Rapid Micronucleus-Sequencing (RaMN-Seq) Reveals Patterns of Bias in Induced and Spontaneous Chromosomal Instability

Abstract

Micronuclei are extra-nuclear structures formed following aberrant chromosome mis-segregation. Due to abnormal membrane features, micronuclei are liable to membrane rupture and are proposed as an important intermediate of extreme genome evolution events in cancer such as chromothripsis. Studying the features and contents of micronuclei can be informative about the underlying defective genomic instability process, but current purification strategies are time-consuming and require specialised methods. We developed Rapid MicroNucleus sequencing (RaMN-seq), a rapid (~2 hour), kit-style micronucleus purification strategy coupled to single micronucleus low-pass DNA sequencing to explore the genomic contents of micronuclei. Using RaMN-Seq we recapitulate the known chromosome bias of nocodazole washout-induced chromosome mis-segregation in diploid cells. We explore the features of ruptured (cGAS-positive) and intact micronuclei, finding no substantial differences in chromosomal contents or sizes between rupture states. Finally, we extend RaMN-Seq to cancer models, demonstrating the ability to rapidly explore features of chromosomal instability in cancer cell lines and fresh-frozen tumour samples. Overall, RaMN-Seq allows simple, rapid purification of micronuclei from a range of sources, and offers an inexpensive alternative to single cell genome sequencing to examine properties and chromosomal identities of genetic material affected by chromosomal instability.