at Jena and Keio indicated that they were largely the same, and that
virtually all differences could be attributable to edge effects (see
Supplementary Information).
Full-length transcripts of known genes contain an average of 9.9
exons, comparable to recently published reports
8–11,19
, have an
average length of 3,056 base pairs (bp), and internal exons have an
average length of 155 bp. There is evidence of extensive alternate
splicing. Gene loci have an average of 4.1 distinct transcripts, with
63% having at least two transcripts, values that are similar to recent
reports
8,9,11,20
. Of the 301 pseudogenes on chromosome 8, ,84% are
processed pseudogenes arising from retrotransposition; the remain-
ing 16% are unprocessed. We also identified 13 tRNA genes (Sup-
plementary Table S6). Examples of genes that represent extremes
from these averages are described in Supplementary Information.
Several aspects of the genome landscape are notable. The overall
gene density is 5.6 genes Mb
21
, below the genome average of
,10 genes Mb
21
. Gene distribution is highly heterogeneous, with
44 gene deserts (500 kb without a coding gene, Supplementar y Table
S7) that together comprise 41.9 Mb or ,29% the total length. The
overall GþC content is 39.2%, but varies substantially across
the chromosome (Fig. 1). Nearly half of the chromosome is com-
posed of repeat sequences, with transposable element fossils
comprising 44.5%, low complexity sequence (including simple
sequence repeats and satellite sequences) comprising 1.8%, and
segmental duplications comprising ,2.1% (with interchromosomal
and intrachromosomal duplications at ,1.5% each, with some
sequence included in both categories) (E. Eichler and X. She,
personal communication).
Chromosome 8 is the first human autosome and one of only two
chromosomes (the other being chromosome X
20
)forwhich
sequences span the entire pericentromeric region. The regions on
both arms stretch from unique euchromatin through pericentro-
meric satellites and into the higher-order alpha-satellite array (Fig. 2).
Three variant higher-order repeat units populate the chromosome 8
higher-order array, D8Z2 (ref. 21 and Supplementary Information).
The proximal termini of both the 8p and 8q sequence contigs are
comprised of nine copies of the 1.9-kb unit. The p and q arm higher-
order units are highly identical to each other (96–98%) and occur in
the same head-to-tail orientation, indicating that these sequences
sample the edges of the chromosome 8-specific array. Analysis of the
finished pericentromeric sequence of chromosome 8 is essential to
test and further develop primate centromere evolution hypotheses
using an autosomal model.
The most striking feature on chromosome 8 emerges from
evolutionary and population genetic comparisons (Fig. 3). The
most distal 15 Mb on chromosome 8p show an extremely high
divergence between human and chimpanzee (0.021 substitutions
per site, 4.0 s.d. above the mean of 0.012). The region also shows a
strikingly high polymorphism rate in the human population (0.0018,
3.2 s.d. above the mean of 0.0010). The peak divergence reaches
0.032 (8.6 s.d.), and diversity 0.0028 (7.1 s.d.), across a 1-Mb region
(3.3–4.3 Mb) overlapping the CSMD1 gene. This is the highest
divergence level seen across all autosomes and chromosome
X. Only regions of chromosome Y may be more rapidly diverging,
driven by the high mutation rate in the male germ line. We
excluded trivial explanations for this observation, such as unresolved
segmental duplications (Supplementary Information). Diversity is
also locally high in the chimpanzee, although the data are more
limited.
The high rate of divergence and diversity at distal 8p might reflect
either an extraordinary mutation rate or population genetic history.
The latter alternative would require an unusually long coalescence
time to the most recent common ancestor over a very large region;
this would be remarkable inasmuch as local coalescence times tend to
be correlated over short distances, as the correlation falls below 0.5
within 20 kb (ref. 22). We sought to resolve the issue by examining
the divergence rates with more distant mammalian species, where the
impact of population genetic history should be negligible.
Comparison of ancestral interspersed repeats in the human, dog
23
Figure 1 |Overview of human chromosome 8. The features are addressed in
the order of top to bottom. In the cartoon, blue shading indicates gene
deserts ($500 kb with no transcript, Supplementary Table S7); telomeres
(pTEL and qTEL), the centromere (CEN) and euchromatic sequence gaps
(red lines) are indicated. The following features are represented in discrete
windows of 100 kb: GþC content (on a scale from 30–70%); densities of
LINEs (long interspersed nucleotide elements; red) and SINEs (short
interspersed nucleotide elements; blue); and densities of transcripts (all are
counts of elements). The box at the bottom shows blocks of conserved
synteny (100-kb resolution) with dog, mouse and rat as determined for this
work. Chromosomes are numbered, and are coloured arbitrarily for ease of
distinction.
LETTERS NATURE|Vol 439|19 January 2006
332
© 2006 Nature Publishing Group
and mouse
24
genomes reveals that the region exhibits above-average
lineage-specific divergence rates on all three lineages across
100 million years of evolution, but that the rate is the most elevated
relative to the genome-wide mean in the lineage leading to humans.
