Search

Within the Netherlands a national network of biobanks has been established (Biobanking and Biomolecular Research Infrastructure-Netherlands (BBMRI-NL)) as a national node of the European BBMRI. One of the aims of BBMRI-NL is to enrich biobanks with different types of molecular and phenotype data. Here, we describe the Genome of the Netherlands (GoNL), one of the projects within BBMRI-NL. GoNL is a whole-genome-sequencing project in a representative sample consisting of 250 trio-families from all provinces in the Netherlands, which aims to characterize DNA sequence variation in the Dutch population. The parent-offspring trios include adult individuals ranging in age from 19 to 87 years (mean=53 years; SD=16 years) from birth cohorts 1910-1994. Sequencing was done on blood-derived DNA from uncultured cells and accomplished coverage was 14-15x. The family-based design represents a unique resource to assess the frequency of regional variants, accurately reconstruct haplotypes by family-based phasing, characterize short indels and complex structural variants, and establish the rate of de novo mutational events. GoNL will also serve as a reference panel for imputation in the available genome-wide association studies in Dutch and other cohorts to refine association signals and uncover population-specific variants. GoNL will create a catalog of human genetic variation in this sample that is uniquely characterized with respect to micro-geographic location and a wide range of phenotypes. The resource will be made available to the research and medical community to guide the interpretation of sequencing projects. The present paper summarizes the global characteristics of the project.

Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and posttranscriptional levels. Here, we integrated in-depth transcriptome and proteome analyses of liver tissues from two rat strains to unravel the interactions within and between these layers. We obtained peptide evidence for 26,463 rat liver proteins. We validated 1,195 gene predictions, 83 splice events, 126 proteins with nonsynonymous variants, and 20 isoforms with nonsynonymous RNA editing. Quantitative RNA sequencing and proteomics data correlate highly between strains but poorly among each other, indicating extensive nongenetic regulation. Our multilevel analysis identified a genomic variant in the promoter of the most differentially expressed gene Cyp17a1, a previously reported top hit in genome-wide association studies for human hypertension, as a potential contributor to the hypertension phenotype in SHR rats. These results demonstrate the power of and need for integrative analysis for understanding genetic control of molecular dynamics and phenotypic diversity in a system-wide manner., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

It has been postulated that aging is the consequence of an accelerated accumulation of somatic DNA mutations and that subsequent errors in the primary structure of proteins ultimately reach levels sufficient to affect organismal functions. The technical limitations of detecting somatic changes and the lack of insight about the minimum level of erroneous proteins to cause an error catastrophe hampered any firm conclusions on these theories. In this study, we sequenced the whole genome of DNA in whole blood of two pairs of monozygotic (MZ) twins, 40 and 100 years old, by two independent next-generation sequencing (NGS) platforms (Illumina and Complete Genomics). Potentially discordant single-base substitutions supported by both platforms were validated extensively by Sanger, Roche 454, and Ion Torrent sequencing. We demonstrate that the genomes of the two twin pairs are germ-line identical between co-twins, and that the genomes of the 100-year-old MZ twins are discerned by eight confirmed somatic single-base substitutions, five of which are within introns. Putative somatic variation between the 40-year-old twins was not confirmed in the validation phase. We conclude from this systematic effort that by using two independent NGS platforms, somatic single nucleotide substitutions can be detected, and that a century of life did not result in a large number of detectable somatic mutations in blood. The low number of somatic variants observed by using two NGS platforms might provide a framework for detecting disease-related somatic variants in phenotypically discordant MZ twins.

Large numbers of inbred laboratory rat strains have been developed for a range of complex disease phenotypes. To gain insights into the evolutionary pressures underlying selection for these phenotypes, we sequenced the genomes of 27 rat strains, including 11 models of hypertension, diabetes, and insulin resistance, along with their respective control strains. Altogether, we identified more than 13 million single-nucleotide variants, indels, and structural variants across these rat strains. Analysis of strain-specific selective sweeps and gene clusters implicated genes and pathways involved in cation transport, angiotensin production, and regulators of oxidative stress in the development of cardiovascular disease phenotypes in rats. Many of the rat loci that we identified overlap with previously mapped loci for related traits in humans, indicating the presence of shared pathways underlying these phenotypes in rats and humans. These data represent a step change in resources available for evolutionary analysis of complex traits in disease models., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Genetic mapping on fully sequenced individuals is transforming understanding of the relationship between molecular variation and variation in complex traits. Here we report a combined sequence and genetic mapping analysis in outbred rats that maps 355 quantitative trait loci for 122 phenotypes. We identify 35 causal genes involved in 31 phenotypes, implicating new genes in models of anxiety, heart disease and multiple sclerosis. The relationship between sequence and genetic variation is unexpectedly complex: at approximately 40% of quantitative trait loci, a single sequence variant cannot account for the phenotypic effect. Using comparable sequence and mapping data from mice, we show that the extent and spatial pattern of variation in inbred rats differ substantially from those of inbred mice and that the genetic variants in orthologous genes rarely contribute to the same phenotype in both species.

Background: The ability to accurately detect DNA copy number variation in both a sensitive and quantitative manner is important in many research areas. However, genome-wide DNA copy number analyses are complicated by variations in detection signal., Results: While GC content has been used to correct for this, here we show that coverage biases are tissue-specific and independent of the detection method as demonstrated by next-generation sequencing and array CGH. Moreover, we show that DNA isolation stringency affects the degree of equimolar coverage and that the observed biases coincide with chromatin characteristics like gene expression, genomic isochores, and replication timing., Conclusion: These results indicate that chromatin organization is a main determinant for differential DNA retrieval. These findings are highly relevant for germline and somatic DNA copy number variation analyses.

Background: Paired-tag sequencing approaches are commonly used for the analysis of genome structure. However, mammalian genomes have a complex organization with a variety of repetitive elements that complicate comprehensive genome-wide analyses., Results: Here, we systematically assessed the utility of paired-end and mate-pair (MP) next-generation sequencing libraries with insert sizes ranging from 170 bp to 25 kb, for genome coverage and for improving scaffolding of a mammalian genome (Rattus norvegicus). Despite a lower library complexity, large insert MP libraries (20 or 25 kb) provided very high physical genome coverage and were found to efficiently span repeat elements in the genome. Medium-sized (5, 8 or 15 kb) MP libraries were much more efficient for genome structure analysis than the more commonly used shorter insert paired-end and 3 kb MP libraries. Furthermore, the combination of medium- and large insert libraries resulted in a 3-fold increase in N50 in scaffolding processes. Finally, we show that our data can be used to evaluate and improve contig order and orientation in the current rat reference genome assembly., Conclusions: We conclude that applying combinations of mate-pair libraries with insert sizes that match the distributions of repetitive elements improves contig scaffolding and can contribute to the finishing of draft genomes.

The use of a priori knowledge in the alignment of targeted sequencing data is investigated using computational experiments. Adapting a Needleman-Wunsch algorithm to incorporate the genomic position information from the targeted capture, we demonstrate that alignment can be done to just the target region of interest. When in addition use is made of direct string comparison, an improvement of up to a factor of 8 in alignment speed compared to the fastest conventional aligner (Bowtie) is obtained. This results in a total alignment time in targeted sequencing of around 7 min for aligning approximately 56 million captured reads. For conventional aligners such as Bowtie, BWA or MAQ, alignment to just the target region is not feasible as experiments show that this leads to an additional 88% SNP calls, the vast majority of which are false positives (≈ 92%).

Chromothripsis represents a novel phenomenon in the structural variation landscape of cancer genomes. Here, we analyze the genomes of ten patients with congenital disease who were preselected to carry complex chromosomal rearrangements with more than two breakpoints. The rearrangements displayed unanticipated complexity resembling chromothripsis. We find that eight of them contain hallmarks of multiple clustered double-stranded DNA breaks (DSBs) on one or more chromosomes. In addition, nucleotide resolution analysis of 98 breakpoint junctions indicates that break repair involves nonhomologous or microhomology-mediated end joining. We observed that these eight rearrangements are balanced or contain sporadic deletions ranging in size between a few hundred base pairs and several megabases. The two remaining complex rearrangements did not display signs of DSBs and contain duplications, indicative of rearrangement processes involving template switching. Our work provides detailed insight into the characteristics of chromothripsis and supports a role for clustered DSBs driving some constitutional chromothripsis rearrangements., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: With the advent of next generation sequencing it has become possible to detect genomic variation on a large scale. However, predicting which genomic variants are damaging to gene function remains a challenge, as knowledge of the effects of genomic variation on gene expression is still limited. Recombinant inbred panels are powerful tools to study the cis and trans effects of genetic variation on molecular phenotypes such as gene expression., Results: We generated a comprehensive inventory of genomic differences between the two founder strains of the rat HXB/BXH recombinant inbred panel: SHR/OlaIpcv and BN-Lx/Cub. We identified 3.2 million single nucleotide variants, 425,924 small insertions and deletions, 907 copy number changes and 1,094 large structural genetic variants. RNA-sequencing analyses on liver tissue of the two strains identified 532 differentially expressed genes and 40 alterations in transcript structure. We identified both coding and non-coding variants that correlate with differential expression and alternative splicing. Furthermore, structural variants, in particular gene duplications, show a strong correlation with transcriptome alterations., Conclusions: We show that the panel is a good model for assessing the genetic basis of phenotypic heterogeneity and for providing insights into possible underlying molecular mechanisms. Our results reveal a high diversity and complexity underlying quantitative and qualitative transcriptional differences.

Background: Structural rearrangements form a major class of somatic variation in cancer genomes. Local chromosome shattering, termed chromothripsis, is a mechanism proposed to be the cause of clustered chromosomal rearrangements and was recently described to occur in a small percentage of tumors. The significance of these clusters for tumor development or metastatic spread is largely unclear., Results: We used genome-wide long mate-pair sequencing and SNP array profiling to reveal that chromothripsis is a widespread phenomenon in primary colorectal cancer and metastases. We find large and small chromothripsis events in nearly every colorectal tumor sample and show that several breakpoints of chromothripsis clusters and isolated rearrangements affect cancer genes, including NOTCH2, EXO1 and MLL3. We complemented the structural variation studies by sequencing the coding regions of a cancer exome in all colorectal tumor samples and found somatic mutations in 24 genes, including APC, KRAS, SMAD4 and PIK3CA. A pairwise comparison of somatic variations in primary and metastatic samples indicated that many chromothripsis clusters, isolated rearrangements and point mutations are exclusively present in either the primary tumor or the metastasis and may affect cancer genes in a lesion-specific manner., Conclusions: We conclude that chromothripsis is a prevalent mechanism driving structural rearrangements in colorectal cancer and show that a complex interplay between point mutations, simple copy number changes and chromothripsis events drive colorectal tumor development and metastasis.

G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies.

A variety of mutational mechanisms shape the dynamic architecture of human genomes and occasionally result in congenital defects and disease. Here, we used genome-wide long mate-pair sequencing to systematically screen for inherited and de novo structural variation in a trio including a child with severe congenital abnormalities. We identified 4321 inherited structural variants and 17 de novo rearrangements. We characterized the de novo structural changes to the base-pair level revealing a complex series of balanced inter- and intra-chromosomal rearrangements consisting of 12 breakpoints involving chromosomes 1, 4 and 10. Detailed inspection of breakpoint regions indicated that a series of simultaneous double-stranded DNA breaks caused local shattering of chromosomes. Fusion of the resulting chromosomal fragments involved non-homologous end joining, since junction points displayed limited or no homology and small insertions and deletions. The pattern of random joining of chromosomal fragments that we observe here strongly resembles the somatic rearrangement patterns--termed chromothripsis--that have recently been described in deranged cancer cells. We conclude that a similar mechanism may also drive the formation of de novo structural variation in the germline.

