Initial sequencing and analysis of the human genome - Vitagenes

articlesthat were used to `seed' the sequencing of new regions. The small®ngerprint clone contigs were extended or merged with others asthe map matured.The clones that make up the draft genome sequence therefore donot constitute a minimally overlapping setÐthere is overlap andredundancy in places. The cost of using suboptimal overlaps wasjusti®ed by the bene®t of earlier availability of the draft genomesequence data. Minimizing the overlap between adjacent cloneswould have required completing the physical map before undertakinglarge-scale sequencing. In addition, the overlaps betweenBAC clones provide a rich collection of SNPs. More than 1.4 millionSNPs have already been identi®ed from clone overlaps and othersequence comparisons 97 .Because the sequencing project was shared among twenty centresin six countries, it was important to coordinate selection of clonesacross the centres. Most centres focused on particular chromosomesor, in some cases, larger regions of the genome. We also maintaineda clone registry to track selected clones and their progress. In laterphases, the global map provided an integrated view of the data fromall centres, facilitating the distribution of effort to maximize coverageof the genome. Before performing extensive sequencing on aFigure 3 The automated production line for sample preparation at the WhiteheadInstitute, Center for Genome Research. The system consists of custom-designed factorystyleconveyor belt robots that perform all functions from purifying DNA from bacterialcultures through setting up and purifying sequencing reactions.Sequence (Mb)5,0004,5004,0003,5003,0002,5002,0001,5001,0005000Jan-96Apr-96Jul-96FinishedUnfinished (draft and pre-draft)Oct-96Jan-97Apr-97Jul-97Oct-97Jan-98Apr-98MonthJul-98Oct-98Jan-99Apr-99Jul-99Oct-99Jan-00Apr-00Jul-00Oct-00Figure 4 Total amount of human sequence in the High Throughput Genome Sequence(HTGS) division of GenBank. The total is the sum of ®nished sequence (red) and un®nished(draft plus predraft) sequence (yellow).clone, several centres routinely examined an initial sample of 96 rawsequence reads from each subclone library to evaluate possibleoverlap with previously sequenced clones.SequencingThe selected clones were subjected to shotgun sequencing. Althoughthe basic approach of shotgun sequencing is well established, thedetails of implementation varied among the centres. For example,there were differences in the average insert size of the shotgunlibraries, in the use of single-stranded or double-stranded cloningvectors, and in sequencing from one end or both ends of each insert.Centres differed in the ¯uorescent labels employed and in the degreeto which they used dye-primers or dye-terminators. The sequencedetectors included both slab gel- and capillary-based devices.Detailed protocols are available on the web sites of many of theindividual centres (URLs can be found at www.nhgri.nih.gov/genome_hub). The extent of automation also varied greatlyamong the centres, with the most aggressive automation effortsresulting in factory-style systems able to process more than 100,000sequencing reactions in 12 hours (Fig. 3). In addition, centresdiffered in the amount of raw sequence data typically obtained foreach clone (so-called half-shotgun, full shotgun and ®nishedsequence). Sequence information from the different centres couldbe directly integrated despite this diversity, because the data wereanalysed by a common computational procedure. Raw sequencetraces were processed and assembled with the PHRED and PHRAPsoftware packages 77,78 (P. Green, unpublished). All assembled contigsof more than 2 kb were deposited in public databases within24 hours of assembly.The overall sequencing output rose sharply during production(Fig. 4). Following installation of new sequence detectors beginningin June 1999, sequencing capacity and output rose approximatelyeightfold in eight months to nearly 7 million samples processed permonth, with little or no drop in success rate (ratio of useable readsto attempted reads). By June 2000, the centres were producing rawsequence at a rate equivalent to onefold coverage of the entirehuman genome in less than six weeks. This corresponded to acontinuous throughput exceeding 1,000 nucleotides per second,24 hours per day, seven days per week. This scale-up resulted in aconcomitant increase in the sequence available in the publicdatabases (Fig. 4).A version of the draft genome sequence was prepared on the basisof the map and sequence data available on 7 October 2000. For thisversion, the mapping effort had assembled the ®ngerprinted BACsinto 1,246 ®ngerprint clone contigs. The sequencing effort hadsequenced and assembled 29,298 overlapping BACs and other largeinsertclones (Table 2), comprising a total length of 4.26 gigabases(Gb). This resulted from around 23 Gb of underlying raw shotgunsequence data, or about 7.5-fold coverage averaged across thegenome (including both draft and ®nished sequence). The variouscontributions to the total amount of sequence deposited in theHTGS division of GenBank are given in Table 3.Table 2 Total genome sequence from the collection of sequenced clones, bysequence statusSequencestatusNumber ofclonesTotal clonelength (Mb)Averagenumber ofsequencereads per kb*Averagesequencedepth²Total amountof rawsequence (Mb)Finished 8,277 897 20±25 8±12 9,085Draft 18,969 3,097 12 4.5 13,395Predraft 2,052 267 6 2.5 667Total 23,147.............................................................................................................................................................................* The average number of reads per kb was estimated based on information provided by eachsequencing centre. This number differed among sequencing centres, based on the actual protocolsused.² The average depth in high quality bases ($99% accuracy) was estimated from informationprovided by each sequencing centre. The average varies among the centres, and the number mayvary considerably for clones with the same sequencing status. For draft clones in the publicdatabases (keyword: HTGS_draft), the number can be computed from the quality scores listed inthe database entry.NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd867