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1.  The Genomic and Transcriptomic Landscape of a HeLa Cell Line 
G3: Genes|Genomes|Genetics  2013;3(8):1213-1224.
HeLa is the most widely used model cell line for studying human cellular and molecular biology. To date, no genomic reference for this cell line has been released, and experiments have relied on the human reference genome. Effective design and interpretation of molecular genetic studies performed using HeLa cells require accurate genomic information. Here we present a detailed genomic and transcriptomic characterization of a HeLa cell line. We performed DNA and RNA sequencing of a HeLa Kyoto cell line and analyzed its mutational portfolio and gene expression profile. Segmentation of the genome according to copy number revealed a remarkably high level of aneuploidy and numerous large structural variants at unprecedented resolution. Some of the extensive genomic rearrangements are indicative of catastrophic chromosome shattering, known as chromothripsis. Our analysis of the HeLa gene expression profile revealed that several pathways, including cell cycle and DNA repair, exhibit significantly different expression patterns from those in normal human tissues. Our results provide the first detailed account of genomic variants in the HeLa genome, yielding insight into their impact on gene expression and cellular function as well as their origins. This study underscores the importance of accounting for the strikingly aberrant characteristics of HeLa cells when designing and interpreting experiments, and has implications for the use of HeLa as a model of human biology.
doi:10.1534/g3.113.005777
PMCID: PMC3737162  PMID: 23550136
genomics; transcriptomics; HeLa cell line; resource; variation
2.  DELLY: structural variant discovery by integrated paired-end and split-read analysis 
Bioinformatics  2012;28(18):i333-i339.
Motivation: The discovery of genomic structural variants (SVs) at high sensitivity and specificity is an essential requirement for characterizing naturally occurring variation and for understanding pathological somatic rearrangements in personal genome sequencing data. Of particular interest are integrated methods that accurately identify simple and complex rearrangements in heterogeneous sequencing datasets at single-nucleotide resolution, as an optimal basis for investigating the formation mechanisms and functional consequences of SVs.
Results: We have developed an SV discovery method, called DELLY, that integrates short insert paired-ends, long-range mate-pairs and split-read alignments to accurately delineate genomic rearrangements at single-nucleotide resolution. DELLY is suitable for detecting copy-number variable deletion and tandem duplication events as well as balanced rearrangements such as inversions or reciprocal translocations. DELLY, thus, enables to ascertain the full spectrum of genomic rearrangements, including complex events. On simulated data, DELLY compares favorably to other SV prediction methods across a wide range of sequencing parameters. On real data, DELLY reliably uncovers SVs from the 1000 Genomes Project and cancer genomes, and validation experiments of randomly selected deletion loci show a high specificity.
Availability: DELLY is available at www.korbel.embl.de/software.html
Contact: tobias.rausch@embl.de
doi:10.1093/bioinformatics/bts378
PMCID: PMC3436805  PMID: 22962449
3.  Nucleotide-resolution analysis of structural variants using BreakSeq and a breakpoint library 
Nature biotechnology  2009;28(1):47-55.
Structural variants (SVs) are a major source of human genomic variation; however, characterizing them at nucleotide resolution remains challenging. Here we assemble a library of breakpoints at nucleotide resolution from collating and standardizing ~2,000 published SVs. For each breakpoint, we infer its ancestral state (through comparison to primate genomes) and its mechanism of formation (e.g., non-allelic homologous recombination, NAHR). We characterize breakpoint sequences with respect to genomic landmarks, chromosomal location, sequence motifs and physical properties, finding that the occurrence of insertions and deletions is more balanced than previously reported and that NAHR-formed breakpoints are associated with relatively rigid, stable DNA helices. Finally, we demonstrate an approach, BreakSeq, for scanning the reads from short-read sequenced genomes against our breakpoint library to accurately identify previously overlooked SVs, which we then validate by PCR. As new data become available, we expect our BreakSeq approach will become more sensitive and facilitate rapid SV genotyping of personal genomes.
doi:10.1038/nbt.1600
PMCID: PMC2951730  PMID: 20037582
4.  Variation in Transcription Factor Binding Among Humans 
Science (New York, N.Y.)  2010;328(5975):232-235.
Differences in gene expression may play a major role in speciation and phenotypic diversity. We examined genome-wide differences in transcription factor (TF) binding in several humans and a single chimpanzee using chromatin immunoprecipitation followed by sequencing (ChIP-Seq). The binding sites of RNA Polymerase II (PolII) and a key regulator of immune responses, NFκB (p65), were mapped in ten lymphoblastoid cell lines and 25% and 7.5% of the respective binding regions were found to differ between individuals. Binding differences were frequently associated with SNPs and genomic structural variants (SVs) and were often correlated with differences in gene expression, suggesting functional consequences of binding variation. Furthermore, comparing PolII binding between human and chimpanzee suggests extensive divergence in TF binding. Our results indicate that many differences in individuals and species occur at the level of TF binding and provide insight into the genetic events responsible for these differences.
doi:10.1126/science.1183621
PMCID: PMC2938768  PMID: 20299548
5.  Distinct Genomic Aberrations Associated With ERG Rearranged Prostate Cancer 
Genes, chromosomes & cancer  2009;48(4):366-380.
Emerging molecular and clinical data suggest that ETS fusion prostate cancer represents a distinct molecular subclass, driven most commonly by a hormonally regulated promoter and characterized by an aggressive natural history. The study of the genomic landscape of prostate cancer in the light of ETS fusion events is required to understand the foundation of this molecularly and clinically distinct subtype. We performed genome-wide profiling of 49 primary prostate cancers and identified 20 recurrent chromosomal copy number aberrations, mainly occurring as genomic losses. Co-occurring events included losses at 19q13.32 and 1p22.1. We discovered 3 genomic events associated with ERG rearranged prostate cancer, affecting 6q, 7q, and 16q. 6q loss in non- rearranged prostate cancer is accompanied by gene expression deregulation in an independent dataset and by protein deregulation of MYO6. To analyze copy number alterations within the ETS genes, we performed a comprehensive analysis of all 27 ETS genes and of the 3Mbp genomic area between ERG and TMPRSS2 (21q) with an unprecedented resolution (30 bp). We demonstrate that high-resolution tiling arrays can be used to pin-point breakpoints leading to fusion events. This study provides further support to defining a distinct molecular subtype of prostate cancer based on the presence of ETS gene rearrangements.
doi:10.1002/gcc.20647
PMCID: PMC2674964  PMID: 19156837
ETS genes; prostate cancer; gain; loss
6.  The current excitement about copy-number variation: how it relates to gene duplication and protein families 
Following recent technological advances there has been an increasing interest in genome structural variation, in particular copy-number variants (CNVs) – large-scale duplications and deletions – in the human genome. Although not immediately evident, CNV surveys make a conceptual connection between the fields of population genetics and protein families, in particular with regard to the stability and expandability of families. The mechanisms giving rise to CNVs can be considered as fundamental processes underlying gene duplication and loss; duplicated genes being the results of “successful” copies, fixed and maintained in the population. Conversely, many “unsuccessful” duplicates remain in the genome as pseudogenes. Here, we survey studies on CNVs, highlighting issues related to protein families. In particular, CNVs tend to affect specific gene functional categories, such as those associated with environmental response, and are depleted in genes related to basic cellular processes. Furthermore, CNVs occur more often at the periphery of the protein interaction network. Thereby, functional categories associated with successful duplicates and unsuccessful duplicates are clearly distinguishable. These trends are likely reflective of CNV formation biases and natural selection, both of which differentially influence distinct protein families.
doi:10.1016/j.sbi.2008.02.005
PMCID: PMC2577873  PMID: 18511261
7.  Paired-End Mapping Reveals Extensive Structural Variation in the Human Genome 
Science (New York, N.Y.)  2007;318(5849):420-426.
Structural variation of the genome involves kilobase- to megabase-sized deletions, duplications, insertions, inversions, and complex combinations of rearrangements. We introduce high-throughput and massive paired-end mapping (PEM), a large-scale genome-sequencing method to identify structural variants (SVs) ~3 kilobases (kb) or larger that combines the rescue and capture of paired ends of 3-kb fragments, massive 454 sequencing, and a computational approach to map DNA reads onto a reference genome. PEM was used to map SVs in an African and in a putatively European individual and identified shared and divergent SVs relative to the reference genome. Overall, we fine-mapped more than 1000 SVs and documented that the number of SVs among humans is much larger than initially hypothesized; many of the SVs potentially affect gene function. The breakpoint junction sequences of more than 200 SVs were determined with a novel pooling strategy and computational analysis. Our analysis provided insights into the mechanisms of SV formation in humans.
doi:10.1126/science.1149504
PMCID: PMC2674581  PMID: 17901297
8.  PEMer: a computational framework with simulation-based error models for inferring genomic structural variants from massive paired-end sequencing data 
Genome Biology  2009;10(2):R23.
Paired-End Mapper (PEMer) enables mapping of genomic structural variants at considerably enhanced sensitivity, specificity and resolution over previous approaches.
Personal-genomics endeavors, such as the 1000 Genomes project, are generating maps of genomic structural variants by analyzing ends of massively sequenced genome fragments. To process these we developed Paired-End Mapper (PEMer; ). This comprises an analysis pipeline, compatible with several next-generation sequencing platforms; simulation-based error models, yielding confidence-values for each structural variant; and a back-end database. The simulations demonstrated high structural variant reconstruction efficiency for PEMer's coverage-adjusted multi-cutoff scoring-strategy and showed its relative insensitivity to base-calling errors.
doi:10.1186/gb-2009-10-2-r23
PMCID: PMC2688268  PMID: 19236709
9.  High-Resolution Copy-Number Variation Map Reflects Human Olfactory Receptor Diversity and Evolution 
PLoS Genetics  2008;4(11):e1000249.
Olfactory receptors (ORs), which are involved in odorant recognition, form the largest mammalian protein superfamily. The genomic content of OR genes is considerably reduced in humans, as reflected by the relatively small repertoire size and the high fraction (∼55%) of human pseudogenes. Since several recent low-resolution surveys suggested that OR genomic loci are frequently affected by copy-number variants (CNVs), we hypothesized that CNVs may play an important role in the evolution of the human olfactory repertoire. We used high-resolution oligonucleotide tiling microarrays to detect CNVs across 851 OR gene and pseudogene loci. Examining genomic DNA from 25 individuals with ancestry from three populations, we identified 93 OR gene loci and 151 pseudogene loci affected by CNVs, generating a mosaic of OR dosages across persons. Our data suggest that ∼50% of the CNVs involve more than one OR, with the largest CNV spanning 11 loci. In contrast to earlier reports, we observe that CNVs are more frequent among OR pseudogenes than among intact genes, presumably due to both selective constraints and CNV formation biases. Furthermore, our results show an enrichment of CNVs among ORs with a close human paralog or lacking a one-to-one ortholog in chimpanzee. Interestingly, among the latter we observed an enrichment in CNV losses over gains, a finding potentially related to the known diminution of the human OR repertoire. Quantitative PCR experiments performed for 122 sampled ORs agreed well with the microarray results and uncovered 23 additional CNVs. Importantly, these experiments allowed us to uncover nine common deletion alleles that affect 15 OR genes and five pseudogenes. Comparison to the chimpanzee reference genome revealed that all of the deletion alleles are human derived, therefore indicating a profound effect of human-specific deletions on the individual OR gene content. Furthermore, these deletion alleles may be used in future genetic association studies of olfactory inter-individual differences.
Author Summary
Copy-number variants (CNVs) are deletions and duplications of DNA segments, responsible for most of the genome variation in mammals. To help elucidate the impact of CNVs on evolution and function, we provide a high-resolution CNV map of the largest gene superfamily in humans, i.e., the olfactory receptor (OR) gene superfamily. Our map reveals twice as many olfactory CNVs per person than previously reported, indicating considerable OR dosage variations in humans. In particular, our findings indicate that CNVs are specifically enriched among evolutionary “young” ORs, some of which originated following the human-chimpanzee split, implying that CNVs may play an important role in the gene-birth and gene-loss processes that continuously shape the human OR repertoire. Furthermore, we describe 15 OR gene loci showing frequent human-specific deletion alleles. Additionally, we present evidence for a recent non-allelic homologous recombination event involving a pair of OR genes, forming a novel fusion OR that may harbor novel odorant-binding properties. Such events may potentially relate to individual functional “holes” in the human smell-detection repertoire, and future studies will address the specific chemosensory impact of our genomic variation map.
doi:10.1371/journal.pgen.1000249
PMCID: PMC2570968  PMID: 18989455
10.  Prediction of effective genome size in metagenomic samples 
Genome Biology  2007;8(1):R10.
A novel computational approach shows a link between genome size and habitat from analysis of environmental metagenomic DNA reads.
We introduce a novel computational approach to predict effective genome size (EGS; a measure that includes multiple plasmid copies, inserted sequences, and associated phages and viruses) from short sequencing reads of environmental genomics (or metagenomics) projects. We observe considerable EGS differences between environments and link this with ecologic complexity as well as species composition (for instance, the presence of eukaryotes). For example, we estimate EGS in a complex, organism-dense farm soil sample at about 6.3 megabases (Mb) whereas that of the bacteria therein is only 4.7 Mb; for bacteria in a nutrient-poor, organism-sparse ocean surface water sample, EGS is as low as 1.6 Mb. The method also permits evaluation of completion status and assembly bias in single-genome sequencing projects.
doi:10.1186/gb-2007-8-1-r10
PMCID: PMC1839125  PMID: 17224063
11.  Systematic Association of Genes to Phenotypes by Genome and Literature Mining 
PLoS Biology  2005;3(5):e134.
One of the major challenges of functional genomics is to unravel the connection between genotype and phenotype. So far no global analysis has attempted to explore those connections in the light of the large phenotypic variability seen in nature. Here, we use an unsupervised, systematic approach for associating genes and phenotypic characteristics that combines literature mining with comparative genome analysis. We first mine the MEDLINE literature database for terms that reflect phenotypic similarities of species. Subsequently we predict the likely genomic determinants: genes specifically present in the respective genomes. In a global analysis involving 92 prokaryotic genomes we retrieve 323 clusters containing a total of 2,700 significant gene–phenotype associations. Some clusters contain mostly known relationships, such as genes involved in motility or plant degradation, often with additional hypothetical proteins associated with those phenotypes. Other clusters comprise unexpected associations; for example, a group of terms related to food and spoilage is linked to genes predicted to be involved in bacterial food poisoning. Among the clusters, we observe an enrichment of pathogenicity-related associations, suggesting that the approach reveals many novel genes likely to play a role in infectious diseases.
The combination of text mining and comparative genomics is shown to be a powerful approach to predicting phenotypes that are associated with particular genes in bacterial genomes
doi:10.1371/journal.pbio.0030134
PMCID: PMC1073694  PMID: 15799710
13.  A Comprehensive Map of Mobile Element Insertion Polymorphisms in Humans 
PLoS Genetics  2011;7(8):e1002236.
As a consequence of the accumulation of insertion events over evolutionary time, mobile elements now comprise nearly half of the human genome. The Alu, L1, and SVA mobile element families are still duplicating, generating variation between individual genomes. Mobile element insertions (MEI) have been identified as causes for genetic diseases, including hemophilia, neurofibromatosis, and various cancers. Here we present a comprehensive map of 7,380 MEI polymorphisms from the 1000 Genomes Project whole-genome sequencing data of 185 samples in three major populations detected with two detection methods. This catalog enables us to systematically study mutation rates, population segregation, genomic distribution, and functional properties of MEI polymorphisms and to compare MEI to SNP variation from the same individuals. Population allele frequencies of MEI and SNPs are described, broadly, by the same neutral ancestral processes despite vastly different mutation mechanisms and rates, except in coding regions where MEI are virtually absent, presumably due to strong negative selection. A direct comparison of MEI and SNP diversity levels suggests a differential mobile element insertion rate among populations.
Author Summary
We embarked on this study to explore the 1000 Genomes Project (1000GP) pilot dataset as a substrate for Mobile Element Insertion (MEI) discovery and analysis. MEI is already well known as a significant component of genetic variation in the human population. However the full extent and effects of MEI can only be assessed by accurate detection in large whole-genome sequencing efforts such as the 1000GP. In this study we identified 7,380 distinct genomic locations of variant MEI and carried out rigorous validation experiments that confirmed the high accuracy of the detected events. We were able to measure the frequency of each variant in three continental population groups and found that inherited MEI variants propagate through populations in much the same way as single nucleotide polymorphisms, except that MEI are more strongly suppressed in protein coding parts of the genome. We also found evidence that the MEI mutation rate has not been constant over human population history, rather that different populations appear to have different characteristic MEI mutation rates.
doi:10.1371/journal.pgen.1002236
PMCID: PMC3158055  PMID: 21876680
14.  Systematic Inference of Copy-Number Genotypes from Personal Genome Sequencing Data Reveals Extensive Olfactory Receptor Gene Content Diversity 
PLoS Computational Biology  2010;6(11):e1000988.
Copy-number variations (CNVs) are widespread in the human genome, but comprehensive assignments of integer locus copy-numbers (i.e., copy-number genotypes) that, for example, enable discrimination of homozygous from heterozygous CNVs, have remained challenging. Here we present CopySeq, a novel computational approach with an underlying statistical framework that analyzes the depth-of-coverage of high-throughput DNA sequencing reads, and can incorporate paired-end and breakpoint junction analysis based CNV-analysis approaches, to infer locus copy-number genotypes. We benchmarked CopySeq by genotyping 500 chromosome 1 CNV regions in 150 personal genomes sequenced at low-coverage. The assessed copy-number genotypes were highly concordant with our performed qPCR experiments (Pearson correlation coefficient 0.94), and with the published results of two microarray platforms (95–99% concordance). We further demonstrated the utility of CopySeq for analyzing gene regions enriched for segmental duplications by comprehensively inferring copy-number genotypes in the CNV-enriched >800 olfactory receptor (OR) human gene and pseudogene loci. CopySeq revealed that OR loci display an extensive range of locus copy-numbers across individuals, with zero to two copies in some OR loci, and two to nine copies in others. Among genetic variants affecting OR loci we identified deleterious variants including CNVs and SNPs affecting ∼15% and ∼20% of the human OR gene repertoire, respectively, implying that genetic variants with a possible impact on smell perception are widespread. Finally, we found that for several OR loci the reference genome appears to represent a minor-frequency variant, implying a necessary revision of the OR repertoire for future functional studies. CopySeq can ascertain genomic structural variation in specific gene families as well as at a genome-wide scale, where it may enable the quantitative evaluation of CNVs in genome-wide association studies involving high-throughput sequencing.
