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Low_input_LC__WGS_

R&D project to develop low input library construction methods.

Type: Other
Archiver: European Genome-Phenome Archive (EGA)

6 Datasets 3 Publications

Click on a Dataset ID in the table below to learn more, and to find out who to contact about access to these data

Dataset ID	Description	Technology	Samples
EGAD00001004192	The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic events and consequent clonal expansions leading to cancer. As for most cancer types, however, understanding of the earliest phases of colorectal neoplastic change, which may occur in morphologically normal tissue, is comparatively limited because of the difficulty of detecting somatic mutations in normal cells. Each colorectal crypt is a small clone of cells derived from a single recently-existing stem cell. Here, we sequenced hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed, some ubiquitous and continuous, others only found in some individuals, in some crypts or during some phases of the cell lineage from zygote to adult cell. Likely driver mutations were present in ~1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium.	HiSeq X Ten	578
EGAD00001004878	R&D project to develop low input library construction methods. . This dataset contains all the data available for this study on 2019-04-01.	HiSeq X Ten Illumina HiSeq 2500	-
EGAD00001006088	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.	HiSeq X Ten Illumina NovaSeq 6000	18
EGAD00001006595	This dataset contains 160 single-cell derived blood colonies from two neonates and 6 adults. It also contains 18 samples that were used as matched normals to call mutations in NanoSeq data (dataset EGAD00001006459).	HiSeq X Ten Illumina NovaSeq 6000	13
EGAD00001008032	The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans. Comparative analyses can shed light on the diversity of mutagenesis across species and on long-standing hypotheses regarding the evolution of somatic mutation rates and their role in cancer and ageing. Here, we used whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found somatic mutagenesis to be dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans, although the relative contribution of each signature varied across species. Remarkably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied displaying a comparable association. Despite widely different life histories among the species surveyed, including ~30-fold variation in lifespan and ~40,000-fold variation in body mass, the somatic mutation burden at the end of lifespan varied only by a factor of ~3. These data unveil common mutational processes across mammals and suggest that somatic mutation rates are evolutionarily constrained and may be a determinant of lifespan.	HiSeq X Ten	36
EGAD00001008764	The single base substitution mutational signatures SBS2 and SBS13, likely caused by APOBEC cytosine deaminases, are common in many human cancer types. However, the stimulus activating APOBEC mutagenesis is unknown and understanding of when it occurs in the progression from normal to cancer cell is limited. Here, as part of a wider survey of human tissues, we whole genome sequenced 342 microdissected normal epithelial crypts from the small intestines of 39 individuals. SBS2/13 mutations were present in 17% normal small intestine crypts and were likely due to APOBEC3A activity. Localised clusters of SBS2/13 mutations (kataegis) were also commonly found. APOBEC mutation burdens were variable between individuals and between crypts from the same individual. Crypts with SBS2/13 often had immediate crypt neighbours without SBS2/13, suggesting that the underlying cause of SBS2/13 is cell-intrinsic rather than a widely distributed microenvironmental exposure, or needs to be permitted by cell-intrinsic conditions. APOBEC mutagenesis occurred throughout the human lifespan, including in young children, and was episodic with a small number of episodes occurring during the life history of a single cell. The results indicate that APOBEC mutagenesis is more common in the small intestine epithelium than in many other cell types, and is an episodic process in vivo initiated or permitted by cell intrinsic factors.	HiSeq X Ten Illumina NovaSeq 6000	408

Publications	Citations
Somatic Evolution in Non-neoplastic IBD-Affected Colon. Olafsson S, McIntyre RE, Coorens T, Butler T, Jung H, Robinson PS, Lee-Six H, Sanders MA, Arestang K, Dawson C, Tripathi M, Strongili K, Hooks Y, Stratton MR, Parkes M, Martincorena I, Raine T, Campbell PJ, Anderson CA. Cell 182: 2020 672-684.e11	96
Somatic mutation rates scale with lifespan across mammals. Cagan A, Baez-Ortega A, Brzozowska N, Abascal F, Coorens THH, Sanders MA, Lawson ARJ, Harvey LMR, Bhosle S, Jones D, Alcantara RE, Butler TM, Hooks Y, Roberts K, Anderson E, Lunn S, Flach E, Spiro S, Januszczak I, Wrigglesworth E, Jenkins H, Dallas T, Masters N, Perkins MW, Deaville R, Druce M, Bogeska R, Milsom MD, Neumann B, Gorman F, Constantino-Casas F, Peachey L, Bochynska D, Smith ESJ, Gerstung M, Campbell PJ, Murchison EP, Stratton MR, Martincorena I. Nature 604: 2022 517-524	138
APOBEC mutagenesis is a common process in normal human small intestine. Wang Y, Robinson PS, Coorens THH, Moore L, Lee-Six H, Noorani A, Sanders MA, Jung H, Katainen R, Heuschkel R, Brunton-Sim R, Weston R, Read D, Nobbs B, Fitzgerald RC, Saeb-Parsy K, Martincorena I, Campbell PJ, Rushbrook S, Zilbauer M, Buczacki SJA, Stratton MR. Nat Genet 55: 2023 246-254	12