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

Peter Ellis, Luiza Moore, Mathijs A. Sanders, Timothy M. Butler, Simon F. Brunner, Henry Lee-Six, Robert Osborne, Ben Farr, Tim H. H. Coorens, Andrew R. J. Lawson, Alex Cagan, Mike R. Stratton, Inigo Martincorena, Peter J. Campbell

Published: 2020-12-13 DOI: 10.1038/s41596-020-00437-6

laser-capture microdissection

low-input DNA sequencing

somatic mutations

genome sequencing

tissue processing

AI 解读

Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail

Profiling native pulmonary basement membrane stiffness using atomic force microscopy

Genetic circuit design automation with Cello 2.0

Synthesis of siRNA nanoparticles to silence plaque-destabilizing gene in atherosclerotic lesional macrophages

Scalable dual-omics profiling with single-nucleus chromatin accessibility and mRNA expression sequencing 2 (SNARE-seq2)

A practical guide to scanning light-field microscopy with digital adaptive optics

Effective high-throughput isolation of fully human antibodies targeting infectious pathogens

Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments

MOF-derived nanoporous carbons with diverse tunable nanoarchitectures

Tutorial: a statistical genetics guide to identifying HLA alleles driving complex disease

查看全部

Sections

Figures

References

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
Introduction
Materials
Procedure
Troubleshooting
Timing
Anticipated results
- References
References
ACKNOWLEDGMENTS

Gawad, C., Koh, W. & Quake, S. R. Single-cell genome sequencing: current state of the science. Nat. Rev. Genet. 17, 175–188 (2016).
Blokzijl, F. et al. Tissue-specific mutation accumulation in human adult stem cells during life. Nature 538, 260–264 (2016).
Lodato, M. A. et al. Aging and neurodegeneration are associated with increased mutations in single human neurons. Science 359, 555–559 (2018).
Fatehullah, A., Tan, S. H. & Barker, N. Organoids as an in vitro model of human development and disease. Nat. Cell Biol. 18, 246–254 (2016).
Lee-Six, H. et al. The landscape of somatic mutation in normal colorectal epithelial cells. Nature 574, 532–537 (2019).
Brunner, S. F. et al. Somatic mutations and clonal dynamics in healthy and cirrhotic human liver. Nature 574, 538–542 (2019).
Moore, L. et al. The mutational landscape of normal human endometrial epithelium. Nature 580, 640–646 (2020).
Olafsson, S. et al. Somatic evolution in non-neoplastic IBD-affected colon. Cell 182, 672–684 (2020).
Telenius, H. et al. Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 13, 718–725 (1992).
Dean, F. B., Nelson, J. R., Giesler, T. L. & Lasken, R. S. Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res 11, 1095–1099 (2001).
Zong, C., Lu, S., Chapman, A. R. & Xie, X. S. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science 338, 1622–1626 (2012).
Deleye, L. et al. Performance of four modern whole genome amplification methods for copy number variant detection in single cells. Sci. Rep. 7, 1–9 (2017).
Mathieson, W. et al. A critical evaluation of the PAXgene tissue fixation system morphology, immunohistochemistry, molecular biology, and proteomics. Am. J. Clin. Pathol. 146, 25–40 (2016).
Lee-Six, H. et al. Population dynamics of normal human blood inferred from somatic mutations. Nature 561, 473–478 (2018).
Chapman, M. S. et al. Lineage tracing of human embryonic development and foetal haematopoiesis through somatic mutations. Preprint at http://www.biorxiv.org/content/10.1101/2020.05.29.088765v1 (2020).
Yoshida, K. et al. Tobacco smoking and somatic mutations in human bronchial epithelium. Nature 578, 266–272 (2020).
Martincorena, I. et al. Somatic mutant clones colonize the human esophagus with age. Science 362, 911–917 (2018).
Martincorena, I. et al. Tumor evolution. High burden and pervasive positive selection of somatic mutations in normal human skin. Science 348, 880–886 (2015).
Picelli, S. et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014).
Hoang, M. L. et al. Genome-wide quantification of rare somatic mutations in normal human tissues using massively parallel sequencing. Proc. Natl Acad. Sci. USA 113, 9846–9851 (2016).
Kennedy, S. R. et al. Detecting ultralow-frequency mutations by Duplex Sequencing. Nat. Protoc. 9, 2586–2606 (2014).
Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 38, e164–e164 (2010).
Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics 26, 589–595 (2010).
Jones, D. et al. cgpCaVEManWrapper: simple execution of CaVEMan in order to detect somatic single nucleotide variants in NGS data. Curr. Protoc. Bioinformatics 56, 15.10.1–15.10.18 (2016).
McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 20, 1297–1303 (2010).
Broad Institute. Picard Tools. http://broadinstitute.github.io/picard/.
Li, H. et al. The sequence alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).
Kozarewa, I. et al. Amplification-free Illumina sequencing-library preparation facilitates improved mapping and assembly of GC-biased genomes. Nat. Methods 6, 291–295 (2009).
Loo, P. V. et al. Allele-specific copy number analysis of tumors. Proc. Natl Acad. Sci. USA 107, 16910–16915 (2010).
Ye, K., Schulz, M. H., Long, Q., Apweiler, R. & Ning, Z. Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics 25, 2865–2871 (2009).

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

Supplementary information

Supplementary Information

推荐阅读