Author Summary
Human individual genome sequencing has recently become affordable, enabling highly detailed genetic sequence comparisons. While the identification and genotyping of single-nucleotide polymorphisms has already been successfully established for different sequencing platforms, the detection, quantification and genotyping of large-scale copy-number variants (CNVs), i.e., losses or gains of long genomic segments, has remained challenging. We present a computational approach that enables detecting CNVs in sequencing data and accurately identifies the actual copy-number at which DNA segments of interest occur in an individual genome. This approach enabled us to obtain novel insights into the largest human gene family – the olfactory receptors (ORs) – involved in smell perception. While previous studies reported an abundance of CNVs in ORs, our approach enabled us to globally identify absolute differences in OR gene counts that exist between humans. While several OR genes have very high gene counts, other ORs are found only once or are missing entirely in some individuals. The latter have a particularly high probability of influencing individual differences in the perception of smell, a question that future experimental efforts can now address. Furthermore, we observed differences in OR gene counts between populations, pointing at ORs that might contribute to population-specific differences in smell.
doi:10.1371/journal.pcbi.1000988
PMCID: PMC2978733  PMID: 21085617
15.  The Baker's Yeast Diploid Genome Is Remarkably Stable in Vegetative Growth and Meiosis 
PLoS Genetics  2010;6(9):e1001109.
Accurate estimates of mutation rates provide critical information to analyze genome evolution and organism fitness. We used whole-genome DNA sequencing, pulse-field gel electrophoresis, and comparative genome hybridization to determine mutation rates in diploid vegetative and meiotic mutation accumulation lines of Saccharomyces cerevisiae. The vegetative lines underwent only mitotic divisions while the meiotic lines underwent a meiotic cycle every ∼20 vegetative divisions. Similar base substitution rates were estimated for both lines. Given our experimental design, these measures indicated that the meiotic mutation rate is within the range of being equal to zero to being 55-fold higher than the vegetative rate. Mutations detected in vegetative lines were all heterozygous while those in meiotic lines were homozygous. A quantitative analysis of intra-tetrad mating events in the meiotic lines showed that inter-spore mating is primarily responsible for rapidly fixing mutations to homozygosity as well as for removing mutations. We did not observe 1–2 nt insertion/deletion (in-del) mutations in any of the sequenced lines and only one structural variant in a non-telomeric location was found. However, a large number of structural variations in subtelomeric sequences were seen in both vegetative and meiotic lines that did not affect viability. Our results indicate that the diploid yeast nuclear genome is remarkably stable during the vegetative and meiotic cell cycles and support the hypothesis that peripheral regions of chromosomes are more dynamic than gene-rich central sections where structural rearrangements could be deleterious. This work also provides an improved estimate for the mutational load carried by diploid organisms.
Author Summary
Mutations result from errors that occur during DNA metabolism. They provide the raw materials for evolution, can affect organism fitness, and have been shown to accumulate in organisms during asexual growth. During a sexual life cycle, mutations can be removed by recombination and mating. While such removal is thought to provide a fitness advantage, studies have shown that recombination itself is mutagenic. To examine if the mutation rate in an organism differs during asexual and sexual cycles, we sequenced the entire nuclear genome of lines of diploid baker's yeast that underwent only asexual growth, or alternating cycles of asexual and sexual growth. The estimated rate of base substitutions in the vegetative lines was extremely low (2.9×10−10 base substitutions per base per cell generation) and the meiotic mutation rate is within the range of being equal to zero to being 55 times higher than the vegetative rate. Interestingly, we observed a large number of changes in the ends of chromosomes in the asexual and sexual cycles that did not affect fitness; changes at other locations were very rare, suggesting a remarkable genome stability of diploid baker's yeast.
doi:10.1371/journal.pgen.1001109
PMCID: PMC2936533  PMID: 20838597

Results 1-15 (15)