A comprehensive analysis of library preparation methods shows high heterogeneity of extrachromosomal circular DNA but distinct chromosomal amount levels reflecting different cell states

• DOI: 10.1039/d3an01300f
• Publication Date: 2023-11-15T09:46:31Z
• Authors: Wenxiang Lu; Fuyu Li; Yunfei Ouyang; Yali Jiang; Weizhong Zhang; Yunfei Bai
• Keywords: Extrachromosomal DNA

Extrachromosomal circular DNA (eccDNA) was discovered several decades ago, but little is known about its function. With the development of sequencing technology, library preparation methods have been developed to elucidate biogenesis and function eccDNA. However, different treatment certain biases that can lead their erroneous interpretation. To address these issues, we compared performance methods. Our investigation revealed utilization rolling-circle amplification (RCA) restriction enzyme linearization mitochondrial (mtDNA) significantly enhanced efficiency enriching extrachromosomal (eccDNA). it also introduced biases, such as an unclear peak in ∼160-200 bp periodicity absence a typical motif pattern. Furthermore, given RCA disproportionate change copy numbers, eccDNA quantification using split discordant reads should be avoided. Analysis genomic elements distribution overall population molecules high correlation between replicates, provided possible stability signature for eccDNA, which could potentially reflect cell lines or states. found only few with identical junction sites each replicate, showing degree heterogeneity The emergence patterns flanking junctional eccDNAs varying sizes suggests involvement multiple potential mechanisms generation. This study comprehensively compares discusses various essential approaches preparation, offering valuable insights practical advice researchers involved characterizing