Coriell Institute for Medical Research
PI: Nahid Turan
The CHD GENES Biorepository, Coriell Institute for Medical Research, provides sample collection, packaging, and shipment materials to the PCGC Centers. DNA is extracted from blood and saliva specimens, aliquoted and stored at -80C. Specimen tracking, manipulation, quality control, and distribution status information is transmitted to the coordinating center, Cincinnati Children's Hospital Medical Center. DNA specimens are prepared and shipped to core facilities and laboratories as instructed by the coordinating center.
PCGC Data Hub:
Cincinnati Children's Hospital Medical Center
PI: Peter White
The PCGC Data Hub provides an IT framework that securely collects, stores, and manages subject and associated genomic data generated by the PCGC. The ability to browse and query the data for combinations of molecular, clinical, and outcome attributes and to construct cohorts based upon these attributes is provided. Genomic data is received from PCGC Molecular Core Facilities and phenotype, outcome, and biomaterials data is received from the coordinating center, Cincinnati Children's Hospital Medical Center.
CvDC Data Hub:
University of Utah
PI: Joe Yost
The Data Sharing Hub updates and maintains GNomEx, a web tool for organizing, annotating, tracking, and distributing raw genomic data and associated downstream analysis. It contains a genomic LIMS, an Analysis Project Center, and Programmatic Data Distribution Server. New reporting systems allow tracking activities, sample annotations, project/experiment reports, and a “related data” tab shows links between experiments, data tracks and analyses. A new query tool, BioMiner, is being developed that internalizes ChIPseq, RNAseq, bisulfite, and variant analysis into a searchable database. The goal of the BioMiner Query Tool is to allow researchers to mine all the data that has been uploaded without the help of a bioinformatician.
CvDC BioInformatics Core:
J. David Gladstone Institutes
PI: Katherine Pollard
The BioInformatics Core focuses on annotation and analysis tools. Projects include RNAseq, ChIPseq, and alignment software vetting, development of methods to allow cross-species comparisons (OrthoRetriever), and development of a standard analysis pipeline to provide a consistent set of analyzed data files to B2B consortium members (modeled after ENCODE project).
Zebrafish Core for Functional Analysis of Candidate Genes: University of Utah
PI: Martin Tristani-Firouzi
The Zebrafish Model Organism Core conducts molecular and functional characterization of candidate genes or pathways identified by CvDC and PCGC centers. The zebrafish orthologue(s) of the candidate gene are identified, sequenced, and antisense morpholinos (MO) directed against the genes of interest are designed and injected into embryos at the 1-2 cell stage. The effects of MO knockdown of candidate genes on heart rate, rhythm, contractility, and gross morphology are assayed. In some cases, MO- knockdown is performed in transgenic fish expressing tissue-specific fluorescent markers in order to improve characterization of cardiovascular phenotype.
Mouse CRISPR/cas for Functional Analysis of Candidate Genes:
The Jackson Laboratory
PI: Laura Reinholdt
The Jackson Laboratory (JAX) will create genetically engineered mouse strains using site directed endonuclease technology (CRISPR/cas), site-specific knock-in or knockout of alleles through microinjection of mouse embryos to produce F0 founder animals that will be screened for CvDC and PCGC projects.
Human iPSCs Generation and CRISPR/cas:
Mount Sinai School of Medicine
PI: Bruce Gelb
A working group was organized and a protocol was established for developing iPSC lines from blood samples obtained from CHD GENES subjects who harbor de novo mutations of interest. Additional blood samples are collected from enrolled subjects, the IPSC Core isolates peripheral erythroblasts and derives iPSCs that can be stored or transported to other sites for study. Two approaches are being used- subjects bearing mutations of interest are recruited and iPSCs derived from peripheral blood cells. Then, CRISPR technology will be used to correct the relevant mutation to provide an isogenic control. Alternatively, control iPSCs are being mutated using CRISPRs to introduce mutations of interest.
Brigham and Women’s Hospital
PI: Christine Seidman
iPS cells generated from individual CHD patients exhibit extensive variability in background genotypes. To overcome the variability inherent in research with human iPS cells, the PCGC elected to introduce selected mutations into isogenic pluripotent human stem cells using CRISPR technology. The iPS/CRISPR Core has identified and selected an iPS cell line that demonstrates consistent robust differentiation into cardiomyoctes for mutagenesis. Multiple lines have been generated with mutation in candidate genes and are being analyzed at iPS, mesoderm, early cardiac progenitor, late cardiac progenitor, and cardiomyocyte stages of differentiation.
Library Generation and Next-Generation Sequencing:
Whole Exome Sequencing:
PI: Rick Lifton
The Whole Exome Sequencing Facility sequences specimens received (from cases of congenital heart disease, parents, and affected relatives) to generate sequence data for all protein coding regions by producing genomic libraries and targeted capture-derived whole exome sequences from genomic libraries. Raw DNA sequence data is distributed to PCGC Centers and the PCGC Data Hub.
PI: Jon Seidman
The RNA Transcriptome Core produces single cell RNAseq libraries from RNA samples submitted by CvDC investigators and from explanted human cardiac tissues provided by PCGC investigators. Analyses of these single cell transcriptomes will provide important new insights into the developmental mechanisms that lead to cardiac formation.
Candidate Gene Evaluation:
PI: Jon Seidman
The Candidate Gene Evaluation Core designs and optimizes subgenome (i.e., candidate genes) target capture methodologies and reagents, constructs genomic DNA libraries and submits captured DNA fragments to DNA sequencing facilities. Raw DNA sequence data (FASTQ files) are produced and delivered to the PCGC Data Hub.
PI: Wendy Chung
Dideoxy sequencing is performed to confirm next generation sequencing variants and quantitative PCR or digital PCR is performed to confirm copy number variants identified either from next generation sequencing or genotyping.