The DNA sequence and comparative analysis of human chromosome 20

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The finished sequence of human chromosome 20 comprises 59,187,298 base pairs (bp) and represents 99.4% of the euchromatic DNA. A single contig of 26 megabases (Mb) spans the entire short arm, and five contigs separated by gaps totalling 320 kb span the long arm of this metacentric chromosome. An additional 234,339 bp of sequence has been determined within the pericentromeric region of the long arm. We annotated 727 genes and 168 pseudogenes in the sequence. About 64% of these genes have a 5′ and a 3′ untranslated region and a complete open reading frame. Comparative analysis of the sequence of chromosome 20 to whole-genome shotgun-sequence data of two other vertebrates, the mouse Mus musculus and the puffer fish Tetraodon nigroviridis, provides an independent measure of the efficiency of gene annotation, and indicates that this analysis may account for more than 95% of all coding exons and almost all genes.


The finished reference sequence of the human genome is now in sight, underpinned by the recently published working draft1,2. From the outset of the Human Genome Project, the plan has been to determine the complete sequence of each chromosome to an accuracy of greater than 99.99%, and to cover more than 95% of the gene-containing part of the genome (the euchromatin). This finished ‘gold’ standard was defined and upheld on completion of the first two human chromosomes, 22 and 21 respectively. Here we report completion of the sequence of the first metacentric human chromosome, chromosome 20, to these standards. Analysis of the finished sequence has benefited from comparison with substantial new data sets that were not available at the time of the previous finished chromosome analyses. These include new collections of human and mouse messenger RNA sequences, the protein indices of fully sequenced model organisms, and extensive sequencing of two vertebrates genomes, those of the mouse and the puffer fish T. nigroviridis. As a result, we were able to assess the quality and completeness of human gene annotation by independent analyses. The application of new analytical tools has also enabled assessment of predictive methods to define transcription start sites and other features of gene structures, although these require further development and calibration with the finished annotated sequence.

Clone map and finished sequence

We identified a set of 629 minimally overlapping clones (the tiling path) that spans the euchromatic regions of the short (p) and long (q) arm of human chromosome 20. The tiling path consists of 455 P1-derived artificial chromosomes (PACs), 169 bacterial artificial chromosomes (BACs), 3 yeast artificial chromosomes (YACs), 1 cosmid and 1 polymerase chain reaction (PCR) product (Fig. 1). The euchromatic portion of the chromosome is represented in six contigs with one contig covering the entire p arm (Table 1). Boundaries between euchromatin and heterochromatin were identified by presence of satellite repeats in the sequence of clones located at the most distal and proximal ends, respectively, of the contigs flanking the centromere, and served as logical termination points for map construction. Clones located at the centromeric boundary of the p arm (Fig. 1) gave an additional signal at 20q11.1 upon fluorescent in situ hybridization (FISH) on metaphase chromosomes. We constructed an additional two-clone contig representing this duplication (Fig. 1) and postulate that it is located in the heterochromatic region of the q arm. In contrast to the p arm, four gaps remain in the clone map of the q arm. Three of them are clustered within a 1.2-Mb region at qtel (Fig. 1). We anticipate that the sequences in these gaps are unclonable to the host–vector systems used in this study, probably owing to the high guanine and cytosine (G+C) content of the sequence in this region. All four euchromatic gaps were sized by FISH of clones immediately flanking each gap to extended DNA fibres. No gap was estimated to be larger than 150 kb and all the gaps together account for no more than 320 kb of DNA (Table 1). Finally, we defined the location of both telomeres. At the end of the p arm (ptel), clone RP11-530N10 (EMBL accession code AL360078; Fig. 1) ends about 10 kb away from the block of subtelomeric repeats, which extends for 40–50 kb on the basis of the telomeric half-YAC yRM2005 (ref. 3 and H. Riethman, personal communication). A larger allelic variant of the subtelomeric repeat block is also known, half-YAC yA35 (ref. 4). At the end of the q arm (qtel), clone RP11-476I15 (AL137028; Fig. 1) contains part of the subtelomeric repeat block. Each clone of the tiling path was subjected to random subcloning and sequencing. On the basis of internal and external5 quality checks, we estimate the accuracy of our finished sequence to exceed 99.99%. Each clone has been finished according to the agreed international finishing standard for the human genome ( In total, we finished 59,421,637 bases in seven sequence contigs. The size of each sequence contig is given in Table 1; the largest one spans the 26,257,626 bp of the entire p arm. The four gaps account for 0.32 Mb (Table 1). Thus, the sequence covers 99.46% of the euchromatic part of chromosome 20, which spans 59.5 Mb. Our estimate for the total size of the chromosome, based on size estimates of 3 Mb for the centromere and 0.2 Mb for subtelomeric repeats, is 62.7 Mb, which is smaller than a previous estimate of 72 Mb (ref. 6).

