Initial sequencing and analysis of the human genome - Vitagenes

articlesultimate goal of a completely ®nished sequence. The results beloware based on the map and sequence data available on 7 October2000, except as otherwise noted. At the end of this section, weprovide a brief update of key data.Clone selectionThe hierarchical shotgun method involves the sequencing of overlappinglarge-insert clones spanning the genome. For the HumanGenome Project, clones were largely chosen from eight large-insertlibraries containing BAC or P1-derived arti®cial chromosome(PAC) clones (Table 1; refs 82±88). The libraries were made bypartial digestion of genomic DNA with restriction enzymes.Together, they represent around 65-fold coverage (redundant sampling)of the genome. Libraries based on other vectors, such ascosmids, were also used in early stages of the project.The libraries (Table 1) were prepared from DNA obtained fromanonymous human donors in accordance with US Federal Regulationsfor the Protection of Human Subjects in Research(45CFR46) and following full review by an Institutional ReviewBoard. Brie¯y, the opportunity to donate DNA for this purpose wasbroadly advertised near the two laboratories engaged in libraryBox 1Genome glossarySequenceRaw sequence Individual unassembled sequence reads, producedby sequencing of clones containing DNA inserts.Paired-end sequence Raw sequence obtained from both ends of acloned insert in any vector, such as a plasmid or bacterial arti®cialchromosome.Finished sequence Complete sequence of a clone or genome, withan accuracy of at least 99.99% and no gaps.Coverage (or depth) The average number of times a nucleotide isrepresented by a high-quality base in a collection of random rawsequence. Operationally, a `high-quality base' is de®ned as one with anaccuracy of at least 99% (corresponding to a PHRED score of at least 20).Full shotgun coverage The coverage in random raw sequenceneeded from a large-insert clone to ensure that it is ready for ®nishing; thisvaries among centres but is typically 8±10-fold. Clones with full shotguncoverage can usually be assembled with only a handful of gaps per100 kb.Half shotgun coverage Half the amount of full shotgun coverage(typically, 4±5-fold random coverage).ClonesBAC clone Bacterial arti®cial chromosome vector carrying a genomicDNA insert, typically 100±200 kb. Most of the large-insert clonessequenced in the project were BAC clones.Finished clone A large-insert clone that is entirely represented by®nished sequence.Full shotgun clone A large-insert clone for which full shotgunsequence has been produced.Draft clone A large-insert clone for which roughly half-shotgunsequence has been produced. Operationally, the collection of draftclones produced by each centre was required to have an averagecoverage of fourfold for the entire set and a minimum coverage ofthreefold for each clone.Predraft clone A large-insert clone for which some shotgunsequence is available, but which does not meet the standards forinclusion in the collection of draft clones.Contigs and scaffoldsContig The result of joining an overlapping collection of sequences orclones.Scaffold The result of connecting contigs by linking information frompaired-end reads from plasmids, paired-end reads from BACs, knownmessenger RNAs or other sources. The contigs in a scaffold are orderedand oriented with respect to one another.Fingerprint clone contigs Contigs produced by joining clonesinferred to overlap on the basis of their restriction digest ®ngerprints.Sequenced-clone layout Assignment of sequenced clones to thephysical map of ®ngerprint clone contigs.Initial sequence contigs Contigs produced by merging overlappingsequence reads obtained from a single clone, in a process calledsequence assembly.Merged sequence contigs Contigs produced by taking the initialsequence contigs contained in overlapping clones and merging thosefound to overlap. These are also referred to simply as `sequence contigs'where no confusion will result.Sequence-contig scaffolds Scaffolds produced by connectingsequence contigs on the basis of linking information.Sequenced-clone contigs Contigs produced by merging overlappingsequenced clones.Sequenced-clone-contig scaffolds Scaffolds produced by joiningsequenced-clone contigs on the basis of linking information.Draft genome sequence The sequence produced by combiningthe information from the individual sequenced clones (by creatingmerged sequence contigs and then employing linking information tocreate scaffolds) and positioning the sequence along the physical map ofthe chromosomes.N50 length A measure of the contig length (or scaffold length)containing a `typical' nucleotide. Speci®cally, it is the maximum length Lsuch that 50% of all nucleotides lie in contigs (or scaffolds) of size at least L.Computer programs and databasesPHRED A widely used computer program that analyses raw sequenceto produce a `base call' with an associated `quality score' for eachposition in the sequence. A PHRED quality score of X corresponds to anerror probability of approximately 10 - X/10 . Thus, a PHRED quality score of30 corresponds to 99.9% accuracy for the base call in the raw read.PHRAP A widely used computer program that assembles rawsequence into sequence contigs and assigns to each position in thesequence an associated `quality score', on the basis of the PHREDscores of the raw sequence reads. A PHRAP quality score of Xcorresponds to an error probability of approximately 10 - X/10 . Thus, aPHRAP quality score of 30 corresponds to 99.9% accuracy for a base inthe assembled sequence.GigAssembler A computer program developed during this projectfor merging the information from individual sequenced clones into a draftgenome sequence.Public sequence databases The three coordinated internationalsequence databases: GenBank, the EMBL data library and DDBJ.Map featuresSTS Sequence tagged site, corresponding to a short (typically lessthan 500 bp) unique genomic locus for which a polymerase chainreaction assay has been developed.EST Expressed sequence tag, obtained by performing a single rawsequence read from a random complementary DNA clone.SSR Simple sequence repeat, a sequence consisting largely of atandem repeat of a speci®c k-mer (such as (CA) 15 ). Many SSRs arepolymorphic and have been widely used in genetic mapping.SNP Single nucleotide polymorphism, or a single nucleotide position inthe genome sequence for which two or more alternative alleles arepresent at appreciable frequency (traditionally, at least 1%) in the humanpopulation.Genetic map A genome map in which polymorphic loci arepositioned relative to one another on the basis of the frequency withwhich they recombine during meiosis. The unit of distance iscentimorgans (cM), denoting a 1% chance of recombination.Radiation hybrid (RH) map A genome map in which STSs arepositioned relative to one another on the basis of the frequency withwhich they are separated by radiation-induced breaks. The frequency isassayed by analysing a panel of human±hamster hybrid cell lines, eachproduced by lethally irradiating human cells and fusing them withrecipient hamster cells such that each carries a collection of humanchromosomal fragments. The unit of distance is centirays (cR), denotinga 1% chance of a break occuring between two loci.NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd865