The International Human Genome Sequencing Consortium (IHGSC) recently completed a sequence of the human genome1. 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 segmentally duplicated, chromosome 8 is distinctly typical in character, being very close to the genome median in each of these aspects. This work describes a finished sequence and gene catalogue 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 selection2—these include the major defensin (DEF) gene cluster3,4 and MCPH15,6, a gene that may have contributed to the evolution of expanded brain size in the great apes. The data from chromosome 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 heterochromatin (Fig. 1 and Supplementary Table S1). These gaps are refractory to current cloning and mapping technology. The estimated 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 average1,7,8,9,10,11. 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 sequence7. Errors in the clone path were detected by identifying discrepancies between the predicted and observed distances between Fosmid ends7. 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 bases12 (J. Schmutz, personal communication).
Several analyses support the idea that nearly the entire euchromatic region of chromosome 8 is present and accurately represented. From the well-curated RefSeq13 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 map14 (Supplementary Fig. S1). Among 247 sequence-based genetic markers (Supplementary 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 population15,16 (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) map17 (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 scheme18, 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 reports8,9,10,11,19, 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 reports8,9,11,20. Of the 301 pseudogenes on chromosome 8, ∼84% are processed pseudogenes arising from retrotransposition; the remaining 16% are unprocessed. We also identified 13 tRNA genes (Supplementary 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-1, below the genome average of ∼10 genes Mb-1. Gene distribution is highly heterogeneous, with 44 gene deserts (500 kb without a coding gene, Supplementary Table S7) that together comprise 41.9 Mb or ∼29% the total length. The overall G + C content is 39.2%, but varies substantially across the chromosome (Fig. 1). Nearly half of the chromosome is composed 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 X20) for which sequences span the entire pericentromeric region. The regions on both arms stretch from unique euchromatin through pericentromeric 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. 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, dog23 and mouse24 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 A + T content25,26. The region on chromosome 8p has all three factors. The mean sex-averaged recombination rate across the first 6 Mb is 2.7 cM Mb-1, 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% A + T, as compared to a genome-wide average of 59%. It is unusual in this regard, because subtelomeric regions with high recombination rates are typically (A + T)-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 peptides crucial to the innate immune response27. Studies2,3 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 number28,29 and haplotype30 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 expansion of brain size throughout the lineage from simian ancestors to the human and chimpanzee4,5.
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 chimpanzees, 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.
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 chimpanzees, 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.
Neighbourhood Quality Standard32 (NQS) scores were computed for all sequenced products using the published constraints32. 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 >3 bases.
International Human Genome Sequencing Consortium. Initial sequencing and analysis of the human genome. Nature 409, 860–921 (2001)
Vallender, E. J. & Lahn, B. T. Positive selection on the human genome. Hum. Mol. Genet. 13 (suppl. 2), R245–R254 (2004)
Maxwell, A. I., Morrison, G. M. & Dorin, J. R. Rapid sequence divergence in mammalian β-defensins by adaptive evolution. Mol. Immunol. 40, 413–421 (2003)
Xiao, Y. et al. A genome-wide screen identifies a single β-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins. BMC Genom. 5, 56 (2004)