Figure 1: The sequence map of human chromosome 20 and its features.

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The short (p) and the long (q) arm of the chromosome are depicted in the top and bottom panels, respectively. The features of each chromosome arm are shown from top to bottom as follows: (1) The finished sequence of each clone in the tiling path as a yellow line. Sequence positions are indicated in megabases along the x-axis of ‘G+C content’ (see 4, below). Eight of the clones were isolated and sequenced elsewhere, namely AC005808 (LBNL H136; BAC 185), AC005914 (LBNL H135; BAC 189), AC006076 (LBNL H133; PAC 12), AC004762 (LBNL H134; PAC 128), AC005220 (LBNL H80; BAC 99), AC004501 (LBNL H144; BAC 121) and AC004505 (LBNL H65; PAC 86C1) at the Joint Genome Institute16 and AC006198 (RP11-3A1) at the Whitehead Institute (Massachusetts Institute of Technology Center for Genome Research). The centromere has been arbitrarily drawn to span 3 Mb. The exact location of contig AL121762–AL441988 in the heterochromatic region of the q arm is not known. Gaps in the map appear as greenish bars. The width of the bar represents the size estimate obtained by fibre FISH. (2) The location of genetic markers. (3) The distribution of the main types of repeats in the sequence. (4) Plot of the G+C content of the sequence. (5) Plot of the SNP density along the sequence. (6) The location of predicted CpG islands. (7) The location of the annotated gene structures. Right and left coloured arrows indicate gene structures on the + and - strand, respectively. The most 3′ end of each gene is drawn halfway along the arrowhead. Only the genes of the annotation group 1 (known; dark blue) and 2 (novel; blue) are named. When no gene symbol is available, the gene name used in the EMBL sequence submission file appears (for example, dJ583P15.4). CDS, protein-coding sequence.

Table 1 Sequence contigs on chromosome 20

Gene index of chromosome 20

The finished genomic sequence was first analysed for G+C content and CpG islands. Interspersed and simple tandem repeats in the sequence were then masked and the masked sequence was compared against protein, DNA and expressed sequence tags (ESTs) using BLASTX and BLASTN7. In parallel, gene structures were predicted ab initio in the masked sequence on a clone-by-clone basis with the programs FGENESH8 and GENSCAN9.

A total of 895 gene structures was annotated in the finished sequence on the basis of human interpretation of the combined supportive evidence generated during sequence analysis (see Fig. 1). The structures were divided into five groups: (1) 335 ‘known’ genes, that is, those that are identical to known human complementary DNA or protein sequences (all known genes were in the LocusLink database,; (2) 222 ‘novel genes’, that is, those that have an open reading frame (ORF), are identical to human ESTs that splice into two or more exons, and/or have homology to known genes or proteins (all species); (3) 23 ‘novel transcripts’, that is, genes as in 2 but for which a unique ORF cannot be determined; (4) 147 ‘putative genes’, that is, sequences identical to human ESTs that splice into two or more exons but without an ORF; and (5) 168 ‘pseudogenes’, that is, sequences homologous to known genes and proteins but with a disrupted ORF.

