Initial sequencing and analysis of the human genome.
TL;DR: The results of an international collaboration to produce and make freely available a draft sequence of the human genome are reported and an initial analysis is presented, describing some of the insights that can be gleaned from the sequence.
Abstract: The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.
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TL;DR: The definition and use of family-specific, manually curated gathering thresholds are explained and some of the features of domains of unknown function (also known as DUFs) are discussed, which constitute a rapidly growing class of families within Pfam.
Abstract: Pfam is a widely used database of protein families and domains. This article describes a set of major updates that we have implemented in the latest release (version 24.0). The most important change is that we now use HMMER3, the latest version of the popular profile hidden Markov model package. This software is approximately 100 times faster than HMMER2 and is more sensitive due to the routine use of the forward algorithm. The move to HMMER3 has necessitated numerous changes to Pfam that are described in detail. Pfam release 24.0 contains 11,912 families, of which a large number have been significantly updated during the past two years. Pfam is available via servers in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/).
14,075 citations
TL;DR: Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems are indicated.
Abstract: A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.
12,098 citations
TL;DR: In a four-genome analysis of 3' UTRs, approximately 13,000 regulatory relationships were detected above the estimate of false-positive predictions, thereby implicating as miRNA targets more than 5300 human genes, which represented 30% of the gene set.
11,624 citations
TL;DR: A mature web tool for rapid and reliable display of any requested portion of the genome at any scale, together with several dozen aligned annotation tracks, is provided at http://genome.ucsc.edu.
Abstract: As vertebrate genome sequences near completion and research refocuses to their analysis, the issue of effective genome annotation display becomes critical. A mature web tool for rapid and reliable display of any requested portion of the genome at any scale, together with several dozen aligned annotation tracks, is provided at http://genome.ucsc.edu. This browser displays assembly contigs and gaps, mRNA and expressed sequence tag alignments, multiple gene predictions, cross-species homologies, single nucleotide polymorphisms, sequence-tagged sites, radiation hybrid data, transposon repeats, and more as a stack of coregistered tracks. Text and sequence-based searches provide quick and precise access to any region of specific interest. Secondary links from individual features lead to sequence details and supplementary off-site databases. One-half of the annotation tracks are computed at the University of California, Santa Cruz from publicly available sequence data; collaborators worldwide provide the rest. Users can stably add their own custom tracks to the browser for educational or research purposes. The conceptual and technical framework of the browser, its underlying MYSQL database, and overall use are described. The web site currently serves over 50,000 pages per day to over 3000 different users.
9,605 citations
TL;DR: Velvet represents a new approach to assembly that can leverage very short reads in combination with read pairs to produce useful assemblies and is in close agreement with simulated results without read-pair information.
Abstract: We have developed a new set of algorithms, collectively called "Velvet," to manipulate de Bruijn graphs for genomic sequence assembly. A de Bruijn graph is a compact representation based on short words (k-mers) that is ideal for high coverage, very short read (25-50 bp) data sets. Applying Velvet to very short reads and paired-ends information only, one can produce contigs of significant length, up to 50-kb N50 length in simulations of prokaryotic data and 3-kb N50 on simulated mammalian BACs. When applied to real Solexa data sets without read pairs, Velvet generated contigs of approximately 8 kb in a prokaryote and 2 kb in a mammalian BAC, in close agreement with our simulated results without read-pair information. Velvet represents a new approach to assembly that can leverage very short reads in combination with read pairs to produce useful assemblies.
9,389 citations
References
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TL;DR: Application of ‘Exofish’, a procedure based on homology searches, to identify human genes quickly and reliably, showed that the human genome contains 28,000–34,000 genes, and that Unigene contains less than 40% of the protein-coding fraction of thehuman genome.
Abstract: The number of genes in the human genome is unknown, with estimates ranging from 50,000 to 90,000 (refs 1, 2), and to more than 140,000 according to unpublished sources. We have developed 'Exofish', a procedure based on homology searches, to identify human genes quickly and reliably. This method relies on the sequence of another vertebrate, the pufferfish Tetraodon nigroviridis, to detect conserved sequences with a very low background. Similar to Fugu rubripes, a marine pufferfish proposed by Brenner et al. as a model for genomic studies, T. nigroviridis is a more practical alternative with a genome also eight times more compact than that of human. Many comparisons have been made between F. rubripes and human DNA that demonstrate the potential of comparative genomics using the pufferfish genome. Application of Exofish to the December version of the working draft sequence of the human genome and to Unigene showed that the human genome contains 28,000-34,000 genes, and that Unigene contains less than 40% of the protein-coding fraction of the human genome.
316 citations
TL;DR: The analysis of excision events for two representatives of this family of short‐inverted‐repeat elements, tagalong and piggysac, provides further validation for their inclusion in a single family of unique transposons.
