eccDNA purification, full-length sequencing, and genomic mapping

Abstract

Extrachromosomal circular DNA (eccDNA) has been discovered for more than half a century1. However, its biogenesis and function have just begun to be elucidated. One hurdle that prevented our understanding of eccDNA is the difficulty in obtaining pure eccDNA from cells. The current eccDNA purification methods mainly rely on exonuclease digestion after alkaline lysis. Due to its low abundance and heterogeneity in size, the current eccDNA purification methods are not efficient in obtaining pure eccDNA. Here, we describe a new 3-step eccDNA purification (3SEP) procedure by adding a new step that allows circular, but not linear, DNA that escaped from exonuclease digestion, bound to silica beads in solution A. Our method allows eccDNA purification with high purity and reproducibility. Additionally, we developed a full-length sequencing and genomic mapping method by combining rolling cycle amplification (RCA) with Nanopore sequencing, through which the consensus sequence of multiple tandem copies of eccDNA contained within the debranched RCA product is derived and mapped to its genomic origin. Collectively, our protocol will facilitate eccDNA identification, characterization, and has the potential for diagnostic and clinic applications.