July 2024
Authors:
Bo Zhou, Carolin Purmann, Hanmin Guo, GiWon Shin, Yiling Huang, Reenal Pattni, Qingxi Meng, Stephanie U. Greer, Tanmoy Roychowdhuryg, Raegan N. Wood, Marcus Ho, Heinrich zu Dohna, Alexej Abyzov, Joachim F. Hallmayer, Wing H. Wong, Hanlee P. Ji, and Alexander E. Urban
Abstract:
“We developed a generally applicable method, CRISPR/Cas9-targeted long-read sequencing (CTLR-Seq), to resolve, haplotype-specifically, the large and complex regions in the human genome that had been previously impenetrable to sequencing analysis, such as large segmental duplications (SegDups) and their associated genome rearrangements. CTLR-Seq combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to isolate intact large (i.e., up to 2,000 kb) genomic regions that encompass previously unresolvable genomic sequences. These targets are then sequenced (amplification-free) at high on-target coverage using long-read sequencing, allowing for their complete sequence assembly. We applied CTLR-Seq to the SegDup-mediated rearrangements that constitute the boundaries of, and give rise to, the 22q11.2 Deletion Syndrome (22q11DS), the most common human microdeletion disorder. We then performed de novo assembly to resolve, at base-pair resolution, the full sequence rearrangements and exact chromosomal breakpoints of 22q11.2DS (including all common subtypes). Across multiple patients, we found a high degree of variability for both the rearranged SegDup sequences and the exact chromosomal breakpoint locations, which coincide with various transposons within the 22q11.2 SegDups, suggesting that 22q11DS can be driven by transposon-mediated genome recombination. Guided by CTLR-Seq results from two 22q11DS patients, we performed three-dimensional chromosomal folding analysis for the 22q11.2 SegDups from patient-derived neurons and astrocytes and found chromosome interactions anchored within the SegDups to be both cell type-specific and patient-specific. Lastly, we demonstrated that CTLR-Seq enables cell-type specific analysis of DNA methylation patterns within the deletion haplotype of 22q11DS.”
Sage Science Products:
SageHLS with the HLS-CATCH process for purifying HMW genomic targets.
Author Affiliations:
Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, CA Stanford Maternal and Child Health Research Institute, Stanford University School of Medicine, Stanford, CA
Department of Genetics, Stanford University School of Medicine, Stanford, CA
Department of Statistics, Stanford University, Stanford, CA
Department of Biomedical Data Science, Stanford University School of Medicine, Stanford, CA
Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA
Division of Computational Biology, Department of Quantitative Health Sciences, Mayo Clinic, Rochester, MN
Department of Biology, American University of Beirut, Lebanon
Program on Genetics of Brain Function, Stanford Center for Genomics and Personalized Medicine, Stanford University School of Medicine, Stanford, CA