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1.  GrabBlur - a framework to facilitate the secure exchange of whole-exome and -genome SNV data using VCF files 
BMC Genomics  2014;15(Suppl 4):S8.
Background
Next Generation Sequencing (NGS) of whole exomes or genomes is increasingly being used in human genetic research and diagnostics. Sharing NGS data with third parties can help physicians and researchers to identify causative or predisposing mutations for a specific sample of interest more efficiently. In many cases, however, the exchange of such data may collide with data privacy regulations. GrabBlur is a newly developed tool to aggregate and share NGS-derived single nucleotide variant (SNV) data in a public database, keeping individual samples unidentifiable. In contrast to other currently existing SNV databases, GrabBlur includes phenotypic information and contact details of the submitter of a given database entry. By means of GrabBlur human geneticists can securely and easily share SNV data from resequencing projects. GrabBlur can ease the interpretation of SNV data by offering basic annotations, genotype frequencies and in particular phenotypic information - given that this information was shared - for the SNV of interest.
Tool description
GrabBlur facilitates the combination of phenotypic and NGS data (VCF files) via a local interface or command line operations. Data submissions may include HPO (Human Phenotype Ontology) terms, other trait descriptions, NGS technology information and the identity of the submitter. Most of this information is optional and its provision at the discretion of the submitter. Upon initial intake, GrabBlur merges and aggregates all sample-specific data. If a certain SNV is rare, the sample-specific information is replaced with the submitter identity. Generally, all data in GrabBlur are highly aggregated so that they can be shared with others while ensuring maximum privacy. Thus, it is impossible to reconstruct complete exomes or genomes from the database or to re-identify single individuals. After the individual information has been sufficiently "blurred", the data can be uploaded into a publicly accessible domain where aggregated genotypes are provided alongside phenotypic information. A web interface allows querying the database and the extraction of gene-wise SNV information. If an interesting SNV is found, the interrogator can get in contact with the submitter to exchange further information on the carrier and clarify, for example, whether the latter's phenotype matches with phenotype of their own patient.
doi:10.1186/1471-2164-15-S4-S8
PMCID: PMC4083413  PMID: 25055742
2.  HomozygosityMapper2012—bridging the gap between homozygosity mapping and deep sequencing 
Nucleic Acids Research  2012;40(Web Server issue):W516-W520.
Homozygosity mapping is a common method to map recessive traits in consanguineous families. To facilitate these analyses, we have developed HomozygosityMapper, a web-based approach to homozygosity mapping. HomozygosityMapper allows researchers to directly upload the genotype files produced by the major genotyping platforms as well as deep sequencing data. It detects stretches of homozygosity shared by the affected individuals and displays them graphically. Users can interactively inspect the underlying genotypes, manually refine these regions and eventually submit them to our candidate gene search engine GeneDistiller to identify the most promising candidate genes. Here, we present the new version of HomozygosityMapper. The most striking new feature is the support of Next Generation Sequencing *.vcf files as input. Upon users’ requests, we have implemented the analysis of common experimental rodents as well as of important farm animals. Furthermore, we have extended the options for single families and loss of heterozygosity studies. Another new feature is the export of *.bed files for targeted enrichment of the potential disease regions for deep sequencing strategies. HomozygosityMapper also generates files for conventional linkage analyses which are already restricted to the possible disease regions, hence superseding CPU-intensive genome-wide analyses. HomozygosityMapper is freely available at http://www.homozygositymapper.org/.
doi:10.1093/nar/gks487
PMCID: PMC3394249  PMID: 22669902
3.  Systematic Comparison of Three Methods for Fragmentation of Long-Range PCR Products for Next Generation Sequencing 
PLoS ONE  2011;6(11):e28240.
