articles partial list of restriction fragments in silico and comparing that list cHromosome ith the experimental database of BAC fingerprints. The compari on was feasible because the experim ing of restriction gments was highly accurate(to within 0.5-1.5% of the true ize, for 95% of fragments from 600 to 12, 000 base pairs(bp)54.ss. Reliable matching scores could be obtained for 16, 193 of the clones e remaining sequenced clones could not be placed on the map by this method because they were too short, or they contained too many small initial sequence contigs to yield enough restriction ragments, or possibly because their sequences were not represented in the fingerprint database. An independent approach to placing sequenced clones on the physical map used the database of end sequences from fingerprint BACs(Table 1). Sequenced clones could typically be reliably mapped if they contained multiple matches to BAC ends, with all corresponding to clones from a single genomic region(multiple matches were required as a safeguard against errors known to exist in the BAC end database and against repeated sequences). Thi approach provided useful placement information for 22, 566 Altogether, we could assign 25, 403 sequenced clones to finger print clone contigs by combining in silico digestion and BAC end sequence match data. To place most of the remaining sequenced clones, we exploited information about sequence overlap or BAC nd paired links of these clones with already positioned clones. This left only a few, mostly small, sequenced clones that could not be laced (152 sequenced clones containing 5. 5 Mb of sequence out of 29, 298 sequenced clones containing more than 4, 260 Mb of equence); these are being localized by radiation hybrid mapi f STSs derived from thei The fingerprint clone contigs were then mapped to chromosomal locations, using sequence matche %o..0 mapped STSs from four human radiation hybrid maps., 0, one YAC and radiation vo genetic maps gether with data from FISH,,o. The mapping was iteratively refined by comparing the order and orientation of the STSs in the fingerprint clone contigs nd the various STS-based maps, to identify and refine discrepan- cies(Fig. 5). Small fingerprint clone contigs(< 1 Mb)were difficult to orient and, sometimes, to order using these methods. In all, 942 fingerprint clone contigs contained sequenced clones. (An addi- tional 304 of the 1, 246 fingerprint clone contigs did not contain Figure 5 Positions of markers on previous maps of the genome(the Genethon'ogenetic lancedclonesbutthesetendedtobeextremelysmallandmapandMarshfieldgeneticmap(http://research.marshfieldclinic.org/genetics/ together contain less than 1% of the mapped clones. About one- genotyping_service/mgsver2 htm), the GeneMap99 radiation hybrid map 00, and the third have been targeted for sequencing. A few derive from the Y Whitehead YAC and radiation hybrid map2) plotted against their derived position on the chromosome, for which the map was constructed separately". Most draft sequence for chromosome 2. The horizontal units are Mb but the vertical units of of the remainder are fragments of other larger contigs or represent each map vary (CM, cR and so on) and thus all were scaled so that the entire map spans other artefacts. These are being eliminated in subsequent versions of the full vertical range Markers that map to other chromosomes are shown in the the database )Of these 942 contigs with sequenced clones, 852 chromosome lines at the top. The data sets generally follow the diagonal, indicating that (90%, containing 99.2% of the total sequence) were localized to order and orientation of the marker sets on the different maps largely agree(note that the specific chromosome locations in this way. An additional 51 two genetic maps are completely superimposed). In a, there are two segments(bars)that fingerprint clone contigs, containing 0.5% of the sequence, could are inverted in an earlier version draft sequence relative to all the other maps. b, The same be assigned to a specific chromosome but not to a precise position. chromosome after the information was used to reorient those two segments end-to-end middle only: not OK Figure 6 The key steps (a-d in assembling individual sequenced clones into the draft genome sequence. A1-A5 represent initial sequence contigs derived from shotgun sequencing of clone A, and B1-B6 are from clone b NatuReVoL409115FebRuAry2001www.nature.comAe2001MacmillanMagazinesLtdpartial list of restriction fragments in silico and comparing that list with the experimental database of BAC ®ngerprints. The compari￾son was feasible because the experimental sizing of restriction fragments was highly accurate (to within 0.5±1.5% of the true size, for 95% of fragments