The greatest elevation is seen in the most distal 6 Mb of 8p, where the
ancestral interspersed repeat divergence rates in the orthologous
sequences have been 0.19 (3.3 s.d. above the mean of 0.14) on the
human lineage and 0.41 (1.0 s.d. above the mean of 0.38) in the
mouse lineage since the primate–rodent split, and 0.24 (1.9 s.d. above
the mean of 0.20) in the dog lineage since the divergence from the
common boreo-eutherian ancestor.
The biological basis for the apparently high mutation rate is
unclear. Three major factors have been associated with high
mutation rates in the human genome: proximity to telomeres, high
recombination rate and high AþTcontent
25,26
. The region on
chromosome 8p has all three factors. The mean sex-averaged recom-
bination rate across the first 6 Mb is 2.7 cM Mb
21
, with a 1-Mb
window peak of 3.5, as compared to the genome-wide average of 1.2.
The region from 2.5–6 Mb is 62% AþT, as compared to a genome-
wide average of 59%. It is unusual in this regard, because sub-
telomeric regions with high recombination rates are typically
(AþT)-poor. Notably, the region is not subtelomeric in the mouse,
where the lowest rate elevation is observed.
The distal region on chromosome 8p also contains at least two loci
that appear to be undergoing positive selection (Fig. 3). The first
locus is the major cluster of defensin genes, which lies within the
region of high mutation (5.5–7.5 Mb), although ,2.5 Mb from
the peak. The defensin genes express small cationic antimicrobial
Table 1 |Chromosome 8 gene content
Category Gene
number
Gene
percentage
Gene length
(bp)*
Number of alternative
transcripts
Transcript
length (bp)†
Number of exons
per transcript‡
Internal exon
length (bp)§
Intron length
(bp)k
CpG-50
association{
Known gene 614 77 81,744 4.1 3,056 9.9 155 (n¼5,725) 9,630 (n¼7,710) 77
Novel transcript 43 5 96,268 1.8 1,116 3.8 146 (n¼127) 27,207 (n¼248) 42
Putative gene 14 2 45,433 1.2 714 2.4 123 (n¼21) 23,787 (n¼57) 36
Novel CDS 109 14 21,890 1.9 1,142 5.0 138 (n¼487) 5,103 (n¼625) 29
Gene fragment 13 2 648 1.0 648 1.0 – – 8
Total 793 – 72,334 – – – – – –
Pseudogene 301 28 1,334 1.0 875 1.3 195 (n¼50) 1,430 (n¼97) 5
*Average chromosomal distance from beginning of 5 0-most exon to 30-most exon in all transcripts in a gene.
†Average length summed across the footprint of all exons in all transcripts in a gene
—
total exon space per gene.
‡Average number of exons in transcripts. Exons common to different transcripts were counted once per transcript.
§Average length of exons using the footprint of all non-terminal exons of all transcripts in a gene. Unique overlapping exons or contained exons are counted separately, making this an average
length of unique exons in a gene.
kAverage length of unique introns in a gene. In the case of exon skipping, both the shorter and longer versions of the overlapping introns were counted towards the average.
{Percentage of genes with a transcript having a CpG island (as assessed by FirstEF) within 22 kb and þ1 kb of transcription start.
Figure 2 |8p and 8q pericentromeric contigs extend into chromosome
8-specific higher-order alpha satellite, D8Z2.The pericentromeric region
of chromosome 8 is shown as a truncated ideogram with the extent of
sequence coverage shown below by black bars. Dotter plots show self–self
alignments of the most proximal ,100 kb from each arm including ,36 kb
of the chromosome-specific alpha satellite array (D8Z2). Junctions between
the arm-specific satellite region and D8Z2 are marked with blue arrows.
Dark blocks indicate the highly repetitive nature of the satellite region and
mark similarity between monomers within each satellite family. Gaps in the
dark blocks occur where interspersed elements (LINEs, SINEs and long
terminal repeats) interrupt the satellite sequences. In the alpha satellite array
dotter plot (bottom), D8Z2 from 8p (,18 kb) is joined with that of 8q
(,18 kb). The plot reveals the periodic nature of the centromeric, higher-
order alpha satellite array with black horizontal lines indicating near identity
of sequences spaced at ,1.9-kb intervals. The regions outlined in blue are
self–self alignments (‘8p’ and ‘8q’), whereas the remaining rectangular
region of the plot is an alignment of 8p versus 8q D8Z2.
