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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, whilst circumventing the use of whole-genome amplification (WGA). The protocol is subdivided broadly into 4 steps: tissue processing, LCM, low-input library generation and mutation calling and filtering. The tissue processing and LCM steps are provided as general guidelines which may require tailoring based on the specific requirements of the study at hand. Our protocol for low-input library generation utilises 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 artefacts 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 a week, though 2-3 weeks would be a more typical turnaround time.

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Studies are experimental investigations of a particular phenomenon, e.g., case-control studies on a particular trait or cancer research projects reporting matching cancer normal genomes from patients.

Study ID Study Title Study Type
EGAS00001001855 Other
EGAS00001002471 Cancer Genomics
EGAS00001003525 Whole Genome Sequencing

This table displays only public information pertaining to the files in the dataset. If you wish to access this dataset, please submit a request. If you already have access to these data files, please consult the download documentation.

ID File Type Size Located in
EGAF00001994161 cram 3.7 GB
EGAF00001994162 cram 3.7 GB
EGAF00001994165 cram 3.5 GB
EGAF00001994169 cram 3.7 GB
EGAF00001994658 cram 4.9 GB
EGAF00001994696 cram 4.8 GB
EGAF00001994705 cram 4.9 GB
EGAF00001994714 cram 4.9 GB
EGAF00001994752 cram 4.9 GB
EGAF00001994869 cram 3.9 GB
EGAF00001994870 cram 3.8 GB
EGAF00001994873 cram 3.7 GB
EGAF00001994877 cram 3.9 GB
EGAF00001994880 cram 3.8 GB
EGAF00001994881 cram 3.7 GB
EGAF00001994884 cram 3.6 GB
EGAF00001994888 cram 3.8 GB
EGAF00001994891 cram 3.9 GB
EGAF00001994892 cram 3.8 GB
EGAF00001994924 cram 3.7 GB
EGAF00001994928 cram 3.9 GB
EGAF00001994931 cram 3.8 GB
EGAF00001994932 cram 3.7 GB
EGAF00001994935 cram 3.6 GB
EGAF00001994939 cram 3.8 GB
EGAF00001994942 cram 3.8 GB
EGAF00001994943 cram 3.7 GB
EGAF00001994946 cram 3.6 GB
EGAF00001994950 cram 3.8 GB
EGAF00002021521 cram 5.2 GB
EGAF00002021522 cram 5.7 GB
EGAF00002027554 cram 4.7 GB
EGAF00002027555 cram 5.3 GB
EGAF00002027563 cram 4.8 GB
EGAF00002027564 cram 5.3 GB
EGAF00002027833 cram 4.8 GB
EGAF00002027834 cram 5.4 GB
EGAF00002027842 cram 4.8 GB
EGAF00002027843 cram 5.5 GB
EGAF00002046821 cram 29.9 GB
EGAF00002046834 cram 28.8 GB
EGAF00002047125 cram 6.8 GB
EGAF00002047131 cram 6.8 GB
EGAF00002047137 cram 6.7 GB
EGAF00002047197 cram 7.1 GB
EGAF00002047321 cram 6.2 GB
EGAF00002047327 cram 6.1 GB
EGAF00002047333 cram 6.1 GB
EGAF00002047339 cram 6.4 GB
EGAF00002047374 cram 6.4 GB
EGAF00002308045 cram 5.6 GB
EGAF00002308046 cram 5.8 GB
EGAF00002308050 cram 5.7 GB
EGAF00002308051 cram 5.9 GB
EGAF00002308055 cram 5.8 GB
EGAF00002308056 cram 6.0 GB
EGAF00002308060 cram 5.8 GB
EGAF00002308061 cram 6.0 GB
EGAF00002308065 cram 5.6 GB
EGAF00002308066 cram 5.8 GB
EGAF00002308466 cram 5.6 GB
EGAF00002308471 cram 5.5 GB
EGAF00002308476 cram 5.6 GB
EGAF00002308481 cram 5.8 GB
EGAF00002308517 cram 6.1 GB
EGAF00002308822 cram 19.0 GB
EGAF00002308825 cram 18.3 GB
EGAF00002308828 cram 18.2 GB
EGAF00003616305 cram 17.5 GB
EGAF00003616308 cram 16.8 GB
EGAF00003623530 cram 35.5 GB
71 Files (494.9 GB)