Principal Investigator

Chia-Lin
Wei
Awardee Organization

Jackson Laboratory
United States

Fiscal Year
2020
Activity Code
R33
Early Stage Investigator Grants (ESI)
Not Applicable
Project End Date

Advancing Ultra Long-read Sequencing and Chromatin Interaction Analyses for Chromosomal and Extrachromosomal Structural Variation Characterization in Cancer

Structural variants (SVs) such as deletions, insertions, inversions, duplications, and translocations in cancer genomes can promote tumor progression by perturbing gene structures and expression. Additionally, extrachromosomal DNA (ecDNA)—an extreme form of SV found in a wide range of cancer types—are a reservoir of oncogene amplification and contribute to the genetic heterogeneity and evolution of tumors. Thus, a complete understanding of the structure and distribution of SVs and ecDNAs in tumors would shed light on their roles in tumor progression. However, the ability to detect and characterize SVs and ecDNAs at the molecular level has been limited by existing short-read sequencing approaches: large and complex SVs thwart efforts to detect them and correctly define their structures; and the multi-copy, heterogenous nature of ecDNAs undermines determination of their primary structures. While ecDNAs can be observed by DAPI-staining of metaphase tumor cells, determining their sequence content has typically relied on fluorescence in situ hybridization (FISH) to probe for candidate oncogenes. To support an unbiased and comprehensive molecular approach to the study of SVs, this project will develop and validate emerging genomic technologies that will enable the detection and characterization of complex SVs and ecDNAs as standard practices in cancer genomics. In Aim 1, the read lengths of the nanopore single-molecule sequencing platform will be further extended by improving genomic DNA quality and optimizing library preparation reactions, with the goal of attaining N50 read lengths of 75-100 Kb. Such long read lengths are expected to span many SVs to more effectively reveal their molecular structures and phasing information. In parallel, the recent SV-detecting computational pipeline, Picky, will be optimized to detect molecular signatures of complex SVs and ecDNAs to allow their accurate and sensitive detection in long read sequencing data to >0.8 precision and recall rates. The active transcription of ecDNAs suggests that they are associated with RNA polymerase II transcription complexes, making them suitable for unsupervised detection by the chromatin interaction assay, ChIA-PET. In Aim 2, this method will be employed to map ecDNAs via their association with RNA polymerase II and reveal transcriptionally relevant interactions between ecDNAs and the chromosomes. Computational methods will be developed to specifically detect ecDNA-amplified sequences in ChIA-PET data and their associated oncogenic genes. Additionally, ecDNAs uncovered by ChIAPET will be targeted by the CRISPR/dCas9-based targeted capture method to physically isolate ecDNA molecules for long-read sequencing and structural characterization. Aim 3 will build on the developed methods to generate a platform for unbiased and unsupervised characterization of SVs and ecDNAs in glioblastoma neurosphere cultures and in xenograft tumor models of glioblastoma, breast, and lung cancer. Taken together, this project will develop methods and tools that will empower the cancer research community to confidently and comprehensively detect SVs and ecDNAs in cancer genomes.