Background: Mutagenesis screens in the mouse have been proven useful for the identification of novel gene functions and generation of interesting mutant alleles. Here we describe a phenotype-based screen for recessive mutations affecting embryonic development., Methodology/principal Findings: Mice were mutagenized with N-ethyl-N-nitrosourea (ENU) and following incrossing the offspring, embryos were analyzed at embryonic day 10.5. Mutant phenotypes that arose in our screen include cardiac and nuchal edema, neural tube defects, situs inversus of the heart, posterior truncation and the absence of limbs and lungs. We isolated amongst others novel mutant alleles for Dll1, Ptprb, Plexin-B2, Fgf10, Wnt3a, Ncx1, Scrib(Scrib, Scribbled homolog [Drosophila]) and Sec24b. We found both nonsense alleles leading to severe protein truncations and mutants with single-amino acid substitutions that are informative at a molecular level. Novel findings include an ectopic neural tube in our Dll1 mutant and lung defects in the planar cell polarity mutants for Sec24b and Scrib., Conclusions/significance: Using a forward genetics approach, we have generated a number of novel mutant alleles that are linked to disturbed morphogenesis during development.

Down's syndrome (DS), resulting from an additional copy of chromosome 21 (trisomy 21), is frequently associated with congenital heart defects (CHDs). Although the increased dosage of chromosome 21 sequences is likely to be part of the etiology of cardiac defects, only a proportion of DS patients exhibit a congenital heart defect (birth prevalence 40-60%). Through a large-candidate gene-sequencing screen in patients with atrioventricular septal defects, substitutions were identified in bone morphogenetic protein (BMP) type I receptor ALK2 and two other genes in a patient with DS and a primum-type atrial septal defect. Structural modeling of the cytoplasmic domain of the ALK2 receptor suggests that H286 is in close proximity to the nucleotide-binding site of the kinase domain. We investigated whether this p.His286Asp substitution altered ALK2 function by using both in vitro as well as in vivo assays. The p.His286Asp variant demonstrated impaired functional activity as measured by BMP-specific transcriptional response assays. Furthermore, mild dominant-interfering activity was observed in vivo compared with wild-type ALK2 as determined by RNA injection into zebrafish embryos. These data indicate that in the context of a DS background, ALK2-mediated reduction of BMP signaling may contribute to CHDs.

Despite considerable genetic and genomic resources the positional cloning of forward mutations remains a slow and manually intensive task, typically using gel based genotyping and sequential rounds of mapping. We have used the latest genetic resources and genotyping technologies to develop two commercially available SNP panels of thousands of markers that can be used to speed up positional cloning., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Immunoprecipitated crosslinked protein-DNA fragments typically range in size from several hundred to several thousand base pairs, with a significant part of chromatin being much longer than the optimal length for next-generation sequencing (NGS) procedures. Because these larger fragments may be non-random and represent relevant biology that may otherwise be missed, but also because they represent a significant fraction of the immunoprecipitated material, we designed a double-fragmentation ChIP-seq procedure. After conventional crosslinking and immunoprecipitation, chromatin is de-crosslinked and sheared a second time to concentrate fragments in the optimal size range for NGS. Besides the benefits of increased chromatin yields, the procedure also eliminates a laborious size-selection step. We show that the double-fragmentation ChIP-seq approach allows for the generation of biologically relevant genome-wide protein-DNA binding profiles from sub-nanogram amounts of TCF7L2/TCF4, TBP and H3K4me3 immunoprecipitated material. Although optimized for the AB/SOLiD platform, the same approach may be applied to other platforms.

The spontaneously hypertensive rat (SHR) is the most widely studied animal model of hypertension. Scores of SHR quantitative loci (QTLs) have been mapped for hypertension and other phenotypes. We have sequenced the SHR/OlaIpcv genome at 10.7-fold coverage by paired-end sequencing on the Illumina platform. We identified 3.6 million high-quality single nucleotide polymorphisms (SNPs) between the SHR/OlaIpcv and Brown Norway (BN) reference genome, with a high rate of validation (sensitivity 96.3%-98.0% and specificity 99%-100%). We also identified 343,243 short indels between the SHR/OlaIpcv and reference genomes. These SNPs and indels resulted in 161 gain or loss of stop codons and 629 frameshifts compared with the BN reference sequence. We also identified 13,438 larger deletions that result in complete or partial absence of 107 genes in the SHR/OlaIpcv genome compared with the BN reference and 588 copy number variants (CNVs) that overlap with the gene regions of 688 genes. Genomic regions containing genes whose expression had been previously mapped as cis-regulated expression quantitative trait loci (eQTLs) were significantly enriched with SNPs, short indels, and larger deletions, suggesting that some of these variants have functional effects on gene expression. Genes that were affected by major alterations in their coding sequence were highly enriched for genes related to ion transport, transport, and plasma membrane localization, providing insights into the likely molecular and cellular basis of hypertension and other phenotypes specific to the SHR strain. This near complete catalog of genomic differences between two extensively studied rat strains provides the starting point for complete elucidation, at the molecular level, of the physiological and pathophysiological phenotypic differences between individuals from these strains.

