Utilizing insights of DNA repair machinery to discover MMEJ deletions and novel mechanisms

We developed Del-read, an algorithm targeting medium-sized deletions (6-100 bp) in short-reads, which are challenging for current variant callers relying on alignment. Our focus was on Micro-Homolog mediated End Joining deletions (MMEJ-dels), prevalent in myeloid malignancies. MMEJ-dels follow a distinct pattern, occurring between two homologies, allowing us to generate a comprehensive list of MMEJ-dels in the exome. Using Del-read, we identified numerous novel germline and somatic MMEJ-dels in BEAT- AML and TCGA-breast datasets. Validation in 672 healthy individuals confirmed their presence. These novel MMEJ-dels were linked to genomic features associated with replication stress, like G-quadruplexes and minisatellite. Additionally, we observed a new category of MMEJ-dels with an imperfect match at the flanking sequences of the homologies, suggesting a mechanism involving mispairing in homology alignment. We demonstrated robustness of the repair system despite CRISPR-Cas9-induced mismatches in the homologies. Further analysis of the canonical ASXL1 deletion revealed a diverse array of these imperfect matches. This suggests a potentially more flexible and error-prone MMEJ repair system than previously understood. Our findings highlight Del-read's potential in uncovering previously undetected deletions and deepen our understanding of repair mechanisms.我们开发了Del-read算法，该算法专门针对短读段测序中的中等长度缺失（6~100碱基对，bp）——这类缺失对于当前依赖序列比对的变异检出工具而言极具挑战性。本研究聚焦于微同源介导末端连接缺失（Micro-Homolog mediated End Joining deletions, MMEJ-dels），这类缺失在髓系恶性肿瘤中较为高发。MMEJ-dels具有独特的发生模式，即形成于两段同源序列之间，借此我们可在外显子组中构建一份全面的MMEJ-dels列表。借助Del-read算法，我们在BEAT-AML数据集与TCGA-breast数据集中鉴定出大量全新的生殖系与体细胞MMEJ-dels。通过对672名健康个体的验证实验，确认了这些变异的真实存在。这些新型MMEJ-dels与复制应激相关的基因组特征密切相关，例如G-四链体（G-quadruplexes）与小卫星序列（minisatellite）。此外，我们还观测到一类新型MMEJ-dels，其同源序列的侧翼区域存在不完美匹配现象，这提示存在一种涉及同源比对错配的修复机制。我们证实，即便CRISPR-Cas9在同源序列中引入错配，该修复系统仍可保持稳健性。针对经典ASXL1缺失的进一步分析，揭示了这类不完美匹配的多样形式。这表明MMEJ修复系统可能比此前认知的更为灵活，且更易出现错误。本研究结果凸显了Del-read算法在挖掘此前未被检出的缺失变异方面的巨大潜力，并加深了我们对DNA修复机制的理解。