The Development and Optimization of Long-Read Sequencing Applications to Cancer Genomics in a Core Setting

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Cancer is a highly complex and heterogeneous disease governed by a multitude of genomic mechanisms. Sequencing technologies have been essential to driving research into new methods of cancer diagnosis and treatment. As sequencing has increased in throughput and decreased in cost, research has revealed genomic complexities not previously appreciated. As these complexities are revealed, new methods must be developed to investigate them. The work of the Next-Generation Shared Resource (NGSSR) Core focuses on making emerging technologies in sequencing available and developing their applications to biological science. In recent years, long-read sequencing has been applied to cancer genomics. The NGSSR has been, and continues to be, on the forefront of long-read sequencing. In 2011, CSHL acquired the first generation of long- read sequencing instruments. This technology was shown to be invaluable to studies of the SK-BR-3 breast cancer cell line, revealing that many structural variations, some with fusion transcripts, are not detected by short-read methods. Given these results, the NGSSR has continued to explore long-read sequencing technologies. These methods are currently being developed to support an array of projects at CSHL, including, high depth Oxford Nanopore and PacBio sequencing of breast cancer organoids to better understand the mechanisms driving tumorigenesis and to validate organoids as molecular models of cancer. By leveraging the unique Oxford Nanopore ability to detect methylation along with sequencing data, possible cancer specific methylation profiles correlated with rearrangement hot spots in breast cancers have been identified. The NGSSR has also developed a pipeline exploiting Oxford Nanopore technology to detect large insertions and deletions in Acute Myeloid Leukemia (AML). These variations are known to be markers for outcome, thus driving treatment choice. This method can facilitate point-of-care testing for AML subtype, providing a new diagnostic tool to oncologists. Full length RNA sequencing and analysis is also being developed by the NGSSR to quantify alternative pre-mRNA splicing events. These splicing events can be used to characterize cancer subtype and to explore the mechanisms of cancer development and progression. The role of the NGSSR in these projects has been to develop methods to work with the technologies and the materials to be examined. This includes all steps from DNA/RNA extraction, library preparation, and data analysis. The services and education/advice the NGSSR provides about these technologies gives CSHL researchers a tremendous boost in their research endeavors. In addition to conducting independent research, the NGSSR core manager will continue to run the day-to-day operation of the NGSSR ensuring that all sequencing related studies at CSHL, using both new and old methods, are of the highest quality to facilitate ground breaking cancer research.