HUMAN GENOME PDF
The Human Genome. Project. From human genome to other genomes and to gene function. June From genome to health. Structural Genomics initiative . human genome at the level of its DNA sequence. These advances have come about in large measure through the applications of molecular genetics and. the human genome was generated by the whole-genome shotgun A random pair of human haploid genomes differed at a rate of 1 bp per.
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Base pairs. • Each turn of the double helix is 10 base pairs (bp). • In one copy of DNA ~ 3,,, bp! • Centimeters. • One chromosome ~. PDF | On Jan 1, , J C Venter and others published The sequence of the human genome. Schematic overview of the inheritance of our genetic information. Each individual has two copies of each chromosome (each harboring one copy of the genes on the chromosome). Assume a particular gene in the DNA which determines the phenotype hair style, and for which two variants.
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Decoding the human genome
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Greven , Brian G. National Institutes of Health. A Design and information in biology: From molecules to systems. Archived from the original on Hum Genomics. A Unique Approach to Genome Sequencing". Hierarchical Shotgun Sequencing v.
Shotgun Sequencing". Department of Biology, Davidson College. Global Coordination in Data Sharing". Center for Biomolecular Science and Engineering. Genome Research. Nature Genetics. Nature News. Annu Rev Med. Genes Dev. Molecular Breeding. The Code of Codes: Cambridge, Massachusetts: Harvard University Press. Living and Working with the New Medical Technologies. Cambridge University Press. Personal genomics.
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Human Genome Project
Reorganized and renamed to Helmholtz Center for Infection Research. Wikinews has related news: Mexico presents first population-wide genome map for a Latin country.
Wikibooks has a book on the topic of: These regions are highly enriched in sequences that are difficult to clone or sequence and thus are not represented even after deep 6—fold coverage with random reads. These regions are also poorly covered by whole-genome shotgun strategies The finishing phase converted this draft assembly into a high-quality continuous sequence by obtaining directed information.
It involved iterative cycles of computational analysis and laboratory work. The first step was to inspect the draft assembly for evidence of mis-assembly, arising from inappropriate merger of repeated sequences. In general, sequence assembly is more straightforward for the clone-based hierarchical shotgun strategy than for the whole-genome shotgun strategy, because the use of clones avoids problems arising from polymorphism and from different copies of repeated regions elsewhere in the genome.
Most clones passed assembly inspection, but some failed due to the presence of very similar local dispersed, tandem or inverted repeats. Careful inspection could resolve the problem in some cases, but specific strategies had to be devised in other cases.
One approach was to isolate distinct copies of the repeat in subclones of intermediate size kb plasmids or fosmids and sequence these subclones. The base of the triangle represents 80 kb of a kb insert. Each dot represents a perfect match of 20 bases. The region between 65 kb and 94 kb contains four copies of a directly repeated sequence of about 3 kb horizontal lines , separated by imperfect short tandemly repeated sequence diamond blocks of dots.
The region between 94 kb and kb contains tandemly repeated imperfect copies of a five-base sequence, unrelated to the previous sequence.
Finishing the euchromatic sequence of the human genome
Two misassemblies resulting from the long direct repeats and the absence of all copies not shown were resolved by manual editing, after which 12 gaps remained in the clone.
Five of these labelled A were spanned by plasmid subclones and were closed by primer walking. Two gaps labelled B were larger; after initial walks failed, these gaps were closed by sequencing short insert libraries prepared from PCR products. Four other gaps labelled C were not spanned by plasmid clones but were closed by primer walks on PCR products. One gap labelled D was closed by primer walks and extensive manual editing.
The second step was gap closure. Because gaps tended to be enriched for problematic sequences, gap closure was challenging; it often required multiple attempts using a variety of alternative methods. Gaps were classified into two types: Spanned gaps were those for which the two flanking contig ends were linked by an end-sequenced plasmid.
Most such gaps could be closed by primer-directed sequencing of the plasmid, serially extending thecontig sequence into the gap. Sequence in the gap was often recalcitrant to the standard sequencing protocol accounting for its absence from the initial shotgun data , making it necessary to use many alternative protocols different buffers, enzymes and temperature conditions.
Some gaps could not be closed by primer walking, because no suitable primer could be found due to repetitive sequence near the end of the contig or because sequencing chemistries were unable to penetrate certain secondary structures such as some inverted repeats. Specialized strategies were used to obtain the missing sequence. Unspanned gaps arose where a contig end was not linked to any other contig.
It was then necessary to infer adjacency and extend the sequence by other means. This battery of techniques succeeded in virtually all cases. In cases, there remains a small region of bases that could not be reliably sequenced; almost all fall in tandem repeat sequences and typically affect tens to hundreds of bases.