Excluding the pseudogenes, chromosome 20 has a gene density of 12.18 per Mb, which is intermediate to 6.71 (low) and 16.31 per Mb (high) reported for chromosome 21 and 22, respectively10,11. We used the gene density of chromosomes 20, 21 and 22 from ref. 12 to adjust the number of genes on each of these chromosomes. The adjusted figures were then used to extrapolate a number of 31,500 genes for the whole genome, which is in agreement with recent estimates1,2.

The analysis of chromosome 20 benefited from the availability of new large data sets to assist the gene annotation. These included human (for example, Genoscope) and mouse ESTs and ‘full-length’ cDNAs (for example, RIKEN mouse cDNA collection) as well as the protein indices of fully sequenced model organisms such as Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana. Some 81% of the 557 genes in groups 1 and 2 (and 64% of all the annotated genes), have a full ORF as defined by a starting ATG codon and the presence of a 5′ and a 3′ untranslated region (UTR). Often a stretch of nucleotides immediately preceding the starting ATG seems to be part of the ORF. When the supporting evidence (for example, ESTs) terminated within such a stretch, we did not annotate a 5′ UTR. Such genes were also included (8.9% of the 557 genes) in the above set.

The transcription start sites of most genes in the human genome are not yet known. We carried out several analyses to assist the annotation of the 5′ ends of as many genes on chromosome 20 as possible. Analysis of the unmasked sequence predicted a total of 660 CpG islands, of which 389 are located near (5 kb upstream or 1 kb downstream) the first exon of an annotated gene structure. Many of the remaining predicted CpG islands have intragenic locations, which in our view does not allow a direct correlation between the observed number of CpG islands and the number of genes on chromosome 20. Among the genes with complete structures, 303 (67%) are associated with a CpG island at their 5′ ends, which is in good agreement with the previously reported figure of 60% (ref. 13). We also scanned the sequence of chromosome 20 for putative transcription start (TS) sites using the probabilistic TS site detector program Eponine (T. Down, unpublished). Eponine is optimized for mammalian genomic DNA sequences and detects likely TS sites on the basis of the surrounding sequence (typically 500 bases upstream to 100 bases downstream). Multiple predictions are often clustered, suggesting alternative TS sites for a gene. Eponine has a detection sensitivity of 40%, on the basis of an analysis of human chromosome 22. We found 1,432 TS sites on chromosome 20, of which 492 (34%) are located within 2 kb of the first exon of an annotated gene. In the set of genes with complete structures, 402 TS sites are associated with 166 genes (37.5%) of which 159 (95.8%) have a CpG island at their 5′ end. So, Eponine predicts multiple TS sites per gene (the mean value is 2.42 in the 166 genes) and has a bias in predicting TS sites in genes associated with a CpG island at their 5′ end.

The 727 genes (that is, introns and exons) extend over a total of 25,213,914 bp (mean 34,682 bp per gene). Excluding expressed pseudogenes, 42.4% of the reported sequence of chromosome 20 is therefore transcribed. Exons account for only 2.43% of the sequence and the mean exon size is 283 bp. A summary per gene group is given in Table 2, which includes figures reported from the analyses of chromosomes 21 and 22 (refs 10, 11). Gene size varies substantially, from 1,234,386 bp (gene C20orf133 (AL117333–AL049633), which is similar to a low-density lipoprotein-related protein, LRP16) to 339 bp (gene C20orf127 (AL121753)). Exon sizes are fairly constant, with the exception of 3′ terminal exons (for example, 8,181 bp in PTPRT), in contrast to intron sizes, which vary from 33 bp (C20orf97 (AL034548)) to 523,790 bp (CDH4).