Publications

  • Varn FS, Johnson KC, Martinek J, Huse JT, Nasrallah MP, Wesseling P, Cooper LAD, Malta TM, Wade TE, Sabedot TS, Brat D, Gould PV, Wöehrer A, Aldape K, Ismail A, Sivajothi SK, Barthel FP, Kim H, Kocakavuk E, Ahmed N, White K, Datta I, Moon HE, Pollock S, Goldfarb C, Lee GH, Garofano L, Anderson KJ, Nehar-Belaid D, Barnholtz-Sloan JS, Bakas S, Byrne AT, D'Angelo F, Gan HK, Khasraw M, Migliozzi S, Ormond DR, Paek SH, Van Meir EG, Walenkamp AME, Watts C, Weiss T, Weller M, Palucka K, Stead LF, Poisson LM, Noushmehr H, Iavarone A, Verhaak RGW, GLASS Consortium, Varn FS, Johnson KC, Martinek J, Huse JT, Nasrallah MP, Wesseling P, Cooper LAD, Malta TM, Wade TE, Sabedot TS, Brat D, Gould PV, Wöehrer A, Aldape K, Ismail A, Sivajothi SK, Barthel FP, Kim H, Kocakavuk E, Ahmed N, White K, Datta I, Moon HE, Pollock S, Goldfarb C, Lee GH, Garofano L, Anderson KJ, Nehar-Belaid D, Barnholtz-Sloan JS, Bakas S, Byrne AT, D'Angelo F, Gan HK, Khasraw M, Migliozzi S, Ryan Ormond D, Ha Paek S, Van Meir EG, Walenkamp AME, Watts C, Weiss T, Weller M, Alfaro KD, Amin SB, Ashley DM, Bock C, Brodbelt A, Bulsara KR, Castro AV, Connelly JM, Costello JF, de Groot JF, Finocchiaro G, French PJ, Golebiewska A, Hau AC, Hong C, Horbinski C, Kannan KS, Kouwenhoven MC, Lasorella A, LaViolette PS, Ligon KL, Lowman AK, Mehta S, Miletic H, Molinaro AM, Ng HK, Niclou SP, Niers JM, Phillips JJ, Rabadan R, Rao G, Reifenberger G, Sanai N, Short SC, Sillevis Smitt P, Sloan AE, Smits M, Snyder JM, Suzuki H, Tabatabai G, Tanner G, Tomaszewski WH, Wells M, Westerman BA, Wheeler H, Xie J, Alfred Yung WK, Zadeh G, Zhao J, Palucka K, Stead LF, Poisson LM, Noushmehr H, Iavarone A, Verhaak RG. Glioma progression is shaped by genetic evolution and microenvironment interactions. Cell. 2022 Jun 9;185(12):2184-2199.e16. Epub 2022 May 31. PMID: 35649412
  • Yi E, Gujar AD, Guthrie M, Kim H, Zhao D, Johnson KC, Amin SB, Costa ML, Yu Q, Das S, Jillette N, Clow PA, Cheng AW, Verhaak RGW. Live-Cell Imaging Shows Uneven Segregation of Extrachromosomal DNA Elements and Transcriptionally Active Extrachromosomal DNA Hubs in Cancer. Cancer discovery. 2022 Feb;12(2):468-483. Epub 2021 Nov 24. PMID: 34819316
  • Chai H, Tjong H, Li P, Liao W, Wang P, Wong CH, Ngan CY, Leonard WJ, Wei CL, Ruan Y. ChIATAC is an efficient strategy for multi-omics mapping of 3D epigenomes from low-cell inputs. Nature communications. 2023 Jan 13;14(1):213. PMID: 36639381
  • Zhu Y, Gong L, Wei CL. Guilt by association: EcDNA as a mobile transactivator in cancer. Trends in cancer. 2022 Sep;8(9):747-758. Epub 2022 Jun 23. PMID: 35753910
  • Zhu Y, Gujar AD, Wong CH, Tjong H, Ngan CY, Gong L, Chen YA, Kim H, Liu J, Li M, Mil-Homens A, Maurya R, Kuhlberg C, Sun F, Yi E, deCarvalho AC, Ruan Y, Verhaak RGW, Wei CL. Oncogenic extrachromosomal DNA functions as mobile enhancers to globally amplify chromosomal transcription. Cancer cell. 2021 May 10;39(5):694-707.e7. Epub 2021 Apr 8. PMID: 33836152
  • Yi E, Chamorro González R, Henssen AG, Verhaak RGW. Extrachromosomal DNA amplifications in cancer. Nature reviews. Genetics. 2022 Dec;23(12):760-771. Epub 2022 Aug 11. PMID: 35953594
  • Yi E. Studying extrachromosomal DNA with the ecTag method. Nature reviews. Cancer. 2022 Jun;22(6):320-321. PMID: 35379949