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Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing


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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Fig. 1: Tissue processing and library preparation workflow.
Fig. 2: Comparison of DNA library performance in targeted sequencing workflows (Agilent SureSelect).
Fig. 3: Genesis of false-positive cruciform DNA-induced variants.
Fig. 4: Effects of the different filtering steps.
Fig. 5: Validation experiments sequencing ‘near-replicate’ samples.
Fig. 6: Library concentrations of epithelial LCM samples.
Fig. 7: Variation of microbiopsy size in samples from the same breast tumor.
Fig. 8: Copy-number variant, SV and small indel calling.
Fig. 9: Example of SNV filtering results.

Data availability

Sequencing data referred to in this study have been deposited in the European Genome-phenome Archive with accession code EGAD00001006088.


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We acknowledge Y. Hooks and K. Roberts (Wellcome Sanger Institute) for assistance in tissue preparation and sectioning, P. Scott (Wellcome Sanger Institute) for advice and training for LCM, D. Rassl (Royal Papworth Hospital) for pathology support and R. Hoogenboezem (Erasmus University Medical Center) for assistance in software development. Finally, we would like to thank L. Apone, K. McKay, V. Panchapakesa and E. Dimalanta (NEB) for assistance in optimizing the library preparation process. This work was supported by the Wellcome Trust. S.F.B. was supported by the Swiss National Science Foundation (P2SKP3-171753 and P400PB-180790). T.M.B. was supported by a Cancer Research UK Grand Challenge Award (C98/A24032). M.A.S. was supported by a Rubicon Fellowship from the Netherlands Organisation for Scientific Research (019.153LW.038). L.M. is a recipient of a CRUK Clinical PhD Fellowship (C20/A20917) and a Pathological Society of Great Britain and Ireland Trainee Small Grant (grant reference no. 1175). I.M. is funded by Cancer Research UK (C57387/A21777). P.J.C. is a Wellcome Trust Senior Clinical Fellow (WT088340MA).

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P.E., L.M., M.A.S., T.M.B., A.R.J.L. and A.C. wrote the manuscript with contributions from all authors. P.E., L.M., R.O., B.F., S.F.B. and H.L.S. devised the protocol for laser-capture microscopy, DNA extraction and sequencing of microbiopsies. M.A.S. developed filters to remove fragmentase-associated artifacts. L.M. and T.M.B. performed LCM experiments, data curation and analysis. T.C. developed the ‘unmatched normal’ filtering strategy. A.R.J.L., M.R.S., I.M. and P.J.C. assisted with data analysis. P.J.C. supervised the study.

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Correspondence to Peter J. Campbell.

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Peer review information Nature Protocols thanks Edwin Cuppen, Subhajyoti De and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Brunner, S. et al. Nature 574, 538–542 (2019):

Lee-Six, H. et al. Nature 574, 532–537 (2019):

Moore, L. et al. Nature 580, 640–646 (2020):

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Ellis, P., Moore, L., Sanders, M.A. et al. Reliable detection of somatic mutations in solid tissues by laser-capture microdissection and low-input DNA sequencing. Nat Protoc 16, 841–871 (2021).

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