Table 2 Structural characteristics of annotated gene structures

For 209 (29%) of the annotated genes, we found alternative splice forms. Alternative splicing can, for example, give rise to two distinct peptides by exclusion of the exons encoding a functional domain from one but not the other transcript. The transcript for the soluble form of attractin (ATRN; AL353193–AL132773) lacks the five exons that encode the transmembrane and cytoplasmic domains and are present in the transcript that encodes the membrane form of the protein. Splice variants may encode structurally unrelated peptides. A complex example of alternative splicing and genetic imprinting is found in the GNAS1 locus (AL132655–AL109840). NESP55 and XLAS are transcribed from distinct mono-allelic promoters located upstream of the bi-allelic promoter that drives the transcription of the gene for the α-subunit of the stimulatory guanine-nucleotide-binding protein Gs. G is encoded by exons 1–13. A large G protein, XLαs, is generated by in-frame splicing of an upstream exon (bp 120,789–121,953; AL132655) to exon 2 (bp 39,075–39,119; AL121917) of G. An additional exon located further upstream (bp 106,368–107,508; AL132655) splices again to exon 2 of G but not in frame, giving rise to a structurally unrelated peptide, NESP55. An antisense transcript (dJ806M20.3.6; AL132655) postulated to regulate this imprinted region has also been reported14. In total, we annotated six isoforms of GNAS1. One gene (PLCB4; AL121898–AL031652) was found to have the most isoforms (eight); in most cases of genes with alternative splicing (130 genes) we observed two isoforms. Typically, we annotated the longest possible terminal exon and did not create entries for alternative splice forms on the basis of alternative polyadenylation sites. If we exclude the putative genes that have mainly incomplete structures, then 35% of the genes (average of 1.65 transcripts per gene) show alternative splicing. This is in agreement with previous estimates15. Analysis of chromosomes 19 and 22 (ref. 1), both gene-rich chromosomes, showed a higher extent of alternative splicing.

Protein index of chromosome 20

We analysed the proteome of chromosome 20 using InterProScan ( to look at the distribution of known protein domains. The InterPro database combines information on protein families, domains and functional sites from the databases Pfam, PRINTS, PROSITE, SMART and SWISS-PROT (see for links). Of all proteins encoded on chromosome 20, 73.5% have an InterPro match and 30% are multidomain with an average of 2.1 distinct InterPro domains. As shown in Table 3, many of the most frequent domains in the chromosome 20 proteome rank in similar order as in the human proteome1. There are, however, five domains for which chromosome 20 seems enriched. Four of them—the cysteine proteases inhibitor (IPR000010), the immunoglobulin subtype (IPR003599), the whey acidic protein (WAP)-type ‘four-disulphide core’ domain (IPR00222), and the pancreatic trypsin inhibitor (Kunitz/Bovine) domain (IPR002223)—are found in proteins encoded by three gene clusters along the chromosome.

Table 3 Most common InterPro domains in the chromosome 20 proteome and their abundance in other species

Functionally related gene clusters indicate probable ancestral gene-duplication events. The first cluster, at 1.5 Mb (Fig. 1), extends from AL109658 to AL034562 and includes genes with immunoglobulin and immunoglobulin-like domains that are involved in signal transduction and cell adhesion (SIRPB1, SIRPB2 and PTPNS1). The annotated genes PTPN1L and PTPNS1L2 and pseudogenes dJ576H24.1 and dJ673D20.1 are new members of this gene family. Interestingly, the apparently functional gene PTPNS1L2 is located within a larger fragment of 33,048 bp (AL049634 and AL592544), which is an insertion type of polymorphism. The insertion allele is represented in the RP4 PAC library but not the RP11 BAC library. Using a panel of 174 Caucasians, we estimated that the frequency of the insertion allele is 37.3%. The second cluster, at 23.5 Mb (AL096677–AL121831; Fig. 1), comprises members of the cystatin gene family, which encode protease inhibitors with antibacterial and antiviral activities. An additional member, CST7, is located about 1 Mb distal of the main cluster (AL035661). Only two of the known cystatins are not on chromosome 20 (IPR003243; Table 3). We annotated three new members (CST8L, CSTL1 and CST9L) and two pseudogenes. The third cluster, at 43.5 Mb (AL049767–AL050348; Fig. 1), includes 11 genes that encode proteins with a WAP-type four-disulphide core domain (IPR002221) and/or a pancreatic trypsin inhibitor (Kunitz/Bovine) domain (IPR002223). A fourth cluster that includes genes for the semenogelins SEMG1 and SEMG2 (semen proteins involved in reproduction) is located within the third gene cluster between members PI3 and SLPI, which have only a WAP-type domain.