Abstract: Transposon mutagenesis of baculoviruses provides an ideal experimental system for analysis of the movement of a unique family of mobile element identified from lepidopteran genomes. Members of this family of short-inverted-repeat elements are characterized by their extreme specificity for TTAA target sites. This report describes the analysis of excision events for two representatives of this family, tagalong (formerly TFP3) and piggyBac (formerly IFP2). These elements were tagged with a polyhedrin/lacZ reporter gene and inserted back into the virus genome either by homologous recombination or by transposition. Revertants were selected based on a white plaque phenotype. Both elements excise in a precise fashion from their positions in the baculovirus genome in either TN-368 cells or IPLB-SF21 AE cells. The precise excision of these elements in infected IPLB-SF21 AE cells occurs in the absence of either tagalong or piggyBac element encoded functions. The common characteristics of both insertion and excision for these elements provides further validation for their inclusion in a single family of unique transposons.
316 citations
University of Pennsylvania1, Roswell Park Cancer Institute2, National Institutes of Health3, J. Craig Venter Institute4, Cedars-Sinai Medical Center5, Fred Hutchinson Cancer Research Center6, California Institute of Technology7, University of California, San Francisco8, Stanford University9, Harvard University10, University of Washington11, Case Western Reserve University12, Los Alamos National Laboratory13, Lawrence Berkeley National Laboratory14, Children's Hospital Oakland Research Institute15, Institute for Systems Biology16, Boston Children's Hospital17, University of California, Santa Cruz18
TL;DR: This resource represents the first comprehensive integration of cytogenetic, radiation hybrid, linkage and sequence maps of the human genome and provides an independent validation of the sequence map and framework for contig order and orientation.
Abstract: We have placed 7,600 cytogenetically defined landmarks on the draft sequence of the human genome to help with the characterization of genes altered by gross chromosomal aberrations that cause human disease. The landmarks are large-insert clones mapped to chromosome bands by fluorescence in situ hybridization. Each clone contains a sequence tag that is positioned on the genomic sequence. This genome-wide set of sequence-anchored clones allows structural and functional analyses of the genome. This resource represents the first comprehensive integration of cytogenetic, radiation hybrid, linkage and sequence maps of the human genome; provides an independent validation of the sequence map and framework for contig order and orientation; surveys the genome for large-scale duplications, which are likely to require special attention during sequence assembly; and allows a stringent assessment of sequence differences between the dark and light bands of chromosomes. It also provides insight into large-scale chromatin structure and the evolution of chromosomes and gene families and will accelerate our understanding of the molecular bases of human disease and cancer.
314 citations
TL;DR: Together, these three species exemplify the known SINE composition of placental mammals, suggesting that mammalian SINEs are similarly regulated and may serve a common function.
Abstract: The abundance of Alu RNA is transiently increased by heat shock in human cell lines. This effect is specific to Alu repeats among Pol III transcribed genes, since the abundance of 7SL, 7SK, 5S and U6 RNAs is essentially unaffected by heat shock. The rapid induction of Alu expression precedes the heat shock induction of mRNAs for the ubiquitin and HSP 70 heat shock genes. Heat shock mimetics also transiently induce Alu expression indicating that increased Alu expression is a general cell-stress response. Cycloheximide treatment rapidly and transiently increases the abundance of Alu RNA. Again, compared with other genes transcribed by Pol III, this increase is specific to Alu. However, as distinguished from the cell stress response, cycloheximide does not induce expression of HSP 70 and ubiquitin mRNAs. Puromycin also increases Alu expression, suggesting that this response is generally caused by translational inhibition. The response of mammalian SINEs to cell stress and translational inhibition is not limited to SINEs which are Alu homologues. Heat shock and cycloheximide each transiently induce Pol III directed expression of B1 and B2 RNAs in mouse cells and C-element RNA in rabbit cells. Together, these three species exemplify the known SINE composition of placental mammals, suggesting that mammalian SINEs are similarly regulated and may serve a common function.
312 citations
TL;DR: In intron 7 of this gene, an insertion/deletion mutation is identified that does not affect intron-exon boundaries, but deletes five G-triplets at the 3' end of the intron.
Abstract: Nonsyndromic hearing impairment is one of the most heterogeneous hereditary conditions, with more than 40 loci mapped on the human genome, however, only a limited number of genes implicated in hearing loss have been identified. We previously reported linkage to chromosome 7p15 for autosomal dominant hearing impairment segregating in an extended Dutch family (DFNA5). Here, we report a further refinement of the DFNA5 candidate region and the isolation of a gene from this region that is expressed in the cochlea. In intron 7 of this gene, we identified an insertion/deletion mutation that does not affect intron-exon boundaries, but deletes five G-triplets at the 3' end of the intron. The mutation co-segregated with deafness in the family and causes skipping of exon 8, resulting in premature termination of the open reading frame. As no physiological function could be assigned, the gene was designated DFNA5.
312 citations