Microarray-based enrichment of selected genomic loci is a powerful method for genome complexity reduction for next-generation sequencing. Since the vast majority of exons in vertebrate genomes are smaller than 150 nt, we explored the use of short fragment libraries (85-110 bp) to achieve higher enrichment specificity by reducing carryover and adverse effects of flanking intronic sequences. High enrichment specificity (60-75%) was obtained with a relative even base coverage. Up to 98% of the target-sequence was covered more than 20x at an average coverage depth of about 200x. To verify the accuracy of SNP/mutation detection, we evaluated 384 known non-reference SNPs in the targeted regions. At approximately 200x average sequence coverage, we were able to survey 96.4% of 1.69 Mb of genomic sequence with only 4.2% false negative calls, mostly due to low coverage. Using the same settings, a total of 1197 novel candidate variants were detected. Verification experiments revealed only eight false positive calls, indicating an overall false positive rate of less than 1 per approximately 200,000 bp. Taken together, short fragment libraries provide highly efficient and flexible enrichment of exonic targets and yield relatively even base coverage, which facilitates accurate SNP and mutation detection. Raw sequencing data, alignment files and called SNPs have been submitted into GEO database http://www.ncbi.nlm.nih.gov/geo/ with accession number GSE18542.

Motivation: ChIP-chip and ChIP-seq technologies provide genome-wide measurements of various types of chromatin marks at an unprecedented resolution. With ChIP samples collected from different tissue types and/or individuals, we can now begin to characterize stochastic or systematic changes in epigenetic patterns during development (intra-individual) or at the population level (inter-individual). This requires statistical methods that permit a simultaneous comparison of multiple ChIP samples on a global as well as locus-specific scale. Current analytical approaches are mainly geared toward single sample investigations, and therefore have limited applicability in this comparative setting. This shortcoming presents a bottleneck in biological interpretations of multiple sample data., Results: To address this limitation, we introduce a parametric classification approach for the simultaneous analysis of two (or more) ChIP samples. We consider several competing models that reflect alternative biological assumptions about the global distribution of the data. Inferences about locus-specific and genome-wide chromatin differences are reached through the estimation of multivariate mixtures. Parameter estimates are obtained using an incremental version of the Expectation-Maximization algorithm (IEM). We demonstrate efficient scalability and application to three very diverse ChIP-chip and ChIP-seq experiments. The proposed approach is evaluated against several published ChIP-chip and ChIP-seq software packages. We recommend its use as a first-pass algorithm to identify candidate regions in the epigenome, possibly followed by some type of second-pass algorithm to fine-tune detected peaks in accordance with biological or technological criteria., Availability: R source code is available at http://gbic.biol.rug.nl/supplementary/2009/ChromatinProfiles/. Access to Chip-seq data: GEO repository GSE17937.

The biological role and structure-function relationship of the Na(+)Ca(2+) exchanger NCX1 have been the subject of much investigation. Subtle mutagenesis to study the function of a protein seems only feasible in in vitro systems, but genetic forward screens have the potential to provide in vivo models to study single amino acid substitutions. In a genetic screen in mouse, we have isolated a mutant line carrying a novel mutant allele of the mouse Ncx1 gene. In this allele, a point mutation causes the substitution of a highly conserved asparagine residue (N874) with lysine. Accepted models for NCX1 structure propose that the affected amino acid is located in one of the reentrant membrane loops and experiments in vitro have identified N874 as critical for the ion transport function of NCX1. We found severe circulation defects and defective placentation in homozygous Ncx1(N87K4) mutant embryos, making the phenotype essentially indistinguishable from those of previously described null mutants. By ex vivo analysis, we demonstrated intrinsic functional abnormalities of cardiomyocytes. Western blot analysis and immunohistochemistry demonstrated normal levels and subcellular localization of the altered protein, ruling out the possibility that the abnormalities are a mere consequence of a major disturbance of protein structure. This study confirms and extends studies in vitro indicating the significance of amino acid N874 for the function of the NCX1 protein. It provides an in vivo model for this mutation and demonstrates the potential of forward genetic screens in a mammalian system.

Metazoan genomes contain thousands of sequence tracts that match the guanine-quadruplex (G4) DNA signature G(3)N(x)G(3)N(x)G(3)N(x)G(3), a motif that is intrinsically mutagenic, probably because it can form secondary structures during DNA replication. Here we show how and to what extent this feature can be used to generate deletion alleles of many Caenorhabditis elegans genes.

Background: Serious congenital heart defects occur as a result of improper atrioventricular septum (AVS) development during embryogenesis. Despite extensive knowledge of the genetic control of AVS development, few genetic lesions have been identified that are responsible for AVS-associated congenital heart defects., Methods and Results: We sequenced 32 genes known to be important in AVS development in patients with AVS defects and identified 11 novel coding single-nucleotide polymorphisms that are predicted to impair protein function. We focused on variants identified in the bone morphogenetic protein receptor, ALK2, and subjected 2 identified variants to functional analysis. The coding single-nucleotide polymorphisms R307L and L343P are heterozygous missense substitutions and were each identified in single individuals. The L343P allele had impaired functional activity as measured by in vitro kinase and bone morphogenetic protein-specific transcriptional response assays and dominant-interfering activity in vivo. In vivo analysis of zebrafish embryos injected with ALK2 L343P RNA revealed improper atrioventricular canal formation., Conclusions: These data identify the dominant-negative allele ALK2 L343P in a patient with AVS defects.