Chromosome landscape

The sequence of chromosome 20 has an average G+C content of 44.1%, which is slightly higher than the genome average of 41%. The distribution of the G+C content fluctuates along the chromosome, and regions with higher G+C have a higher gene density (Fig. 1). For example, the sequence from 49.5 to 54 Mb has an average G+C content of 41.1% and a gene density of only 4.9 genes per Mb, in contrast to the region between 60 and 62.5 Mb, which has 56.6% G+C content and a gene density of 28 genes per Mb. Given a sequence length and gene ratio of 1.25 and 1.65, respectively, between the q and the p arm, the q arm seems rich in genes. Gene density can drop as low as 1.54, for instance in a 1.9-Mb region between AL136990 and AL139163. Interestingly, the largest genes, such as PTPRT, PLCB1 and dJ631M13.5 are located adjacent to or within gene-poor regions.

The repeat content of chromosome 20 is 42%. The distribution of the main classes of repeats (detailed in Supplementary Information) is shown in Fig. 1. Regions of high gene density seem enriched in short interspersed elements (SINEs).

Segmental duplications are another interesting feature of the genome. We compared the masked sequence of chromosome 20 with the rest of the genome and with itself to identify inter- and intrachromosomal duplications, respectively. The segments of chromosome 20 involved in interchromosomal duplications (Fig. 2) often contain pseudogenes; for example, at 6 Mb, AL359954 contains a pseudogene similar to TRDBP that maps to 1p36 (AL109811). The region at 53.9 Mb (Fig. 2) that is duplicated in chromosomes 21 and 22 was recently described as part of a breast cancer amplicon16. A region of about 500 kb between 25.8 and 26.3 Mb is implicated in both types of segmental duplications. A core region of 100 kb that harbours a copy of exon 7 of the CFTR gene is duplicated on chromosome 20. The second copy is located in AL121762–AL441988 at the pericentromeric region of the q arm. Copies of the extended region seem to be present on chromosomes 9, 12, 15, 17 and 19 (Fig. 2). Secondary signals in the pericentromeric regions of these and other chromosomes were also observed on FISH analysis of clones AL078587 and AL121762. It will be interesting to investigate whether the gene structures annotated in AL121762 and AL441988 are expressed genes, particularly C20orf80, which is similar to the FRG1 gene. FRG1 is located 100 kb centromeric of the repeat units on chromosome 4q35, which are deleted in facioscapulohumeral muscular dystrophy. The region from 48.2 to 48.8 Mb is bordered by two copies of a 60-kb intrachromosomal duplication.

Figure 2: Duplication landscape of chromosome 20.

Intrachromosomal and interchromosomal duplications are shown in blue and red, respectively. Each horizontal line represents 1 Mb of the sequence from the telomeric end of the short arm (top left) to the telomeric end of the long arm (bottom right). The gap indicates the centromeric region. Pairwise alignments generated by Exonerate and longer than 1 kb are shown.

The integrated Marshfield male, female and sex-averaged genetic maps of chromosome 20 (ref. 17) were aligned to the physical map (Fig. 3). The steepest increase in recombination frequency is observed between markers D20S178 and D20S176, which are both located in the region of duplication described above. A region of very low recombination extends for about 20 Mb between markers D20S432 and D20S859. The rate of recombination in specific loci differs between the two sexes. Compared with that of the female, the rate of male recombination is higher along the p arm up to marker D20S432 and lower across the rest of the chromosome (Fig. 3).

