Initial sequencing and analysis of the human genome - Vitagenes

articlesbetween ®sh and human). These analyses also suggest around30,000 human genes.Extrapolations have also been made from the gene counts forchromosomes 21 and 22 (refs 93, 94), adjusted for differencesin gene densities on these chromosomes, as inferred from ESTmapping. These estimates are between 30,500 and 35,500, dependingon the precise assumptions used 286 .Insights from invertebrates. The worm and ¯y genomes contain alarge proportion of novel genes (around 50% of worm genes and30% of ¯y genes), in the sense of showing no signi®cant similarity toorganisms outside their phylum 293±295 . Such genes may have beenpresent in the original eukaryotic ancestor, but were subsequentlylost from the lineages of the other eukaryotes for which sequence isavailable; they may be rapidly diverging genes, so that it is dif®cult torecognize homologues solely on the basis of sequence; they mayrepresent true innovations developed within the lineage; or theymay represent acquisitions by horizontal transfer. Whatever theirorigin, these genes tend to have different biological properties fromhighly conserved genes. In particular, they tend to have low expressionlevels as assayed both by direct studies and by a paucity ofcorresponding ESTs, and are less likely to produce a visible phenotypein loss-of-function genetic experiments 294,296 .Gene prediction. Current gene prediction methods employ combinationsof three basic approaches: direct evidence of transcriptionprovided by ESTs or mRNAs 297±299 ; indirect evidence based onsequence similarity to previously identi®ed genes and proteins 300,301 ;and ab initio recognition of groups of exons on the basis of hiddenMarkov models (HMMs) that combine statistical informationabout splice sites, coding bias and exon and intron lengths (forexample, Genscan 275 , Genie 302,303 and FGENES 304 ).The ®rst approach relies on direct experimental data, but issubject to artefacts arising from contaminating ESTs derived fromunspliced mRNAs, genomic DNA contamination and nongenictranscription (for example, from the promoter of a transposableelement). The ®rst two problems can be mitigated by comparingtranscripts with the genomic sequence and using only those thatshow clear evidence of splicing. This solution, however, tends todiscard evidence from genes with long terminal exons or singleexons. The second approach tends correctly to identify gene-derivedsequences, although some of these may be pseudogenes. However, itobviously cannot identify truly novel genes that have no sequencesimilarity to known genes. The third approach would suf®ce alone ifone could accurately de®ne the features used by cells for generecognition, but our current understanding is insuf®cient to doso. The sensitivity and speci®city of ab initio predictions are greatlyaffected by the signal-to-noise ratio. Such methods are moreaccurate in the ¯y and worm than in human. In ¯y, ab initiomethods can correctly predict around 90% of individual exons andcan correctly predict all coding exons of a gene in about 40% ofcases 303 . For human, the comparable ®gures are only about 70% and20%, respectively 94,305 . These estimates may be optimistic, owing tothe design of the tests used.In any collection of gene predictions, we can expect to see variouserrors. Some gene predictions may represent partial genes, becauseof inability to detect some portions of a gene (incomplete sensitivity)or to connect all the components of a gene (fragmentation);some may be gene fusions; and others may be spurious predictions(incomplete speci®city) resulting from chance matches or pseudogenes.Creating an initial gene index. We set out to create an initialintegrated gene index (IGI) and an associated integrated proteinindex (IPI) for the human genome. We describe the results obtainedfrom a version of the draft genome sequence based on the sequencedata available in July 2000, to allow time for detailed analysis of thegene and protein content. The additional sequence data that hassince become available will affect the results quantitatively, but areunlikely to change the conclusions qualitatively.We began with predictions produced by the Ensembl system 306 .Ensembl starts with ab initio predictions produced by Genscan 275and then attempts to con®rm them by virtue of similarity toproteins, mRNAs, ESTs and protein motifs (contained in thePfam database 307 ) from any organism. In particular, it con®rmsintrons if they are bridged by matches and exons if they are ¯ankedby con®rmed introns. It then attempts to extend protein matchesusing the GeneWise computer program 308 . Because it requirescon®rmatory evidence to support each gene component, it frequentlyproduces partial gene predictions. In addition, when thereis evidence of alternative splicing, it reports multiple overlappingtranscripts. In total, Ensembl produced 35,500 gene predictionswith 44,860 transcripts.To reduce fragmentation, we next merged Ensembl-based genepredictions with overlapping gene predictions from anotherprogram, Genie 302 . Genie starts with mRNA or EST matches andemploys an HMM to extend these matches by using ab initiostatistical approaches. To avoid fragmentation, it attempts to linkinformation from 59 and 39 ESTs from the same cDNA clone andthereby to produce a complete coding sequence from an initial ATGto a stop codon. As a result, it may generate complete genes moreaccurately than Ensembl in cases where there is extensive ESTsupport. (Genie also generates potential alternative transcripts,but we used only the longest transcript in each group.) Wemerged 15,437 Ensembl predictions into 9,526 clusters, and thelongest transcript in each cluster (from either Genie or Ensembl)was taken as the representative.Next, we merged these results with known genes contained in theRefSeq (version of 29 September 2000), SWISSPROT (release 39.6of 30 August 2000) and TrEMBL databases (TrEMBL release 14.17of 1 October 2000, TrEMBL_new of 1 October 2000). Incorporatingthese sequences gave rise to overlapping sequences because ofalternative splice forms and partial sequences. To construct anonredundant set, we selected the longest sequence from eachoverlapping set by using direct protein comparison and by mappingthe gene predictions back onto the genome to construct the overlappingsets. This may occasionally remove some close paralogues inthe event that the correct genomic location has not yet beensequenced, but this number is expected to be small.Finally, we searched the set to eliminate any genes derived fromcontaminating bacterial sequences, recognized by virtue of nearidentity to known bacterial plasmids, transposons and chromosomalgenes. Although most instances of such contamination hadbeen removed in the assembly process, a few cases had slippedthrough and were removed at this stage.The process resulted in version 1 of the IGI (IGI.1). Thecomposition of the corresponding IPI.1 protein set, obtained bytranslating IGI.1, is given in Table 22. There are 31,778 proteinpredictions, with 14,882 from known genes, 4,057 predictions fromEnsembl merged with Genie and 12,839 predictions from Ensemblalone. The average lengths are 469 amino acids for the knownproteins, 443 amino acids for protein predictions from theEnsembl±Genie merge, and 187 amino acids for those fromEnsembl alone. (The smaller average size for the predictions fromEnsembl alone re¯ects its tendency to predict partial genes wherethere is supporting evidence for only part of the gene; the remainderof the gene will often not be predicted at all, rather than included aspart of another prediction. Accordingly, the smaller size cannot beused to estimate the rate of fragmentation in such predictions.)The set corresponds to fewer than 31,000 actual genes, becausesome genes are fragmented into more than one partial predictionand some predictions may be spurious or correspond to pseudogenes.As discussed below, our best estimate is that IGI.1 includesabout 24,500 true genes.Evaluation of IGI/IPI. We used several approaches to evaluate thesensitivity, speci®city and fragmentation of the IGI/IPI set.Comparison with `new' known genes. One approach was to examineNATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com 899© 2001 Macmillan Magazines Ltd