The presence of copy number variants in normal genomes poses a challenge to identify small genuine somatic copy number changes in high-resolution cancer genome profiling studies due to the use of unpaired reference DNA. Another problem is the well-known rearrangements of immunoglobulin and T-cell receptor genes in lymphocytes (a commonly used reference), which may misdirect the researcher to a locus with no relevance in tumorigenesis. We here show real gains of the IgG heavy chain V gene region in carcinogen-induced rat mammary tumor samples after normalization to paired mammary gland, a tissue without lymphocyte infiltration. We further show that the segmental duplication region encompassing the IgG heavy chain V genes is a copy number variant between the susceptible (SS) and the resistant (BN) to mammary tumor development inbred rat strains. Our data suggest that the already inherently unstable genomic region is a convenient target for additional structural rearrangements (gains) at the somatic level when exposed to a carcinogen (7,12-dimethylbenz[a]anthracene), which subsequently seem to benefit tumor development in the mammary gland of the susceptible strain. Thus, the selection of an appropriate reference DNA enabled us to identify immunoglobulin genes as novel cancer targets playing a role in mammary tumor development. We conclude that control DNA in array-based comparative genomic hybridization experiments should be selected with care, and DNA from pooled spleen (contains immature lymphocytes and is used as reference in animal studies) or blood may not be the ideal control in the study of primary tumors.

Rapid advances in DNA sequencing improve existing techniques and enable new approaches in genetics and functional genomics, bringing about unprecedented coverage, resolution and sensitivity. Enhanced toolsets can facilitate the untangling of connections between genomic variation, environmental factors and phenotypic effects, providing novel opportunities, but may also pose challenges in data interpretation, especially in highly heterogeneous human populations. Laboratory rodent strains, however, offer a variety of tailored model systems with controlled genetic backgrounds, facilitating complex genotype/phenotype relationship studies. In this review we discuss the advent of massively parallel sequencing, its methodological advantage for molecular analysis in model organisms and the expectation of increased understanding of biologically relevant consequences of human genetic variation.

The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt target Achaete scute-like 2 (Ascl2). Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic crypts on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the Lgr5 stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate.

Background: The laboratory rat (Rattus norvegicus) is one of the preferred model organisms in physiological and pharmacological research, although the availability of specific genetic models, especially gene knockouts, is limited. N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis is currently the most successful method in rats, although it is still very laborious and expensive., Results: As ENU-induced DNA damage is normally recognized by the mismatch repair (MMR) system, we hypothesized that the effectiveness of the target-selected mutagenesis approach could be improved by using a MMR-deficient genetic background. Indeed, Msh6 knockout rats were found to be more sensitive to ENU treatment and the germ line mutation rate was boosted more than two-fold to 1 mutation per 585 kb. In addition, the molecular mutation spectrum was found to be changed in favor of generating knockout-type alleles by approximately 20%, resulting in an overall increase in efficiency of approximately 2.5 fold. The improved effectiveness was demonstrated by high throughput mutation discovery in 70 Mb of sequence in a set of only 310 mutant F1 rats. This resulted in the identification of 89 mutations of which four introduced a premature stopcodon and 64 resulted in amino acid changes., Conclusion: Taken together, we show that the use of a MMR-deficient background considerably improves ENU-driven target-selected mutagenesis in the rat, thereby reducing animal use as well as screening costs. The use of a mismatch repair-deficient genetic background for improving mutagenesis and target-selected knockout efficiency is in principle applicable to any organism of interest.

To safeguard genetic integrity, cells have evolved an accurate but not failsafe mechanism of DNA replication. Not all DNA sequences tolerate DNA replication equally well [1]. Also, genomic regions that impose structural barriers to the DNA replication fork are a potential source of genetic instability [2, 3]. Here, we demonstrate that G4 DNA-a sequence motif that folds into quadruplex structures in vitro [4, 5]-is highly mutagenic in vivo and is removed from genomes that lack dog-1, the C. elegans ortholog of mammalian FANCJ [6, 7], which is mutated in Fanconi anemia patients [8-11]. We show that sequences that match the G4 DNA signature G3-5N1-3G3-5N1-3G3-5N1-3G3-5 are deleted in germ and somatic tissues of dog-1 animals. Unbiased aCGH analyses of dog-1 genomes that were allowed to accumulate mutations in >100 replication cycles indicate that deletions are found exclusively at G4 DNA; deletion frequencies can reach 4% per site per animal generation. We found that deletion sizes fall short of Okazaki fragment lengths [12], and no significant microhomology was observed at deletion junctions. The existence of 376,000 potentially mutagenic G4 DNA sites in the human genome could have major implications for the etiology of hereditary FancJ and nonhereditary cancers.

The abundance and dynamics of copy number variants (CNVs) in mammalian genomes poses new challenges in the identification of their impact on natural and disease phenotypes. We used computational and experimental methods to catalog CNVs in rat and found that they share important functional characteristics with those in human. In addition, 113 one-to-one orthologous genes overlap CNVs in both human and rat, 80 of which are implicated in human disease. CNVs are nonrandomly distributed throughout the genome. Chromosome 18 is a cold spot for CNVs as well as evolutionary rearrangements and segmental duplications, suggesting stringent selective mechanisms underlying CNV genesis or maintenance. By exploiting gene expression data available for rat recombinant inbred lines, we established the functional relationship of CNVs underlying 22 expression quantitative trait loci. These characteristics make the rat an excellent model for studying phenotypic effects of structural variation in relation to human complex traits and disease.

The laboratory rat is one of the most extensively studied model organisms. Inbred laboratory rat strains originated from limited Rattus norvegicus founder populations, and the inherited genetic variation provides an excellent resource for the correlation of genotype to phenotype. Here, we report a survey of genetic variation based on almost 3 million newly identified SNPs. We obtained accurate and complete genotypes for a subset of 20,238 SNPs across 167 distinct inbred rat strains, two rat recombinant inbred panels and an F2 intercross. Using 81% of these SNPs, we constructed high-density genetic maps, creating a large dataset of fully characterized SNPs for disease gene mapping. Our data characterize the population structure and illustrate the degree of linkage disequilibrium. We provide a detailed SNP map and demonstrate its utility for mapping of quantitative trait loci. This community resource is openly available and augments the genetic tools for this workhorse of physiological studies.