These cases are annotated in the accessioned clones. The third step which proceeded in parallel to gap closure was the resolution of low-quality regions.
This was accomplished by obtaining additional sequence reads from resequencing of existing shotgun subclones or from primer-directed sequencing.
The final step involved quality control. To confirm the accuracy of the overall assembly, the restriction digestion pattern of the BAC predicted from the finished sequence was compared with the pattern observed experimentally. To confirm accuracy at the nucleotide level, the finished sequence and supporting data were reviewed by human inspection and computational analysis.
The finished sequence was then annotated and deposited in public databases. The human sequence reported here consists of 2,,, nucleotides, lying almost entirely within the euchromatic portion of the genome Table 2. Mb are euchromatic.
The long-range continuity of the current genome sequence is high by various measures Table 3. The N50 length is Mb and the N -average length is Focusing on individual chromosome arms, the N50 length exceeds half the length of the arm in three-quarters of cases Table 3. The analyses reported here were performed on Build 35 or, in a few cases, its immediate predecessor, Build 34 which differed only slightly.
The poster accompanying this paper displays the 24 human chromosomes, together with various biological annotations. These include GC content, repeat content, segmental duplications, protein-coding genes, sequence similarity and synteny conservation with mouse, sequence similarity with the pufferfish, and density of single-nucleotide polymorphisms in the human genome.
Many additional annotations can be found on public genome browsers http: The near-complete sequence is a great improvement over the earlier draft sequence. It has substantially fewer gaps versus , and greater continuity 38, kilobases kb versus 81? The draft sequence contained regions in which the local order and orientation were unknown; these have now been resolved.
The case of chromosome 7 is illustrated in Fig. Additionally, the draft sequence contained substantial artefactual duplication, including local events caused by errors in merging some adjacent BAC-based sequences, made by the first-generation global assembly program, and global events caused by contamination of shotgun assemblies of some BACs with data from other clones.
These artefacts have now been eliminated. At large scale, there was good collinearity between draft and near-complete sequence, although some inversions were present in the draft due to lack of sufficient anchors in some regions. At finer scale, the draft sequence contained some sequence contigs for which order and orientation were not known. The inset shows a region of ? BACs at each end were finished at the time of draft assembly, whereas the middle BAC was at an early stage of shotgun coverage in which contigs were not yet ordered and oriented.
Because the human genome sequence is intended to serve as a permanent foundation for biomedical research, it was important to assess its quality and to characterize its remaining defects. For this purpose, we used a number of comparisons and consistency checks.
Tests of accuracy were designed to detect potential problems that may have occurred in clone-based sequencing. This may include errors in assembling the finished sequence within individual clones, and errors in concatenating adjacent finished clones to create the final product.
The analysis was complicated by the presence of polymorphism in the human population, because differences between sequence clones may reflect either errors or polymorphism. Independent quality assessment. In the final stages, an independent group examined a random sample of finished clones by generating additional data and generating new assemblies Mb and found an error rate of 1.
The small events consisted largely of single-base substitutions, whereas the remaining small and large events primarily concerned the number of consecutive copies of a tandem repeat Analysis of clone overlap. Mb , by examining overlapping sequence between consecutive finished large-insert clones.
If two such clones derive from the same copy of the human genome, any sequence differences in the overlap must reflect an error in one of the two clones. By comparing independent clones, this quality assessment method also has the ability to detect cloning artefacts. We examined 4, substantially overlapping clones derived from the same library; half are expected to be derived from the same haplotype and half from a different haplotype.
The resulting distribution Fig. The number of single-base differences in overlaps for clones from the same library and from different libraries is plotted. The results are consistent with half of the clones from the same library representing identical underlying DNA sequence with low error rate, and half representing different haplotypes as expected.
The number of indels per? We then examined overlapping clones likely to be from the same haplotype with no single-base mismatches and counted the discrepancy rate for indels Fig. By contrast, clones from different libraries show a discrepancy rate that is at least fold higher. Overall, the analysis indicates that the overall error rate reflecting both sequence error and cloning artefacts is 20—fold lower than the human polymorphism rate.
Analysis of junctions. We assessed longer-range integrity of the genome sequence by studying read pairs from large insert clones. Fosmid clones are particularly useful because their insert sizes cluster tightly around 40? We aligned the fosmid end sequences to the genome sequence.
Some fosmids could not be uniquely placed because one or both ends consisted almost entirely of repeat sequence. Using the uniquely placed fosmids which provide about eightfold clone coverage of the euchromatic genome , we sought to obtain independent confirmation of the order, orientation and adjacency of the junction between consecutive finished large-insert clones used to construct the genome sequence.
