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Single-cell whole-genome sequencing has facilitated the detection of somatic single-nucleotide variant (sSNV) mutations in individual, non-clonal cells. However, accurate detection of sSNV mutations is impeded by single-stranded DNA damage arising from both intracellular processes and artefactual contributors. As such, conventional methods of whole-genome amplification (WGA) are susceptible to false positives, potentially contributing to an overestimation of sSNV mutational burden. Duplex-strand genome sequencing exploits DNA complementarity to mitigate false positive calls, thereby enhancing burden estimates by effectively discriminating genuine mutations from sequencing errors or artifacts at the single-cell level. By identifying and comparing complementary sequences, duplex sequencing effectively discriminates genuine double-stranded mutations from sequencing errors or artefacts, providing a robust framework for accurate mutation detection at the single-cell level.
We have built on a recently developed method of transposon-based whole-genome amplification of complimentary strands for use on single neurons (1). Our method utilizes enhanced approaches to accessing the genome of each single cell and to the calibration of genome-wide mutation rates. The application of duplex-strand whole-genome sequencing –optimized to limit artefacts introduced by WGA methods – to single neurons facilitates a more accurate assessment of the mutational landscape of neurodegeneration. Previous work by our group utilizing single-cell whole-genome sequencing has demonstrated the increase of somatic DNA alterations in neurons with normal aging, as well as the accelerated accumulation of somatic changes in Alzheimer’s disease (2,3). We have now employed duplex-strand sequencing to determine the burden of double-stranded and single-stranded somatic mutations across the human lifespan, in neurotypical individuals ranging from infancy to centenarian. We observe that double-stranded somatic SNV and single-stranded DNA lesions both accumulate consistently with age, but with distinct kinetics and signature elements. The accurate assessment of somatic DNA alterations provides insight to the genomic damage that occurs in neurons to ultimately impair function; thus, duplex-strand sequencing provides a powerful tool to perturb aging and neurodegeneration in the human brain.
References:
1 Xing D, Tan L, Chang CH, Li H, Xie XS. Accurate SNV detection in single cells by transposon-based whole-genome amplification of complementary strands. Proc Natl Acad Sci U S A. 2021;118(8). Epub 2021/02/18. doi: 10.1073/pnas.2013106118. PubMed PMID: 33593904; PMCID: PMC7923680.
2 Miller MB, Huang AY, Kim J, Zhou Z, Kirkham SL, Maury EA, Ziegenfuss JS, Reed HC, Neil JE, Rento L, Ryu SC, Ma CC, Luquette LJ, Ames HM, Oakley DH, Frosch MP, Hyman BT, Lodato MA, Lee EA, Walsh CA. Somatic genomic changes in single Alzheimer’s disease neurons. Nature. 2022;604(7907):714-22. Epub 2022/04/22. doi: 10.1038/s41586-022-04640-1. PubMed PMID: 35444284; PMCID: PMC9357465.
3 Luquette LJ, Miller MB, Zhou Z, Bohrson CL, Zhao Y, Jin H, Gulhan D, Ganz J, Bizzotto S, Kirkham S, Hochepied T, Libert C, Galor A, Kim J, Lodato MA, Garaycoechea JI, Gawad C, West J, Walsh CA, Park PJ. Single-cell genome sequencing of human neurons identifies somatic point mutation and indel enrichment in regulatory elements. Nat Genet. 2022;54(10):1564-71. Epub 2022/09/27. doi: 10.1038/s41588-022-01180-2. PubMed PMID: 36163278; PMCID: PMC9833626.