Wnt signaling activates gene expression through the induced formation of complexes between DNA-binding T-cell factors (TCFs) and the transcriptional coactivator beta-catenin. In colorectal cancer, activating Wnt pathway mutations transform epithelial cells through the inappropriate activation of a TCF7L2/TCF4 target gene program. Through a DNA array-based genome-wide analysis of TCF4 chromatin occupancy, we have identified 6,868 high-confidence TCF4-binding sites in the LS174T colorectal cancer cell line. Most TCF4-binding sites are located at large distances from transcription start sites, while target genes are frequently "decorated" by multiple binding sites. Motif discovery algorithms define the in vivo-occupied TCF4-binding site as evolutionarily conserved A-C/G-A/T-T-C-A-A-A-G motifs. The TCF4-binding regions significantly correlate with Wnt-responsive gene expression profiles derived from primary human adenomas and often behave as beta-catenin/TCF4-dependent enhancers in transient reporter assays.

Background: The laboratory rat (Rattus norvegicus) is an important model for human disease, and is extensively used for studying complex traits for example in the physiological and pharmacological fields. To facilitate genetic studies like QTL mapping, genetic makers that can be easily typed, like SNPs, are essential., Results: A genome-wide set of 820 SNP assays was designed for the KASPar genotyping platform, which uses a technique based on allele specific oligo extension and energy transfer-based detection. SNPs were chosen to be equally spread along all chromosomes except Y and to be polymorphic between Brown Norway and SS or Wistar rat strains based on data from the rat HapMap EU project. This panel was tested on 38 rats of 34 different strains and 3 wild rats to determine the level of polymorphism and to generate a phylogenetic network to show their genetic relationships. As a proof of principle we used this panel to map an obesity trait in Zucker rats and confirmed significant linkage (LOD 122) to chromosome 5: 119-129 Mb, where the leptin receptor gene (Lepr) is located (chr5: 122 Mb)., Conclusion: We provide a fast and cost-effective platform for genome-wide SNP typing, which can be used for first-pass genetic mapping and association studies in a wide variety of rat strains.

Reverse genetic or gene-driven knockout approaches have contributed significantly to the success of model organisms for fundamental and biomedical research. Although various technologies are available for C. elegans, none of them scale very well for genome-wide application. To address this, we implemented a target-selected knockout approach that is based on random chemical mutagenesis and detection of single nucleotide mutations in genes of interest using high-throughput resequencing. A clonal library of 6144 EMS-mutagenized worms was established and screened, resulting in the identification of 1044 induced mutations in 109 Mbp, which translates into an average spacing between exonic mutations in the library of only 17 bp. We covered 25% of the open reading frames of 32 genes and identified one or more inactivating mutations (nonsense or splice site) in 84% of them. Extrapolation of our results indicates that nonsense mutations for >90% of all C. elegans genes are present in the library. To identify all of these mutations, one only needs to inspect those positions that--given the known specificity of the mutagen--can result in the introduction of a stop codon. We define these positions as nonsense introducing mutations (NIMs). The genome-wide collection of possible NIMs can be calculated for any organism with a sequenced genome and reduces the screening complexity by 200- to 2000-fold, depending on the organism and mutagen. For EMS-mutagenized C. elegans, there are only approximately 500,000 NIMs. We show that a NIM genotyping approach employing high-density microarrays can, in principle, be used for the genome-wide identification of C. elegans knockouts.

Prediction of transcription factor binding sites (TFBS) is commonly used to formulate working hypotheses for experimental studies on gene regulation. Computational identification of functional TFBS is complicated because of short length and degeneracy of sequence motifs recognized by transcription factors. Information on conservation of predicted sites in orthologous sequences from different species (phylogenetic footprinting) can be used to distinguish potentially functional elements from background predictions. Results of phylogenetic footprinting may substantially depend on the algorithm used to construct an alignment of orthologous sequences, from which conservation of predicted TFBS is estimated. The CONREAL web server allows prediction and comparison of conserved TFBS based on AVID, BLASTZ, CONREAL, and LAGAN alignments. The web tool is particularly suited for the analysis of individual genes or genomic regions, although the underlying algorithm can also be used in high-throughput promoter analysis.

MicroRNAs are 20- to 23-nucleotide RNA molecules that can regulate gene expression. Currently > 400 microRNAs have been experimentally identified in mammalian genomes, whereas estimates go up to 1000 and beyond. Here we show that many more mammalian microRNAs exist. We discovered novel microRNA candidates using two approaches: testing of computationally predicted microRNAs by a modified microarray-based detection system, and cloning and sequencing of large numbers of small RNAs from different human and mouse tissues. Together these efforts experimentally identified 348 novel mouse and 81 novel human microRNA candidate genes. Most novel microRNAs candidates are not conserved beyond mammals, and ~10% are taxon-specific. Our analyses indicate that the entire microRNA repertoire is not remotely exhausted.