Evans, P. D., Anderson, J. R., Vallender, E. J., Choi, S. S. & Lahn, B. T. Reconstructing the evolutionary history of microcephalin, a gene controlling human brain size. Hum. Mol. Genet. 13, 1139–1145 (2004)
Evans, P. D. et al. Microcephalin, a gene regulating brain size, continues to evolve adaptively in humans. Science 309, 1717–1720 (2005)
International Human Genome Sequencing Consortium. Finishing the euchromatic sequence of the human genome. Nature 431, 931–945 (2004)
Grimwood, J. et al. The DNA sequence and biology of human chromosome 19. Nature 428, 529–535 (2004)
Deloukas, P. et al. The DNA sequence and comparative analysis of human chromosome 10. Nature 429, 375–381 (2004)
Martin, J. et al. The sequence and analysis of duplication-rich human chromosome 16. Nature 432, 988–994 (2004)
Nusbaum, C. et al. DNA sequence and analysis of human chromosome 18. Nature 437, 551–555 (2005)
Schmutz, J. et al. Quality assessment of the human genome sequence. Nature 429, 365–368 (2004)
Pruitt, K. D., Tatusova, T. & Maglott, D. R. NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 33, D501–D504 (2005)
Kong, A. et al. A high-resolution recombination map of the human genome. Nature Genet. 31, 225–226 (2002)
Giglio, S. et al. Olfactory receptor-gene clusters, genomic-inversion polymorphisms, and common chromosome rearrangements. Am. J. Hum. Genet. 68, 874–883 (2001)
Shimokawa, O. et al. Molecular characterization of inv dup del(8p): analysis of five cases. Am. J. Med. Genet. A 128, 133–137 (2004)
Deloukas, P. et al. A physical map of 30,000 genes. Science 282, 744–746 (1998)
Ashurst, J. L. et al. The Vertebrate Genome Annotation (Vega) database. Nucleic Acids Res. 33, D459–D465 (2005)
Hillier, L. W. et al. Generation and annotation of the DNA sequences of human chromosomes 2 and 4. Nature 434, 724–731 (2005)
Ross, M. T. et al. The DNA sequence of the human X chromosome. Nature 434, 325–337 (2005)
Ge, Y., Wagner, M. J., Siciliano, M. & Wells, D. E. Sequence, higher order repeat structure, and long-range organization of alpha satellite DNA specific to human chromosome 8. Genomics 13, 585–593 (1992)
Reich, D. E. et al. Human genome sequence variation and the influence of gene history, mutation and recombination. Nature Genet. 32, 135–142 (2002)
Lindblad-Toh, K. et al. Genome sequence, comparative analysis and haplotype structure of the domestic dog. Nature 438, 803–819 (2005)
Mouse Genome Sequencing Consortium, Initial sequencing and comparative analysis of the mouse genome. Nature 420, 520–562 (2002)
The Chimpanzee Sequencing and Analysis Consortium. Initial sequence of the chimpanzee genome and comparison with the human genome. Nature 437, 69–87 (2005)
Hellmann, I. et al. Why do human diversity levels vary at a megabase scale? Genome Res. 15, 1222–1231 (2005)
Lehrer, R. I. Primate defensins. Nature Rev. Microbiol. 2, 727–738 (2004)
Hollox, E. J., Armour, J. A. & Barber, J. C. Extensive normal copy number variation of a β-defensin antimicrobial-gene cluster. Am. J. Hum. Genet. 73, 591–600 (2003)
Mars, W. M. et al. Inheritance of unequal numbers of the genes encoding the human neutrophil defensins HP-1 and HP-3. J. Biol. Chem. 270, 30371–30376 (1995)
Taudien, S. et al. Polymorphic segmental duplications at 8p23.1 challenge the determination of individual defensin gene repertoires and the assembly of a contiguous human reference sequence. BMC Genom. 5, 92 (2004)
Wyckoff, G. J., Malcom, C. M., Vallender, E. J. & Lahn, B. T. A highly unexpected strong correlation between fixation probability of nonsynonymous mutations and mutation rate. Trends Genet. 21, 381–385 (2005)
Altshuler, D. et al. An SNP map of the human genome generated by reduced representation shotgun sequencing. Nature 407, 513–516 (2000)
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 Thüringer Kultusministerium.
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.
This file contains Supplementary Methods (mapping, sequencing and annotation; Defensin gene sequencing), Supplementary Results (gaps in the clone path, extreme genes, CSMD gene family, structure of the chromosome 8 pericentromeric region) and a Supplementary Discussion of annotation rules and methods, comparison of manual annotations of selected regions of human chromosome 8 and alternative explanations for apparent rapid divergence on 8p22-23. (DOC 56 kb)
This file contains Supplementary Tables 1–8. (DOC 2643 kb)
Relationship of the finished sequence map to genetic and radiation hybrid maps of chromosome 8. (PDF 286 kb)
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