SBCapSeq Protocol: a method for selective cloning of Sleeping Beauty transposon insertions using liquid capture hybridization and Ion Torrent semiconductor sequencing

Mann, Karen; Guzman-Rojas, Liliana; Amaya-Manzanares, Felipe; Jones, Devin J.; Newberg, Justin Y.; Jenkins, Nancy A.; Copeland, Neal G.; Mann, Michael B.

doi:10.1038/protex.2016.053

Cited by 5 publications

(5 citation statements)

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“…Full details for the SBCapSeq protocol, an enhanced SBCapSeq method optimized for sequencing from solid tumors (Mann et al, 2019), will be published elsewhere (Mann, Mann et al, in preparation; for general protocol and concept, see also refs. (Mann et al, 2016a;Mann et al, 2016b)). Briefly, for selective SB insertion site sequencing by liquid hybridization capture, gDNA (0.5 µg per sample) of bulk tumor genomes was used for library construction using the AB Library Builder™ System, including random fragmentation and ligation of barcoded Ion Xpress™ sequencing adapters.…”

Section: Ras-gef1 Ph2mentioning

confidence: 99%

Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation

Aiderus

Contreras-Sandoval

Meshey

et al. 2020

Preprint

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A central challenge in cancer genomics is the systematic identification of single and cooperating tumor suppressor genes driving cellular transformation and tumor progression in the absence of oncogenic driver mutation(s). Multiple in vitro and in vivo gene inactivation screens have enhanced our understanding of the tumor suppressor gene landscape in various cancers. However, these studies are limited to single or combination gene effects, specific organs, or require sensitizing mutations. In this study, we developed and utilized a Sleeping Beauty transposon mutagenesis system that functions only as a gene trap to exclusively inactivate tumor suppressor genes. Using whole body transposon mobilization in wild type mice, we observed that cumulative gene inactivation can drive tumorigenesis of solid cancers. We provide a quantitative landscape of the tumor suppressor genes inactivated in these cancers, and show that despite the absence of oncogenic drivers, these genes converge on key biological pathways and processes associated with cancer hallmarks.

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Section: Ras-gef1 Ph2mentioning

confidence: 99%

Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation

Aiderus

Contreras-Sandoval

Meshey

et al. 2020

Preprint

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“…Mapping transposon insertion sites using the SBCapSeq method. Full details for the SBCapSeq protocol (Mann et al, 2016b) optimized for sequencing from solid tumors, will be published elsewhere (Mann et al, in preparation; for general protocol and concept, see (Mann et al, 2016a;Mann et al, 2016b;Mann et al, 2019b) million reads per barcode. Reads containing the transposon IRDR element were processed using the SBCapSeq bioinformatic workflow as described (Mann et al, 2016b).…”

Section: Keratoacanthoma 20xmentioning

confidence: 99%

Transposon mutagenesis identifies cooperating genetic drivers during keratinocyte transformation and cutaneous squamous cell carcinoma progression

Aiderus

Newberg

Guzman-Rojas

et al. 2019

Preprint

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30The systematic identification of genetic events driving cellular transformation and tumor progression in the absence 31 of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell 32 carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular 33 landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically 34 define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified 35 established tumor suppressor genes, as well as previously unknown oncogenic drivers of cuSCC. Functional analysis 36 confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the 37 initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely 38 heterogeneous genetic landscape of cuSCC initiation and progression, which could be harnessed to better 39 understand skin oncogenic etiology and prioritize therapeutic candidates. 40

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“…Full details for the SBCapSeq protocol (9) optimized for sequencing from solid tumors, will be published elsewhere (Mann et al ., in review; for general protocol and concept, see [ 9 , 71 , 72 ]). Briefly, for selective SB insertion site sequencing by liquid hybridization capture, gDNA (0.5 μg per sample) of either bulk tumor specimens or single cell WGA genomes was used for library construction using the AB Library Builder System, including random fragmentation and ligation of barcoded Ion Xpress sequencing adapters.…”

Section: Methodsmentioning

confidence: 99%

Transposon mutagenesis identifies cooperating genetic drivers during keratinocyte transformation and cutaneous squamous cell carcinoma progression

et al. 2021

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The systematic identification of genetic events driving cellular transformation and tumor progression in the absence of a highly recurrent oncogenic driver mutation is a challenge in cutaneous oncology. In cutaneous squamous cell carcinoma (cuSCC), the high UV-induced mutational burden poses a hurdle to achieve a complete molecular landscape of this disease. Here, we utilized the Sleeping Beauty transposon mutagenesis system to statistically define drivers of keratinocyte transformation and cuSCC progression in vivo in the absence of UV-IR, and identified both known tumor suppressor genes and novel oncogenic drivers of cuSCC. Functional analysis confirms an oncogenic role for the ZMIZ genes, and tumor suppressive roles for KMT2C, CREBBP and NCOA2, in the initiation or progression of human cuSCC. Taken together, our in vivo screen demonstrates an extremely heterogeneous genetic landscape of cuSCC initiation and progression, which can be harnessed to better understand skin oncogenic etiology and prioritize therapeutic candidates.

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SBCapSeq Protocol: a method for selective cloning of Sleeping Beauty transposon insertions using liquid capture hybridization and Ion Torrent semiconductor sequencing

Cited by 5 publications

References 0 publications

Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation

Promoterless Transposon Mutagenesis Drives Solid Cancers via Tumor Suppressor Inactivation

Transposon mutagenesis identifies cooperating genetic drivers during keratinocyte transformation and cutaneous squamous cell carcinoma progression

Transposon mutagenesis identifies cooperating genetic drivers during keratinocyte transformation and cutaneous squamous cell carcinoma progression

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