About half of the remaining junctions were supported by fosmids with unique placement at one end but multiple placements at the other end. Overall, the analysis provided strong support for accuracy of the junctions underlying the current genome sequence. Search for deletions. We next scanned the genome sequence for evidence of deletions of several kilobases in size, using the same fosmid data set. Such differences could reflect either an error in the genome sequence, a deletion in the fosmid clone, or a deletion polymorphism between the DNA sources.
Because the methodology cannot detect deletions larger than a fosmid, we also analysed discrepant fosmid links, which could reflect deletions.
See Methods in Supplementary Information. The top portion shows fosmids along a region of chromosome 10 centred at nucleotide 46,, , mapped by virtue of their paired-end sequences. The difference between inferred length, calculated from the location of fosmid ends in finished sequence, and average length for the entire library, is shown to the right of each clone.
For each point, the standard deviation of the local average difference for all spanning fosmids is plotted below; the threshold of 3. The region from 45 to 55? Comparison with available chimpanzee sequence further localized the difference vertical line. The majority of length differences detected by this analysis appear to represent polymorphisms, not sequence errors. These regions were then scrutinized by alignment with the recently obtained draft sequence of the chimpanzee genome R.
Waterston, personal communication. Roughly two-thirds appear to represent polymorphic deletions in the human population and one-third represent actual errors in the current genome sequence.
Analysis of a larger collection of fosmids could probably pinpoint the majority of these errors, allowing them to be corrected. Tests of coverage were designed to measure the proportion of the euchromatic genome missing from the current genome sequence, by assessing the presence of independently sampled human sequences such as complementary DNA clones and random genomic clones.
Analysis of cDNAs. The analysis 35 involved 17, distinct gene loci spanning ? Mb of genomic sequence. The vast majority A few of these 0. A few others 0. We examined the remaining cases 0.
The cDNA sequence appeared to be completely absent in 0. For almost all of completely absent cDNAs, the genomic location of the gene was known or could be inferred and corresponds to a gap in the current genome sequence.
For the partially absent cDNAs, more than half of the cases lie adjacent to gaps.
Decoding the human genome
The remainder may represent either errors in the current genome sequence or polymorphic deletions; these are being investigated further.
Overall, the proportion of cDNA sequence that is missing from the genome sequence is only 0. This may underestimate the proportion of genome missing from the finished sequence, however, because focused efforts were made to capture genomic sequence containing missing messenger RNAs.
Analysis of random genomic plasmids. As an additional and broader test of coverage, we analysed paired end-sequences from 5, small-insert 3—4? After excluding heterochromatic repeats and other artefacts, we found that For 0. For another 0. The current genome sequence contains gaps, which could not be closed with available techniques. We briefly describe the nature of these gaps and discuss the prospects for eventual closure. See Supplementary Information Notes 2 and 4.
Heterochromatic regions 33 gaps. The heterochromatic regions of the human genome were not targeted by the HGP, because their highly repetitive properties make them largely refractory to current cloning and sequencing strategies. There are 33 heterochromatic regions falling into four types. The three secondary constrictions are immediately adjacent to the centromere on chromosome arms 1q, 9q and 16q and contain various satellite repeats beta, gamma, satellite I, II, III.
Finally, there is a single large region on distal Yq composed primarily of thousands of copies of several repeat families. The heterochromatic regions all tend to be highly polymorphic in length in the human population.
Euchromatic boundary regions 35 gaps. The euchromatic regions of the human genome are bounded proximally by heterochromatin and distally by a telomere consisting of several kilobases of the hexamer repeat TTAGGG. We examined the current genome sequence for evidence of the expected boundaries on the 43 euchromatic arms. See Supplementary Information Note 4.
At the proximal ends, 30 of the 43 cases show sequence characteristic of either heterochromatin or immediately flanking regions such as higher-order centromeric repeats, stretches of at least 10? We cannot exclude the possibility that there is additional unique sequence between this point and the proximal heterochromatin; but efforts to extend the finished sequence further were unsuccessful. In the remaining 13 cases, the finished sequence contains no evidence of heterochromatin-related sequence.
At the telomeric ends, 21 of the 43 cases show continuous sequence extending to the telomeric repeat. This sequence was typically obtained by isolation and sequencing of half-YAC clones spanning to the telomere An additional 18 cases are sequence gaps, in which half-YACs reaching to the telomere were isolated but finished sequence could not be obtained.
The remaining four cases are physical gaps, in which large-insert clones extending to the telomere could not be obtained. Euchromatic interior regions gaps. The remaining gaps are located within the current genome sequence. These consist of physical gaps for which no clones could be isolated, and 58 sequence gaps for which clones were found but reliable finished sequence could not be obtained.
