Long Read Pilot Projects

The goal of the Kids First long-read pilot is to leverage rapidly advancing long-read sequencing technologies to uncover genetic structural variation underlying childhood cancers and structural birth defects.

Below is a list of Kids First cohorts approved for long-read sequencing conducted by Kids First sequencing centers (Broad Institute and HudsonAlpha Institute for Biotechnology).

Project Title: Long-read whole genome sequencing of nonsyndromic craniosynostosis
PI: Boyd, Simeon 
Sequencing Center: HudsonAlpha Institute for Biotechnology 
Project Narrative: Nonsyndromic craniosynostosis (NCS) is a common, major structural birth defect that occurs due to the premature fusion of one or more cranial sutures. It requires extensive surgical correction and is associated with considerable ongoing medical problems and health care costs. Because little is known about the causes of NCS, with the support from the Gabriella Miller Kids First Pediatric Research Program we performed whole genome sequencing (WGS) of more than 300 affected families with the goal to find the genetic factors contributing to the etiology of NCS. However, conventional short-read WGS can miss disease causing structural genomic variation. Long-read WGS technologies, such as the Oxford Nanopore Technologies, offer complementary strengths to short-read WGS by producing sequencing read lengths of 1000 bp or longer. We will perform 114 long-read WGS of affected NCS families for a more comprehensive analysis of the genomes of the affected individuals and their parents, to identify genes and loci that could better explain the etiology and pathogenesis of NCS.

Project Title: Long-read whole genome sequencing of a congenital diaphragmatic hernia cohort
PI: Chung, Wendy 
Sequencing Center: Broad Institute
Project Narrative: We have a well-characterized cohort of individuals with congenital diaphragmatic hernia (CDH) with detailed clinical phenotypes.  The cohort has been analyzed by short read genome sequencing, and we have identified plausible genetic causes in about 30% of the CDH cases. We will now extend the genomic analysis to long-read sequencing in trios with multiple congenital anomalies to identify cryptic genetic causes of CDH and other anomalies.

Project Title: Kids First X01 Long-Read Sequencing Pilot: Structural Heart and Other Defects 
PI: Gelb, Bruce
Sequencing Center: Broad Institute
Project Narrative: While congenital heart defects (CHD) are believed to arise predominantly from genetic variation, only about 1/2 of cases are accounted for with genetic variation altering coding and non-coding regions of the genome. While implementation of next generation sequencing-based approaches to exome and genome sequencing for elucidating causal variants underlying CHD has been highly successful, discovery of structural variants and repeat expansions has proven more difficult using short-read DNA sequencing. For this Gabriella Miller Kids First pilot study, we will explore the hypothesis that long-reading genome sequencing will identify causal structural variants and repeat expansions in a modest fraction of 200 individuals with unsolved CHD.

Project Title: Long-read whole genome sequencing of Bladder Exstrophy Epispadias Complex (BEEC)
PI: Jelin, Angie 
Sequencing Center: Broad Institute
Project Narrative: Bladder Exstrophy Epispadias Complex (BEEC) describes a subset of anomalies ranging from a mild form of epispadias to moderate classic bladder exstrophy to OEIS. We propose that BEEC is an embryopathy that evolves secondary to abnormalities in signaling pathways involved in protein-DNA and protein-protein interactions. Structural variants (SVs) can influence gene dosage or disrupt regulatory element interactions and can exert effects such as haploinsufficiency, partial loss of function, gain of function, epistatic interaction, or can alter the level or pattern of gene expression during development. Although we have identified several missense variants via whole genome sequencing, none have yet been proven to be causative. Using long-read sequencing we will be able to identify SVs, such as deletions, duplications, inversions or translocations, which were not previously identified.

Project Title: Deep Characterization of -Omic Cold Cases in Pediatric Acute Myeloid Leukemia through Long Read Genome and Transcriptome Sequencing
PI: Meshinchi, Soheil 
Sequencing Center: HudsonAlpha Institute for Biotechnology
Project Narrative: Large scale sequencing efforts in childhood AML have identified numerous structural and sequence variants that either cause or contribute to clonal expansion of the malignant clone. However, a large cohort of children with AML lack identifiable genomic or transcriptomic variants. This may in part be due to the limitation of technologies used to identify disease-associated variants. Recent advances in long-read sequencing provides an opportunity to identify structural alterations that are not amenable to detection by other methods. We propose to interrogate the genome and transcriptome of a cohort of 100 affected children without disease-associated structural alterations. We hypothesize that long-read sequencing will provide detailed knowledge of the splice isoform variants that may be linked to AML pathogenesis which will not only lead to better diagnostics, but also the discovery of new therapeutic targets.

Project Title: Kids First X01 Long-read Sequencing Pilot - Sequencing of the BASIC3 cohort
PI: Plon, Sharon 
Sequencing Center: HudsonAlpha Institute for Biotechnology
Project Narrative: The Baylor Advancing Sequencing in Childhood Cancer Care (BASIC3) cohort consists of ethnically diverse trios with pediatric solid tumor patients and their unaffected parents originally enrolled as part of our NHGRI/NCI clinical sequencing grant. We are seeking to leverage this cohort to improve our understanding of pediatric cancer susceptibility through the analysis of germline structural variation using long-read WGS. We propose to fill current gaps in our knowledge by identifying (1) the proportion and nature of pathogenic or likely pathogenic germline structural variants which will be identified through long-read WGS evaluation of the patient and parent sequencing that are missed by more standard proband-only short-read WGS or exome methods and (2) new cancer susceptibility genes particularly when this pilot data is combined with other Gabriela Miller Kids First sequencing projects.

Project Title: Identification of germline structural variants in patients with Ollier disease and Maffucci syndrome using long read sequencing
PI: Sobreira, Nara 
Sequencing Center: HudsonAlpha Institute for Biotechnology
Project Narrative: Ollier disease (OD) and Maffucci syndrome (MS) are untreatable, poorly characterized, newly recognized cancer susceptibility syndromes. Both syndromes are characterized by enchondromas (cartilage tumors) and severe bone deformities. Our recent work shows that ~50% of patients develop a malignancy. The cancer spectrum is broad, but commonly include chondrosarcomas, gliomas, and vascular malignancies.  There is a critical need to define the genetic bases of OD and MS; without this information, effective treatments will remain limited for these patients. Here we will use germline long-read WGS to identify structural variants (SVs) that cause OD and MS. Long-read WGS will allow investigation of coding and non-coding SVs including large insertions/deletions, copy number variations (CNVs), and repeat expansions missed by short-read WGS.

Kids First Sequencing Center Investigators

Broad Institute:

• PI: Stacey Gabriel
• Kiran Garimella
• Stacy-Ann Mano
• Kayla Delano 

HudsonAlpha Institute for Biotechnology:

• PI: Shawn Levy
• Nripesh Prasad
• Salina Hall
• Kimberleigh Fedi

Data Resource Center Investigators

• Adam Resnick
• Allison Heath
• Deanne Taylor
• David Higgins