Next Generation Sequencing (NGS) technologies are gaining importance in the routine clinical diagnostic setting. It is thus desirable to simplify the workflow for high-throughput diagnostics. Fragmentation of DNA is a crucial step for preparation of template libraries and various methods are currently known. Here we evaluated the performance of nebulization, sonication and random enzymatic digestion of long-range PCR products on the results of NGS. All three methods produced high-quality sequencing libraries for the 454 platform. However, if long-range PCR products of different length were pooled equimolarly, sequence coverage drastically dropped for fragments below 3,000 bp. All three methods performed equally well with regard to overall sequence quality (PHRED) and read length. Enzymatic fragmentation showed highest consistency between three library preparations but performed slightly worse than sonication and nebulization with regard to insertions/deletions in the raw sequence reads. After filtering for homopolymer errors, enzymatic fragmentation performed best if compared to the results of classic Sanger sequencing. As the overall performance of all three methods was equal with only minor differences, a fragmentation method can be chosen solely according to lab facilities, feasibility and experimental design.
doi:10.1371/journal.pone.0028240
PMCID: PMC3227650  PMID: 22140562
5.  Fatal Cardiac Arrhythmia and Long-QT Syndrome in a New Form of Congenital Generalized Lipodystrophy with Muscle Rippling (CGL4) Due to PTRF-CAVIN Mutations 
PLoS Genetics  2010;6(3):e1000874.
We investigated eight families with a novel subtype of congenital generalized lipodystrophy (CGL4) of whom five members had died from sudden cardiac death during their teenage years. ECG studies revealed features of long-QT syndrome, bradycardia, as well as supraventricular and ventricular tachycardias. Further symptoms comprised myopathy with muscle rippling, skeletal as well as smooth-muscle hypertrophy, leading to impaired gastrointestinal motility and hypertrophic pyloric stenosis in some children. Additionally, we found impaired bone formation with osteopenia, osteoporosis, and atlanto-axial instability. Homozygosity mapping located the gene within 2 Mbp on chromosome 17. Prioritization of 74 candidate genes with GeneDistiller for high expression in muscle and adipocytes suggested PTRF-CAVIN (Polymerase I and transcript release factor/Cavin) as the most probable candidate leading to the detection of homozygous mutations (c.160delG, c.362dupT). PTRF-CAVIN is essential for caveolae biogenesis. These cholesterol-rich plasmalemmal vesicles are involved in signal-transduction and vesicular trafficking and reside primarily on adipocytes, myocytes, and osteoblasts. Absence of PTRF-CAVIN did not influence abundance of its binding partner caveolin-1 and caveolin-3. In patient fibroblasts, however, caveolin-1 failed to localize toward the cell surface and electron microscopy revealed reduction of caveolae to less than 3%. Transfection of full-length PTRF-CAVIN reestablished the presence of caveolae. The loss of caveolae was confirmed by Atomic Force Microscopy (AFM) in combination with fluorescent imaging. PTRF-CAVIN deficiency thus presents the phenotypic spectrum caused by a quintessential lack of functional caveolae.
Author Summary
Patients with generalized lipodystrophy have a marked lack of body fat. Several gene defects have been described that impede fat synthesis and maturation of fat cells. Here we report on mutations in a novel gene, called PTRF-CAVIN, causing congenital generalized lipodystrophy type 4 (CGL4) that is additionally associated with muscle disease. Patients' muscles are large but weak and show an involuntary, rolling contraction pattern called “rippling.” Further symptoms comprise life-threatening cardiac arrhythmias and a disorder of bone formation. We searched for shared segments in the genome of seven patients and found the responsible gene, called PTRF-CAVIN, on chromosome 17. This gene is crucial for caveolae (latin for “small caves”) formation. These small indentations of the cell membrane are found on the surface of muscle, bone, fat, and immune cells and facilitate cell-to-cell communication and the absorption of substances from the extracellular space. Patients lack more than 97% of caveolae and artificial insertion of the correct gene into patient skin cells led to the reappearance of caveolae. As cardiac arrhythmia is a severe and potentially life-threatening condition, patients with CGL4 should be closely monitored by ECG and, if necessary, fitted with an implanted pacemaker and cardioverter defibrillator (ICD) device.
doi:10.1371/journal.pgen.1000874
PMCID: PMC2837386  PMID: 20300641
6.  Individuals with mutations in XPNPEP3, which encodes a mitochondrial protein, develop a nephronophthisis-like nephropathy  
The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1–NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are “ciliopathies”. Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.
doi:10.1172/JCI40076
PMCID: PMC2827951  PMID: 20179356
7.  HomozygosityMapper—an interactive approach to homozygosity mapping 
Nucleic Acids Research  2009;37(Web Server issue):W593-W599.