Genetic variation in genomes is organized in haplotype blocks, and species-specific block structure is defined by differential contribution of population history effects in combination with mutation and recombination events. Haplotype maps characterize the common patterns of linkage disequilibrium in populations and have important applications in the design and interpretation of genetic experiments. Although evolutionary processes are known to drive the selection of individual polymorphisms, their effect on haplotype block structure dynamics has not been shown. Here, we present a high-resolution haplotype map for a 5-megabase genomic region in the rat and compare it with the orthologous human and mouse segments. Although the size and fine structure of haplotype blocks are species dependent, there is a significant interspecies overlap in structure and a tendency for blocks to encompass complete genes. Extending these findings to the complete human genome using haplotype map phase I data reveals that linkage disequilibrium values are significantly higher for equally spaced positions in genic regions, including promoters, as compared to intergenic regions, indicating that a selective mechanism exists to maintain combinations of alleles within potentially interacting coding and regulatory regions. Although this characteristic may complicate the identification of causal polymorphisms underlying phenotypic traits, conservation of haplotype structure may be employed for the identification and characterization of functionally important genomic regions., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

Although zebrafish was introduced as a laboratory model organism several decades ago and now serves as a primary model for developmental biology, there is only limited data on its genetic variation. An establishment of a dense polymorphism map becomes a requirement for effective linkage analysis and cloning approaches in zebrafish. By comparing ESTs to whole-genome shotgun data, we predicted >50,000 high-quality candidate SNPs covering the zebrafish genome with average resolution of 41 kbp. We experimentally validated approximately 65% of a randomly sampled subset by genotyping 16 samples from seven commonly used zebrafish strains. The analysis reveals very high nucleotide diversity between zebrafish isolates. Even with the limited number of samples that we genotyped, zebrafish isolates revealed considerable interstrain variation, ranging from 7% (inbred) to 37% (wild-derived) of polymorphic sites being heterozygous. The increased proportion of polymorphic over monomorphic sites results in five times more frequent observation of a three allelic variant compared with human or mouse. Phylogenetic analysis shows that comparisons between even the least divergent strains used in our analysis may provide one informative marker approximately every 500 nucleotides. Furthermore, the number of haplotypes per locus is relatively large, reflecting independent establishment of the different lines from wild isolates. Finally, our results suggest the presence of prominent C-to-U and A-to-I RNA editing events in zebrafish. Overall, the levels and organization of genetic variation between and within commonly used zebrafish strains are markedly different from other laboratory model organisms, which may affect experimental design and interpretation.

Objective: The rat is one of the most important model organisms for biomedical and pharmacological research. However, the generation of novel models for studying specific aspects of human diseases largely depends on selection for specific traits using existing rat strains, thereby solely depending on naturally occurring variation. This study aims to provide the tools to manipulate the rat genome in a more directed way., Methods: We developed robust, automated, and scaleable reverse genetic methodology based on ENU (N-ethyl-N-nitrosourea)-driven target-selected mutagenesis. Optimal mutagenesis conditions have been determined in three different rat strains and a universal, rapid, and cost-effective dideoxy resequencing-based screening setup was established for mutation discovery. The effectiveness of the approach is illustrated by the identification of 120 induced mutations in a set of genes of interest, including six that result in unique rat knockout models due to the introduction of premature stop codons. In addition, 56 mutations were found that change amino acids, including critical residues in transmembrane domains of receptors and channels., Conclusions: The approach described here allows for the systematic generation of knockout and protein function altering alleles in the rat. The resulting induced rat models will be powerful tools for studying many aspects of a wide variety of human diseases.

Background: The laboratory rat (Rattus norvegicus) is an important model for studying many aspects of human health and disease. Detailed knowledge on genetic variation between strains is important from a biomedical, particularly pharmacogenetic point of view and useful for marker selection for genetic cloning and association studies., Results: We show that Single Nucleotide Polymorphisms (SNPs) in commonly used rat strains are surprisingly well represented in wild rat isolates. Shotgun sequencing of 814 Kbp in one wild rat resulted in the identification of 485 SNPs as compared with the Brown Norway genome sequence. Genotyping 36 commonly used inbred rat strains showed that 84% of these alleles are also polymorphic in a representative set of laboratory rat strains., Conclusion: We postulate that shotgun sequencing in a wild rat sample and subsequent genotyping in multiple laboratory or domesticated strains rather than direct shotgun sequencing of multiple strains, could be the most efficient SNP discovery approach. For the rat, laboratory strains still harbor a large portion of the haplotypes present in wild isolates, suggesting a relatively recent common origin and supporting the idea that rat inbred strains, in contrast to mouse inbred strains, originate from a single species, R. norvegicus.

The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis to increase genetic variation in laboratory rats and identified a recessive mutant, named tornado, showing aberrant circling behavior, hyperactivity, and stereotypic head shaking. More detailed analysis revealed profound deafness due to disorganization and degeneration of the organ of Corti that already manifests at the onset of hearing. We set up a single nucleotide polymorphism (SNP)-based mapping strategy to identify the affected gene, revealing strong linkage to the central region of chromosome 1. Candidate gene resequencing identified a point mutation that introduces a premature stopcodon in Myo7a. Mutations in human MYO7A result in Usher syndrome type 1B, a severe autosomal inherited recessive disease that involves deafness and vestibular dysfunction. Here, we present the first characterized rat model for this disease. In addition, we demonstrate proof of principle for the generation and cloning of human disease models in rat using ENU mutagenesis, providing good perspectives for systematic phenotypic screens in the rat.

Background: With the recent progress made in large-scale genome sequencing projects a vast amount of novel data is becoming available. A comparative sequence analysis, exploiting sequence information from various resources, can be used to uncover hidden information, such as genetic variation. Although there are enormous amounts of SNPs for a wide variety of organisms submitted to NCBI dbSNP and annotated in most genome assembly viewers like Ensembl and the UCSC Genome Browser, these platforms do not easily allow for extensive annotation and incorporation of experimental data supporting the polymorphism. However, such information is very important for selecting the most promising and useful candidate polymorphisms for use in experimental setups., Description: The CASCAD database is designed for presentation and query of candidate SNPs that are retrieved by in silico mining of high-throughput sequencing data. Currently, the database provides collections of laboratory rat (Rattus norvegicus) and zebrafish (Danio rerio) candidate SNPs. The database stores detailed information about raw data supporting the candidate, extensive annotation and links to external databases (e.g. GenBank, Ensembl, UniGene, and LocusLink), verification information, and predictions of a potential effect for non-synonymous polymorphisms in coding regions. The CASCAD website allows search based on an arbitrary combination of 27 different parameters related to characteristics like candidate SNP quality, genomic localization, and sequence data source or strain. In addition, the database can be queried with any custom nucleotide sequences of interest. The interface is crosslinked to other public databases and tightly coupled with primer design and local genome assembly interfaces in order to facilitate experimental verification of candidates., Conclusions: The CASCAD database discloses detailed information on rat and zebrafish candidate SNPs, including the raw data underlying its discovery. An advanced web-based search interface http://cascad.niob.knaw.nl allows universal access to the database content and allows various queries supporting many types of research utilizing single nucleotide polymorphisms.