Figure 3: Alignment of the genetic map of chromosome 20 to the physical map.

The two maps are aligned from the telomeric end of the short arm to the telomeric end of the long arm. The position of each genetic marker on the female, the male and the sex-averaged genetic map is indicated.

Sequence variation

The definition of the common ancestral haplotypes that are present in the population relies on the availability of an extensive collection of single nucleotide polymorphisms (SNPs). We first placed 26,678 SNPs (deposited in the dbSNP database, on the sequence of chromosome 20. Of those, 13,016 were derived from sequence analysis of clone overlaps by the program ssahaSNP18. To recover additional SNPs in clone overlaps, we realigned all available clone-based shotgun sequences from chromosome 20 (including unfinished sequence in clone overlaps that was previously archived and therefore excluded from the earlier analysis) onto the finished sequence with ssahaSNP, and detected 11,050 SNPs (submitted to dbSNP). Merging the two data sets resulted in 32,763 unique SNPs on chromosome 20 (Fig. 1), of which 6,085 are new. In the unique set, there are 14,211 SNPs (43.4%) located within annotated genes and 3,061 of them are in exons.

Comparative analysis

Functional features such as exons and regulatory elements have been conserved through evolution and there is compelling evidence that comparative genomic sequence analysis is a powerful tool in the quest to complete the structural annotation of the human genome. Two data sets were available in the public domain at the time of analysis: about 13 million sequence reads of a mouse whole-genome shotgun giving an estimated genome coverage of 2.3-fold (, released by the Mouse Sequencing Consortium on 8 May 2001; and 816,262 single sequence reads from BAC and plasmid ends of the T. nigroviridis genome, totalling 663,839,518 bases and corresponding to 1.72 genome equivalents, generated at Genoscope. Thus we undertook the comparative analysis of the finished and annotated sequence of an entire human chromosome against two vertebrate genomes.

Mouse sequences were aligned to the sequence of chromosome 20 using Exonerate version 0.3d (Guy StC. Slater, unpublished). We obtained matches with 63,644 mouse sequences representing 12,041 regions of sequence conservation (RSC) along chromosome 20. Tetraodon sequences were aligned at Genoscope by Exofish (‘exon finding by sequence homology’), which generates ecores (evolutionary conserved regions)19. Matches were obtained with 2,992 ecores (available at We first examined the annotated gene structures; 77.4% of the 727 genes and 89% of the 168 pseudogenes have at least one exon matched by a mouse RSC or Tetraodon ecore. This figure is much higher for the 557 genes in groups 1 and 2 (94%) than it is for the ‘putative’ gene structures in group 4 (33%). Furthermore, the two sets differ in the ratio of genes with only a mouse RSC to genes with both an RSC and ecore match: 1:3.8 and 1:0.2, respectively. These observations suggest that the putative gene structures may represent largely UTRs, which have sequences known to be less well conserved between species, and possibly genes that appeared later in evolution.

We then looked at matches outside annotated exons as a way to assess the completeness of the current annotation. Such matches may correspond to exonic sequences that have not been annotated in the present study owing to lack of supporting evidence (for example, EST, cDNA and protein homologies). Note that we did not use RSCs and ecores during the annotation process. We found 5,447 RSC and 207 ecore matches, and 60 of these non-exonic regions are conserved in all three species. Of all annotated exons (including pseudogenes), 2,050 (36.3%) contain a region conserved in all three species (in contrast to 0.2% of the annotated introns). Thus, we postulate that about 97.2% (2,050 / (2,050 + 60)) of all coding exons of chromosome 20 have been annotated in this study. A caveat is whether the set of annotated genes used in this analysis is representative of genes that appeared recently in evolution, as ecores are biased to more conserved genes; however, we consider that such an effect cannot be substantial.