Homozygosity mapping is a common method for mapping recessive traits in consanguineous families. In most studies, applications for multipoint linkage analyses are applied to determine the genomic region linked to the disease. Unfortunately, these are neither suited for very large families nor for the inclusion of tens of thousands of SNPs. Even if less than 10 000 markers are employed, such an analysis may easily last hours if not days. Here we present a web-based approach to homozygosity mapping. Our application stores marker data in a database into which users can directly upload their own SNP genotype files. Within a few minutes, the database analyses the data, detects homozygous stretches and provides an intuitive graphical interface to the results. The homozygosity in affected individuals is visualized genome-wide with the ability to zoom into single chromosomes and user-defined chromosomal regions. The software also displays the underlying genotypes in all samples. It is integrated with our candidate gene search engine, GeneDistiller, so that users can interactively determine the most promising gene. They can at any point restrict access to their data or make it public, allowing HomozygosityMapper to be used as a data repository for homozygosity-mapping studies. HomozygosityMapper is available at http://www.homozygositymapper.org/.
doi:10.1093/nar/gkp369
PMCID: PMC2703915  PMID: 19465395
8.  FragIdent – Automatic identification and characterisation of cDNA-fragments 
BMC Genomics  2009;10:95.
Background
Many genetic studies and functional assays are based on cDNA fragments. After the generation of cDNA fragments from an mRNA sample, their content is at first unknown and must be assigned by sequencing reactions or hybridisation experiments.
Even in characterised libraries, a considerable number of clones are wrongly annotated. Furthermore, mix-ups can happen in the laboratory. It is therefore essential to the relevance of experimental results to confirm or determine the identity of the employed cDNA fragments. However, the manual approach for the characterisation of these fragments using BLAST web interfaces is not suited for larger number of sequences and so far, no user-friendly software is publicly available.
Results
Here we present the development of FragIdent, an application for the automatic identification of open reading frames (ORFs) within cDNA-fragments. The software performs BLAST analyses to identify the genes represented by the sequences and suggests primers to complete the sequencing of the whole insert. Gene-specific information as well as the protein domains encoded by the cDNA fragment are retrieved from Internet-based databases and included in the output. The application features an intuitive graphical interface and is designed for researchers without any bioinformatics skills. It is suited for projects comprising up to several hundred different clones.
Conclusion
We used FragIdent to identify 84 cDNA clones from a yeast two-hybrid experiment. Furthermore, we identified 131 protein domains within our analysed clones. The source code is freely available from our homepage at .
doi:10.1186/1471-2164-10-95
PMCID: PMC2672089  PMID: 19254371
9.  A Systematic Approach to Mapping Recessive Disease Genes in Individuals from Outbred Populations 
PLoS Genetics  2009;5(1):e1000353.
The identification of recessive disease-causing genes by homozygosity mapping is often restricted by lack of suitable consanguineous families. To overcome these limitations, we apply homozygosity mapping to single affected individuals from outbred populations. In 72 individuals of 54 kindred ascertained worldwide with known homozygous mutations in 13 different recessive disease genes, we performed total genome homozygosity mapping using 250,000 SNP arrays. Likelihood ratio Z-scores (ZLR) were plotted across the genome to detect ZLR peaks that reflect segments of homozygosity by descent, which may harbor the mutated gene. In 93% of cases, the causative gene was positioned within a consistent ZLR peak of homozygosity. The number of peaks reflected the degree of inbreeding. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations within a single ZLR peak of homozygosity as short as 2 Mb, containing an average of only 16 candidate genes. As many specialty clinics have access to cohorts of individuals from outbred populations, and as our approach will result in smaller genetic candidate regions, the new strategy of homozygosity mapping in single outbred individuals will strongly accelerate the discovery of novel recessive disease genes.