NATURE|Vol 439|19 January 2006 LETTERS
333
© 2006 Nature Publishing Group
peptides crucial to the innate immune response
27
. Studies
2,3
have
suggested that defensins have been under positive selection, with a
high ratio of non-synonymous to synonymous changes detected in
the mature peptide coding exon. Moreover, gene and segmental
duplication within the cluster have led to extensive copy number
28,29
and haplotype
30
polymorphism within and across populations,
which are thought to influence variation in disease susceptibility
and contribute to ongoing adaptive evolution in both the human and
chimpanzee species. The second locus showing positive selection is
MCPH1, mutations in which cause microcephaly (Online Mendelian
Inheritance in Man (OMIM): 251200); there is clear evidence of
accelerated non-synonymous divergence correlating with the expan-
sion of brain size throughout the lineage from simian ancestors to the
human and chimpanzee
4,5
.
To investigate the diversity of copy number in the defensin clusters,
we resequenced several dozen polymerase chain reaction (PCR)
products from representative intervals from DEFB105A (beta-
defensin cluster) and DEFA1 (alpha-defensin cluster) in 14 chim-
panzees, 1 gibbon, 1 macaque and 4 breeds of dog (see Methods and
Supplementary Information). In all species studied, the gene family
has multiple members, and the members are more similar within a
species than across species. Thus, the defensin clusters have either
independently duplicated in each species or have undergone gene
conversion events within species.
Finally, we note that the majority of the genes in the region of high
divergence in distal 8p play important roles in development or
signalling in the nervous system. Notably, the extremely large
CSMD1 gene, which lies at the peak of divergence and diversity, is
widely expressed in brain tissues. High regional mutation rates and
positive selection are generally assumed to be distinct, but it is
possible that the former may facilitate the latter by increasing the
rate of appearance of potentially advantageous single, or interacting,
alleles (see also ref. 31). It is intriguing to speculate whether the
accelerated divergence rate of this region has contributed to the rapid
expansion and evolution of the primate brain.
METHODS
See Supplementary Information for details on clone path building, generation of
sequence map, sizing of gaps and gene annotation. The final version of the clone
path is available in AGP format (see http://www.ncbi.nlm.nih.gov/genome/
guide/glossary.htm) at http://www.broad.mit.edu/tools/data/data-human.html.
Gene amplification and sequencing. TBLASTN (http://www.ncbi.nlm.nih.gov/
BLAST) was used to identify DEFB105 and DEFA1 orthologues in 16 chimpan-
zees, 1 gibbon, 1 macaque and 4 dog breeds (akita, golden retriever, greyhound
and mastiff). PCR primers for gene amplification were designed using Primer3
(http://frodo.wi.mit.edu/primer3) based on the species reference sequence.
Human and macaque primers were used for gibbon. Amplified products were
cloned, and for each individual/gene combination, 48 or 96 clones were
sequenced.
Haplotype analysis. Neighbourhood Quality Standard
32
(NQS) scores were
computedfor all sequenced products using the published constraints
32
. Reads were
trimmed to the first and last three consecutive NQS bases, and aligned to the
reference sequence using PatternHunter (http://www.bioinformaticssolutions.
com). Multiple sequence alignments were built from the pairwise alignments
and inspected to find SNPs that were: at NQS bases, supported by at least two
reads, and in a ten base window where not more than two other variations were
observed. To minimize false positives due to errors during PCR amplification, we
restricted our analysis to haplotypes that differed in .3bases.
Received 5 August; accepted 6 October 2005.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank L. Gaffney for help with figures and tables;
L. French and her group at the Sanger Institute for attempting fibre FISH
analysis to size some clone gaps in the tiling path of chromosome 8; E. Eichler
and X. She for sharing their data on segmental duplications; T. Furey for help
with lists of genetic markers and placement of RefSeq genes; M. Kamal for
assistance and advice with synteny analysis; and K. Lindblad-Toh for sharing
data from the dog genome project. We also acknowledge the HUGO Gene
Nomenclature Committee (S. Povey, chair) for assigning official gene symbols.
We are deeply grateful to all the members, present and past, of the Genome
Sequencing Platform of the Broad Institute (and Whitehead Center for Genome
Research), Keio University School of Medicine and the Institute of Molecular
Biology at Jena for their dedication and for the consistent high quality of their
data that made this work possible. This work was supported by grants from the
National Human Genome Research Institute, RIKEN, the ‘Research for the
Future’ Program from the Japan Society for the Promotion of Science (JSPS), the
Ministry of Education, Culture, Sports, Science and Technology of Japan
(MEXT), the Federal German Ministry of Education, Research and Technology,
and the Thu¨ringer Kultusministerium.
Author Information Accession numbers for all clones contributing to the
finished sequence of human chromosome 8 can be found in Supplementary
Table S2. The updated human chromosome 8 sequence can be accessed
through GenBank accession number NC_000008. Reprints and permissions
information is available at npg.nature.com/reprintsandpermissions. The authors
declare no competing financial interests. Correspondence and requests for
materials should be addressed to C.N. (chad@broad.mit.edu) or N.S.
(shimizu@dmb.med.keio.ac.jp).
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