The 639 and 4,808 RSC matches in annotated introns and intergenic regions, respectively, suggest that although the mouse data set provides better coverage (70% of all exons) than the ecores, exonic sequences cannot be readily identified by simple comparison at the DNA level.

GENSCAN can be used on small segments of genomic sequence to effectively evaluate the likelihood of that segment containing an exon. We performed a GENSCAN analysis on ‘extended RSCs’, which included 100 bp of human sequence either side of the RSC match, to divide them into those that were more likely to be coding regions of sequence conservation (cRSC) and those more likely to be noncoding. This predicted 3,299 cRSCs and 8,836 noncoding RSCs and found that 65.7% of cRSCs match annotated exons (3.8% are within introns). As a result, the 874 cRSCs found between annotated genes is a set enriched in regions that may represent non-annotated exons (there are 4,748 RSC matches in intergenic regions).

Conclusion and medical implications

We sequenced the euchromatic portion of human chromosome 20 leaving four small gaps that account for no more than 320 kb. In the 59,421,637 bp of sequence, we annotated 727 gene structures of which 64% are complete and 168 pseudogenes. A comparison of this product with the draft assembly of chromosome 20 reported earlier this year2 clearly shows the importance of generating a contiguous finished reference sequence for each human chromosome. Both the G+C and gene density plots of chromosome 20 peak between 60 and 62.5 Mb (Fig. 1) at the qtel region, which is in sharp contrast to the corresponding plots shown in Fig. 11 in ref. 2, which peak at least 12 Mb proximal of the telomere. Furthermore, the order in which genes are shown in the magnified part of Fig. 13 in ref. 2 is incorrect. For example, the gene OSBPL2 (oxysterol binding protein 2) and bB379O24.1 (GATA5 related) are located at 60.2–60.6 Mb and cannot map between PTPRT (protein tyrosine phosphatase, receptor type) at 41 Mb and ZNF217 (Kruppel-like transcription factor) at 51.7 Mb. The use of the clone map information was instrumental in resolving similar problems during the assembly of the chromosome 20 draft sequence.

The output of the comparative analysis of chromosome 20 from the mouse whole-genome shotgun and the ecores generated from the Tetraodon genomic sequence suggests that the current sets of human and mouse ESTs and ‘full-length’ cDNAs together with the proteomes of model organisms are adequate to allow the identification of the vast majority of human genes in the sequence. As expected, we found that comparative analysis can be used to reliably identify exonic sequences. The mouse shotgun data alone cannot be used reliably to postulate the number of non-annotated exons, owing to the overall higher degree of sequence conservation. The use of the two data sets together, however, provides an excellent tool for assisting the identification of new, and the completion of existing, gene structures. In the present study, the ability to identify regulatory elements in the sequence of chromosome 20 by comparison to the mouse sequence data can be substantiated only by anecdotal evidence. A three-way comparison with the addition of the genome sequence of a species more closely related to humans may hold the key in this endeavour20.

Chromosome 20 is best known for harbouring the genes that cause Creutzfeldt–Jakob disease (PRNP) and severe combined immunodeficiency (ADA). However, the causes of the sporadic cases of Creutzfeldt–Jakob disease (80% of all cases) remain unknown, and no mutation in the ADA gene has been identified to explain the phenotype of ADA excess in haemolytic anaemia. Furthermore, there are still single-gene disorders mapped to chromosome 20 ( for which the underlying genetic defect is not known. The resources generated by the Human Genome Project have already been used to accelerate the cloning of disease genes on chromosome 20; the Alagille (JAG1)21, McKusick–Kaufman (MKKS)22, ICF (DNMT3B)23 and Hallervorden–Spatz (PANK2)24 syndromes are recent examples. The reported finished and annotated sequence and its variation will be a valuable tool in tackling not only the remaining single-gene diseases but also the multifactorial diseases that have been linked to chromosome 20, such as type 2 diabetes, obesity, cataract, eczema and Grave's disease. Evidence for a susceptibility locus for hereditary prostate cancer on 20q13 has also been reported25,26. In addition to the sequence itself, the isolated clones used in the sequencing process constitute a unique resource in studying chromosome loss and/or amplification in various types of cancer. We have recently reported the refinement of a commonly deleted region (CDR) of 20q12-13.1 found in patients with myeloproliferative disorders and myelodisplastic syndromes27. Others have reported the characterization of a breast cancer amplicon at 20q13.2 (ref. 16), whereas several studies have reported loss of heterozygosity across regions of 20q using comparative genomic hybridization28,29.