Author Summary
Many childhood diseases are caused by single-gene mutations of recessive genes, in which a child has inherited one mutated gene copy from each parent causing disease in the child, but not in the parents who are healthy heterozygous carriers. As the two mutations represent the disease cause, gene mapping helped understand disease mechanisms. “Homozygosity mapping” has been particularly useful. It assumes that the parents are related and that a disease-causing mutation together with a chromosomal segment of identical markers (i.e., homozygous markers) is transmitted to the affected child through the paternal and the maternal line from an ancestor common to both parents. Homozygosity mapping seeks out those homozygous regions to map the disease-causing gene. Homozygosity mapping requires families, in which the parents are knowingly related, and have multiple affected children. To overcome these limitations, we applied homozygosity mapping to single affected individuals from outbred populations. In 72 individuals with known homozygous mutations in 13 different recessive disease genes, we performed homozygosity mapping. In 93% we detected the causative gene in a segment of homozygosity. We demonstrate that disease-causing homozygous mutations can be detected in single cases from outbred populations. This will strongly accelerate the discovery of novel recessive disease genes.
doi:10.1371/journal.pgen.1000353
PMCID: PMC2621355  PMID: 19165332
10.  GeneDistiller—Distilling Candidate Genes from Linkage Intervals 
PLoS ONE  2008;3(12):e3874.
Background
Linkage studies often yield intervals containing several hundred positional candidate genes. Different manual or automatic approaches exist for the determination of the gene most likely to cause the disease. While the manual search is very flexible and takes advantage of the researchers' background knowledge and intuition, it may be very cumbersome to collect and study the relevant data. Automatic solutions on the other hand usually focus on certain models, remain “black boxes” and do not offer the same degree of flexibility.
Methodology
We have developed a web-based application that combines the advantages of both approaches. Information from various data sources such as gene-phenotype associations, gene expression patterns and protein-protein interactions was integrated into a central database. Researchers can select which information for the genes within a candidate interval or for single genes shall be displayed. Genes can also interactively be filtered, sorted and prioritised according to criteria derived from the background knowledge and preconception of the disease under scrutiny.
Conclusions
GeneDistiller provides knowledge-driven, fully interactive and intuitive access to multiple data sources. It displays maximum relevant information, while saving the user from drowning in the flood of data. A typical query takes less than two seconds, thus allowing an interactive and explorative approach to the hunt for the candidate gene.
Access
GeneDistiller can be freely accessed at http://www.genedistiller.org
doi:10.1371/journal.pone.0003874
PMCID: PMC2587712  PMID: 19057649
11.  d-matrix – database exploration, visualization and analysis 
BMC Bioinformatics  2004;5:168.
Background
Motivated by a biomedical database set up by our group, we aimed to develop a generic database front-end with embedded knowledge discovery and analysis features. A major focus was the human-oriented representation of the data and the enabling of a closed circle of data query, exploration, visualization and analysis.
Results
We introduce a non-task-specific database front-end with a new visualization strategy and built-in analysis features, so called d-matrix. d-matrix is web-based and compatible with a broad range of database management systems. The graphical outcome consists of boxes whose colors show the quality of the underlying information and, as the name suggests, they are arranged in matrices. The granularity of the data display allows consequent drill-down. Furthermore, d-matrix offers context-sensitive categorization, hierarchical sorting and statistical analysis.
Conclusions
d-matrix enables data mining, with a high level of interactivity between humans and computer as a primary factor. We believe that the presented strategy can be very effective in general and especially useful for the integration of distinct data types such as phenotypical and molecular data.
doi:10.1186/1471-2105-5-168
PMCID: PMC533865  PMID: 15511298

Results 1-11 (11)