The physical gaps are greatly enriched in regions of segmental duplication Fig. Such segmental duplications are especially frequent in pericentromeric regions, and gaps are notably more frequent in these regions. The association of gaps with segmental duplications is examined in detail elsewhere Large duplications are shown to approximate scale; smaller ones are indicated as ticks.
Sequence gaps are indicated above the chromosomes in red. Unfinished clones are indicated as black ticks. The blue bars show the result of direct analysis of near-complete sequence. The gold bars show an independent estimate 65 using whole-genome shotgun data to correct for potential mis-assembly of such segmental duplications.
The strong agreement suggests that most segmental duplications are properly represented in near-complete genome sequence. The discrepancy for chromosome X is probably a result of errors in the independent estimate, due to limited coverage and diversity of data from this chromosome The most extreme case occurs near the centromere of chromosome 9.
The most proximal 5? Mb on 9p and 4? Mb on 9q comprise a mere 0. These two pericentric regions are unique in the genome with respect to density of segmental duplication and the average degree of intrachromosomal sequence identity Other proximal regions also show a higher-than-average density of gaps.
For example, the proximal 2? Mb on the remaining 41 euchromatic arms comprise 2. Nearly all of these proximal gaps are flanked by segmental duplications Fig.
There is also a clustering of such gaps in subtelomeric regions. The terminal 1? Mb on the 43 euchromatic arms represents 1. The most proximal regions are crowded with alpha satellite sequences and other centromeric repeats; composition, density and order may vary considerably between chromosome arms Just outside this region, there is usually a high density of inter- and intra-chromosomal duplication.
For details, see text and refs 39, 40, 66 and The terminal repeat tract consists of 2—15? Short 50—? Proximal to the Srpt region is chromosome-specific genomic DNA, typically with a high GC content and high gene density. Stretches of segmentally duplicated DNA that occur only once within subtelomeric regions tan are interspersed with 1-copy subtelomeric DNA yellow in a telomere-specific fashion. Closing the remaining gaps. These represent regions that could not be reliably mapped, cloned and sequenced with current methods.
Rather than applying further brute force, it is now time to develop focused strategies to resolve the regions. The remaining euchromatic gaps probably reflect two major issues. The first pertains to regions harbouring segmentally duplicated sequence. Such regions are challenging to map because it can be extremely difficult to discern whether two clones with small sequence differences represent different loci or different alleles at a single locus.
This challenge was eventually resolved for chromosome Y ref. By using DNA from a single haploid source, it was possible to rely on differences at only a handful of nucleotides to distinguish repeated sequences. This approach could be applied to the rest of the genome by using appropriate haploid sources, such as a hydatidiform mole or monochromosomal hybrids. In both instances, use of parental controls to guard against being misled by somatic rearrangements would be well advised.
It may be useful to test these approaches on individual chromosomes. The second issue is that some gaps are likely to correspond to regions that cannot be efficiently propagated in current large-insert vectors and hosts. It may be useful to test new kinds of large-insert libraries for clones containing unique sequences not contained in the current human genome sequence perhaps seeded by probes derived from random small-insert genomic plasmids, as discussed above.
In addition, genome completion may benefit from long-range mapping techniques such as optical mapping 38 , which may provide independent information about difficult regions. Completing the euchromatic sequence is an important goal, but is clearly now a research effort rather than a high-throughput project. Sequencing the human heterochromatin poses an even greater challenge. The current human sequence penetrates only the periphery of the heterochromatin—for example, the pericentric regions on a few chromosome arms 39 , This progress has required concerted efforts with specialized mapping techniques and painstaking assembly.
The fundamental issue is that current shotgun strategies are poorly suited to assembling large, highly repetitive regions. The hierarchical shotgun strategy faces the challenge of accurate assembly of individual BACs and accurate overlap of BAC clones, with the underlying data consisting of nearly identical sequence; the whole-genome shotgun strategy compounds these problems.
Conceivably, the hierarchical strategy could be adapted as was done for repetitive regions of chromosome Y. Approaches might include the use of the following: Such an approach will also require ensuring accurate recovery and stability of heterochromatic regions in large-insert clones. Even so, the path is likely to be arduous and expensive to obtain regions of uncertain information content. Alternatively, it may be possible to develop new approaches.
These might include methods to obtain much longer effective read lengths, directed reads from known locations and long-range mapping information about the location of rare base differences among repeat copies such as optical mapping 38 or padlock probes The present genome sequence enables far more precise analyses of the human genome, especially those that depend sensitively on high accuracy and near-completeness.
Rather than revisit all of the analyses in our initial analysis of the human genome, we have chosen four examples that illustrate the utility of the current near-complete sequence. The human genome is notable for its high proportion of recent segmental duplications.
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