The midge Chironomus riparius is distributed all over the Palearctic region and is well characterized both at the morphological and cytogenetic levels. Here we describe a population study based on the insertional polymorphism of the retroposon NLRCth1, by means of a S-SAP (sequence-specific amplification polymorphism) derived technique (transposon insertion display; TID). While a previous study of allozyme polymorphism in Russian samples showed little variability, all the amplicons we identified are polymorphic. Genetic distances between 6 natural populations were calculated according to Nei and did not show a positive correlation with geographic distances. The genetic diversity detected among individuals of a given population was one order of magnitude higher than that among populations. However, the value of phi(ST) was significant (p < 0.001) and indicates that natural populations are more genetically differentiated than random samples of individuals.

Single nucleotide polymorphisms (SNPs) are the most common source of genetic variation in populations and are thus most likely to account for the majority of phenotypic and behavioral differences between individuals or strains. Although the rat is extensively studied for the latter, data on naturally occurring polymorphisms are mostly lacking. We have used publicly available sequences consisting of whole-genome shotgun (WGS), expressed sequence tag (EST), and mRNA data as a source for the in silico identification of SNPs in gene-coding regions and have identified a large collection of 33,305 high-quality candidate SNPs. Experimental verification of 471 candidate SNPs using a limited set of rat isolates revealed a confirmation rate of approximately 50%. Although the majority of SNPs were identified between Sprague-Dawley (EST data) and Brown Norway (WGS data) strains, we found that 66% of the verified variations are common among different rat strains. All SNPs were extensively annotated, including chromosomal and genetic map information, and nonsynonymous SNPs were analyzed by SIFT and PolyPhen prediction programs for their potential deleterious effect on protein function. Interestingly, we retrieved three SNPs from the database that result in the introduction of a premature stop codon and that could be confirmed experimentally. Two of these "in silico-identified knockouts" reside in interesting QTL regions. Data are publicly available via a Web interface (http://cascad.niob.knaw.nl), allowing simple and advanced search queries., (Copyright 2004 Cold Spring Harbor Laboratory Press ISSN)

Prediction of transcription-factor target sites in promoters remains difficult due to the short length and degeneracy of the target sequences. Although the use of orthologous sequences and phylogenetic footprinting approaches may help in the recognition of conserved and potentially functional sequences, correct alignment of the short transcription-factor binding sites can be problematic for established algorithms, especially when aligning more divergent species. Here, we report a novel phylogenetic footprinting approach, CONREAL, that uses biologically relevant information, that is, potential transcription-factor binding sites as represented by positional weight matrices, to establish anchors between orthologous sequences and to guide promoter sequence alignment. Comparison of the performance of CONREAL with the global alignment programs LAGAN and AVID using a reference data set, shows that CONREAL performs equally well for closely related species like rodents and human, and has a clear added value for aligning promoter elements of more divergent species like human and fish, as it identifies conserved transcription-factor binding sites that are not found by other methods. CONREAL is accessible via a Web interface at http://conreal.niob.knaw.nl/.

Two mitochondrial genes, Cytochrome b (Cytb) and Cytochrome c oxidase subunit I (COI), have been used as phylogenetic markers in Chironomids. The nucleotide sequences of 685 bp from Cytb and 596 bp from COI have been determined for 36 Chironomus species from the Palearctic, or Holarctic, and Australasia. The concatenated sequence of 1281 bp from both genes was used to investigate the phylogenetic relationships among these species. The nucleotide sequence alignments were used for construction of phylogenetic trees based on maximum-parsimony and neighbor-joining methods. Both techniques produced similar phylogenies. Monophyly of the genus Chironomus is supported by a bootstrap value of 100% at the basal branch. Six clusters of species have been revealed with high bootstrap values supporting both monophyly of each cluster and the validity of the branching order within each cluster. Four species, C. circumdatus, C. nepeanensis, C. dorsalis, and C. crassiforceps, cannot be placed into any cluster. Cytological phylogenies were constructed using the same set of species, except for C. biwaprimus. These trees showed many similarities to that obtained from the mitochondrial (mt) sequence analysis, but also a number of significant differences. When compared with the tree constructed from the sequence of 23 species available for one of the globin genes, globin 2b (gb2b), there was better support for the mt tree than for the cytological trees. An intron, which varies in its occurrence and position in gb2b, was also investigated and the distribution of the introns supports the phylogenetic history of the genus Chironomus obtained with mt data. The differences observed in the cytological trees seem to be attributable more to the retention of the same chromosome banding sequence across several species, rather than convergent evolutionary events. An important question is the determination of the position of the subgenus Camptochironomus in relation to the representatives of the nominal subgenus Chironomus, since it has been suggested that this is a separate genus. The Camptochironomus species are internal to the trees and have arisen more recently than some of the species of the subgenus Chironomus, indicating that they are not sufficiently differentiated to be considered more than a subgenus., (Copyright 2001 Academic Press.)