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

Nature Protocols
Peter EllisPeter J Campbell

Abstract

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 o...Continue Reading

References

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Sep 7, 2018·Nature·Henry Lee-SixPeter J Campbell
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Oct 28, 2019·Nature·Simon F BrunnerPeter J Campbell
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Jul 23, 2020·Cell·Sigurgeir OlafssonCarl A Anderson

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Citations

Apr 30, 2021·Nature·Federico AbascalIñigo Martincorena
May 14, 2021·Nature·Michael Spencer ChapmanAna Cvejic
Jul 7, 2021·Trends in Genetics : TIG·Sigurgeir Olafsson, Carl A Anderson
Aug 27, 2021·Nature·Luiza MooreRaheleh Rahbari
Aug 27, 2021·Nature·Tim H H CoorensMichael R Stratton
Oct 2, 2021·Nature Genetics·Philip S RobinsonMichael R Stratton
Oct 15, 2021·Nature·Stanley W K NgPeter J Campbell
Oct 12, 2021·Nucleic Acids Research·Shixiang SunJan Vijg

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Methods Mentioned

BETA
single-cell sequencing
MDA
PCR
microbiopsies
microbiopsy
exome sequencing
dissection
chip

Software Mentioned

GATK package
Pindel
CaVEMan
LCM
ASCAT
microbiopsy
MEM
ANNOVAR
AnnotateBAMStatistics
HiSeqX

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