Dataset Information

Global gene disruption in human cells to assign genes to phenotypes by deep sequencing.

ABSTRACT: Insertional mutagenesis in a haploid background can disrupt gene function. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones enables rapid assessment of the spectrum of genes involved in the phenotypes under study. This facilitates comparative screens as illustrated here for the family of cytolethal distending toxins (CDTs). CDTs are virulence factors secreted by a variety of pathogenic Gram-negative bacteria responsible for tissue damage at distinct anatomical sites. We identify 743 mutations distributed over 12 human genes important for intoxication by four different CDTs. Although related CDTs may share host factors, they also exploit unique host factors to yield a profile characteristic for each CDT.

SUBMITTER: Carette JE

PROVIDER: S-EPMC3111863 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Global gene disruption in human cells to assign genes to phenotypes by deep sequencing.

Carette Jan E JE Guimaraes Carla P CP Wuethrich Irene I Blomen Vincent A VA Varadarajan Malini M Sun Chong C Bell George G Yuan Bingbing B Muellner Markus K MK Nijman Sebastian M SM Ploegh Hidde L HL Brummelkamp Thijn R TR

Nature biotechnology 20110529 6

Insertional mutagenesis in a haploid background can disrupt gene function. We extend our earlier work by using a retroviral gene-trap vector to generate insertions in >98% of the genes expressed in a human cancer cell line that is haploid for all but one of its chromosomes. We apply phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing. Analysis of pools of cells, rather than individual clones enables rapid assessmen ...[more]

PMID: 21623355

Similar Datasets

Project description:BackgroundEST-PCR markers normally represent specific products from target genes, and are therefore effective tools for genetic analysis. However, because wheat is an allohexaploid plant, PCR products derived from homoeologous genes are often simultaneously amplified. Such products may be easier to differentiate if they include intron sequences, which are more polymorphic than exon sequences. However, genomic sequence data for wheat are limited; therefore it is difficult to predict the location of introns. By using the similarities in gene structures between rice and wheat, we developed a system called PLUG (PCR-based Landmark Unique Gene) to design primers so that PCR products include intron sequences. We then investigated whether products amplified using such primers could serve as markers able to distinguish multiple products derived from homoeologous genes.ResultsThe PLUG system consists of the following steps: (1) Single-copy rice genes (Landmark Unique Gene loci; LUGs) exhibiting high degrees of homology to wheat UniGene sequences are extracted; (2) Alignment analysis is carried out using the LUGs and wheat UniGene sequences to predict exon-exon junctions, and LUGs which can be used to design wheat primers flanking introns (TaEST-LUGs) are extracted; and (3) Primers are designed in an interactive manner. From a total of 4,312 TaEST-LUGs, 24 loci were randomly selected and used to design primers. With all of these primer sets, we obtained specific, intron-containing products from the target genes. These markers were assigned to chromosomes using wheat nullisomic-tetrasomic lines. By PCR-RFLP analysis using agarose gel electrophoresis, 19 of the 24 markers were located on at least one chromosome.ConclusionIn the development of wheat EST-PCR markers capable of efficiently sorting products derived from homoeologous genes, it is important to design primers able to amplify products that include intron sequences with insertion/deletion polymorphisms. Using the PLUG system, wheat EST sequences that can be used for marker development are selected based on comparative genomics with rice, and then primer sets flanking intron sequences are prepared in an interactive, semi-automatic manner. Hence, the PLUG system is an effective tool for large-scale marker development.

Project description:BackgroundCongenital myopathies are severe genetic diseases with a strong impact on patient autonomy and often on survival. A large number of patients do not have a genetic diagnosis, precluding genetic counseling and appropriate clinical management. Our objective was to find novel pathogenic variants and genes associated with congenital myopathies and to decrease diagnostic odysseys and dead-end.MethodsTo identify pathogenic variants and genes implicated in congenital myopathies, we established and conducted the MYOCAPTURE project from 2009 to 2018 to perform exome sequencing in a large cohort of 310 families partially excluded for the main known genes.ResultsPathogenic variants were identified in 156 families (50%), among which 123 families (40%) had a conclusive diagnosis. Only 44 (36%) of the resolved cases were linked to a known myopathy gene with the corresponding phenotype, while 55 (44%) were linked to pathogenic variants in a known myopathy gene with atypical signs, highlighting that most genetic diagnosis could not be anticipated based on clinical-histological assessments in this cohort. An important phenotypic and genetic heterogeneity was observed for the different genes and for the different congenital myopathy subtypes, respectively. In addition, we identified 14 new myopathy genes not previously associated with muscle diseases (20% of all diagnosed cases) that we previously reported in the literature, revealing novel pathomechanisms and potential therapeutic targets.ConclusionsOverall, this approach illustrates the importance of massive parallel gene sequencing as a comprehensive tool for establishing a molecular diagnosis for families with congenital myopathies. It also emphasizes the contribution of clinical data, histological findings on muscle biopsies, and the availability of DNA samples from additional family members to the diagnostic success rate. This study facilitated and accelerated the genetic diagnosis of congenital myopathies, improved health care for several patients, and opened novel perspectives for either repurposing of existing molecules or the development of novel treatments.

Dataset Information

Global gene disruption in human cells to assign genes to phenotypes by deep sequencing.

Publications

Global gene disruption in human cells to assign genes to phenotypes by deep sequencing.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets