ena-STUDY-JOHNS HOPKINS-06-04-2019-19:53:20:343-6
We have used Cas9 enrichment for targeted nanopore sequencing to study DNA methylation, structural variants and mutations
We used the CRISPR/Cas9 system to introduce breaks for targeted ligation of nanopore sequencing adaptors, and subsequently evaluate methylation at gene promoters, decreasing the relatively high cost/bp of nanopore sequencing while retaining its sensitivity and long-reads. We have demonstrated the ability of this method to generate greater than 300X coverage at 10 genomic loci simultaneously from a single flow cell, which represents a several hundred fold improvement over the 2-3X coverage achieved without enrichment. Together, our methods provide a valuable and efficient approach with applications in clinical and research labs--using nanopore sequencing for analysis of DNA methylation, evaluation of structural variation and chromatin state, and segregation of assignment of reads to allele of origin.
We used the CRISPR/Cas9 system to introduce breaks for targeted ligation of nanopore sequencing adaptors, and subsequently evaluate methylation at gene promoters, decreasing the relatively high cost/bp of nanopore sequencing while retaining its sensitivity and long-reads. We have demonstrated the ability of this method to generate greater than 300X coverage at 10 genomic loci simultaneously from a single flow cell, which represents a several hundred fold improvement over the 2-3X coverage achieved without enrichment. Together, our methods provide a valuable and efficient approach with applications in clinical and research labs--using nanopore sequencing for analysis of DNA methylation, evaluation of structural variation and chromatin state, and segregation of assignment of reads to allele of origin.