| Autor: |
Ellis P; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK.; Inivata Limited, The Glenn Berge Building, Babraham Research Campus, Babraham, UK., Moore L; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK., Sanders MA; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK.; Department of Hematology, Erasmus University Medical Center, Rotterdam, the Netherlands., Butler TM; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK., Brunner SF; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK., Lee-Six H; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK., Osborne R; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK.; Inivata Limited, The Glenn Berge Building, Babraham Research Campus, Babraham, UK., Farr B; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK., Coorens THH; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK., Lawson ARJ; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK., Cagan A; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK., Stratton MR; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK., Martincorena I; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK., Campbell PJ; Cancer, Ageing and Somatic Mutation (CASM), Wellcome Sanger Institute, Hinxton, UK. pc8@sanger.ac.uk. |
| Abstrakt: |
Somatic mutations accumulate in healthy tissues as we age, giving rise to cancer and potentially contributing to ageing. To study somatic mutations in non-neoplastic tissues, we developed a series of protocols to sequence the genomes of small populations of cells isolated from histological sections. Here, we describe a complete workflow that combines laser-capture microdissection (LCM) with low-input genome sequencing, while circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into four steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines that might require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation uses enzymatic rather than acoustic fragmentation to generate WGA-free whole-genome libraries. Finally, the mutation calling and filtering strategy has been adapted from previously published protocols to account for artifacts introduced via library creation. To date, we have used this workflow to perform targeted and whole-genome sequencing of small populations of cells (typically 100-1,000 cells) in thousands of microbiopsies from a wide range of human tissues. The low-input DNA protocol is designed to be compatible with liquid handling platforms and make use of equipment and expertise standard to any core sequencing facility. However, obtaining low-input DNA material via LCM requires specialized equipment and expertise. The entire protocol from tissue reception through whole-genome library generation can be accomplished in as little as 1 week, although 2-3 weeks would be a more typical turnaround time. |
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