Clone map and sequence assembly

Clone map construction is described in ref. 30. Mapped sequence tagged sites (STSs) for screening genomic PAC and BAC libraries were selected from the integrated radiation hybrid map constructed for chromosome 20 (, which harbours 1,493 STS-based markers. In regions with no clone coverage, screening was extended to the CEPH (Centre d’Etude du Polymorphisme Humain), ICRF (Imperial Cancer Research Fund) and ICI (Imperial Chemical Industries) YAC libraries and the LANL (Los Alamos National Laboratory) chromosome-20-specific cosmid library (links for the libraries can be found at For the shotgun phase, pUC plasmids with inserts of 1.4–2 kb were sequenced from both ends by the dideoxy chain termination method31 with big dye terminator chemistry32. Most of the reactions were analysed on ABI3700 capillary sequencing machines. The resulting data were processed by a suite of in-house programs ( before assembly with the PHRED33,34 and PHRAP ( algorithms. For the finishing phase, we used the GAP4 program35 to help assess, edit and select reactions, eliminate ambiguities and close sequence gaps. Sequence gaps were closed by a combination of primer walking, PCR, short/long insert sublibraries36, sublibrary screening with oligonucleotides and, in rare cases, transposon sublibraries.

Sequence analysis tools

Interspersed and simple tandem repeats were identified with Repeatmasker ( and etandem (, respectively. BLAST 1.4 (default parameters and matrix; was used to identify initial matches, which were then re-aligned by EST_GENOME37. BLASTN was used with a 65% similarity cutoff in the comparison against the RIKEN mouse cDNA set38 instead of 95%, which is used when searching human ESTs, to find significant matches. In the unmasked sequence, CpG islands were predicted by searching for sequence segments that are at least 400 bp, have a G+C content greater than 50%, and an expected/observed CpG count of greater than 0.6. The completed analysis was assembled into contigs and visualiszed in AceDB (, whereas an Ensembl ( database of the sequence assembly and the annotated genes was constructed and used for calculation of statistics and producing Fig. 1. In SNP analysis, only those regions of chromosome 20 where a SNP was detected by at least four reads was considered valid, since the depth of shotgun sequencing for these clones was greater than 4×. The phred-quality value of at least four of the reads at the SNP location had to be at least 30 (error probability of phred base calling 0.001 or less). Exonerate was run with an initial word length of 14 bp, gap penalties of 8 for opening a gap and 4 for extending one, and a score of 5 and -4 for DNA matches and mismatches, respectively.


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All the reported DNA sequences have been deposited to the EMBL, GenBank or DDBJ databases. We thank H. Roest Crollius, O. Jaillon and the Tetraodon group at Genoscope for the Exofish data; E. Zdobnov for the InterPro analysis; the Ensembl team for assistance in the computational analysis; and the group of M. S. Jackson at the University of Newcastle upon Tyne for markers and clones that assisted the mapping of the pericentromeric duplication. Finally, we thank H. Wain, E. Bruford, M. Lush, R. Lovering, M. Wright, P. Shetty and S. Povey for help with gene nomenclature and the Wellcome Trust for financial support.

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Correspondence to P. Deloukas.

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