from 600 to 12,000 base pairs (bp))84,85. Reliable matching scores could be obtained for 16,193 of the clones. The remaining sequenced clones could not be placed on the map by this method because they were too short, or they contained too many small initial sequence contigs to yield enough restriction fragments, or possibly because their sequences were not represented in the ®ngerprint database. An independent approach to placing sequenced clones on the physical map used the database of end sequences from ®ngerprinted BACs (Table 1). Sequenced clones could typically be reliably mapped if they contained multiple matches to BAC ends, with all corresponding to clones from a single genomic region (multiple matches were required as a safeguard against errors known to exist in the BAC end database and against repeated sequences). This approach provided useful placement information for 22,566 sequenced clones. Altogether, we could assign 25,403 sequenced clones to ®nger￾print clone contigs by combining in silico digestion and BAC end sequence match data. To place most of the remaining sequenced clones, we exploited information about sequence overlap or BAC￾end paired links of these clones with already positioned clones. This left only a few, mostly small, sequenced clones that could not be placed (152 sequenced clones containing 5.5 Mb of sequence out of 29,298 sequenced clones containing more than 4,260 Mb of sequence); these are being localized by radiation hybrid mapping of STSs derived from their sequences. The ®ngerprint clone contigs were then mapped to chromosomal locations, using sequence matches to mapped STSs from four human radiation hybrid maps95,99,100, one YAC and radiation hybrid map29, and two genetic maps101,102, together with data from FISH86,90,103. The mapping was iteratively re®ned by comparing the order and orientation of the STSs in the ®ngerprint clone contigs and the various STS-based maps, to identify and re®ne discrepan￾cies (Fig. 5). Small ®ngerprint clone contigs (, 1 Mb) were dif®cult to orient and, sometimes, to order using these methods. In all, 942 ®ngerprint clone contigs contained sequenced clones. (An addi￾tional 304 of the 1,246 ®ngerprint clone contigs did not contain sequenced clones, but these tended to be extremely small and together contain less than 1% of the mapped clones. About one￾third have been targeted for sequencing. A few derive from the Y chromosome, for which the map was constructed separately89. Most of the remainder are fragments of other larger contigs or represent other artefacts. These are being eliminated in subsequent versions of the database.) Of these 942 contigs with sequenced clones, 852 (90%, containing 99.2% of the total sequence) were localized to speci®c chromosome locations in this way. An additional 51 ®ngerprint clone contigs, containing 0.5% of the sequence, could be assigned to a speci®c chromosome but not to a precise position. articles NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com 869 50 100 150 200 250 Chromosome 2 50 100 150 200 250 Map location Map location Chromosome 2 Chromosome 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y b Chromosome 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y a Genethon map Gene map Marshfield map YAC map Genethon map Gene map Marshfield map YAC map Figure 5 Positions of markers on previous maps of the genome (the Genethon101 genetic map and Marsh®eld genetic map (http://research.marsh®eldclinic.org/genetics/ genotyping_service/mgsver2.htm), the GeneMap99 radiation hybrid map100, and the Whitehead YAC and radiation hybrid map29) plotted against their derived position on the draft sequence for chromosome 2. The horizontal units are Mb but the vertical units of each map vary (cM, cR and so on) and thus all were scaled so that the entire map spans the full vertical range. Markers that map to other chromosomes are shown in the chromosome lines at the top.The data sets generally follow the diagonal, indicating that order and orientation of the marker sets on the different maps largely agree (note that the two genetic maps are completely superimposed). In a, there are two segments (bars) that are inverted in an earlier version draft sequence relative to all the other maps. b, The same chromosome after the information was used to reorient those two segments. A1 A1 A2 A2 A1 B1 A3 B3 A4 B6A5 B2 B4 B5 A2 A3 A4 A4 A5 A5 B1 B1 A3 B2 B2 B3 B3 B4 B4 B5 B5 B6 B6 a d b c end-to-end alignment : OK alignment in middle only : not OK Figure 6 The key steps (a±d) in assembling individual sequenced clones into the draft genome sequence. A1±A5 represent initial sequence contigs derived from shotgun sequencing of clone A, and B1±B6 are from clone B. © 2001 Macmillan Magazines Ltd