Human naive small circular DNA (nscDNA) sequencing
Extrachromosomal circular DNA (eccDNA) contributes to genetic variation within eukaryotic genomes (Cohen, Regev and Lavi 1997, Verhaak, Bafna and Mischel 2019). There are various classes of eccDNA that vary by size and chromosomal content, including the largely studied double minute (DM) chromosomes and more recently discovered, microDNA (Paulsen et al. 2018). We developed a technique to enrich for a newly discovered class of eccDNA, naive small circular DNA (nscDNA), in four human cell lines (Colo320DM, HeLa S3, GM12878, IMR-90), frozen human semen samples (Innovative Research, IRHUSM1ML), fresh mouse sperm, and human/mouse tissues without the use rolling circle amplification. After nscDNA isolation using a plasmid purification kit (Qiagen, 12143) and enrichment using Plasmid-safe DNase (Lucigen, E3101K) to deplete residual linear dsDNA, we utilized qPCR to measure ratios of circular DNA controls (mtDNA or spiked pUC18) over linear dsDNA controls (Chr17 target or spiked pEGFP-C1-BamHI-EcoRI) as a way to verify enrichment. We created nscDNA libraries from samples processed in this manner and gDNA libraries for next generation sequencing (NGS) platforms (MiSeq, 2x300 PE/HiSeq, 2x150 PE/NextSeq 2x75 PE) and analyzed their chromosomal content. Samples were sequenced by Cedars-Sinai's Applied Genomics, Computation and Translational Core or Novogene Co., Ltd.Cohen, S., A. Regev &amp; S. Lavi (1997) Small polydispersed circular DNA (spcDNA) in human cells: association with genomic instability. Oncogene, 14, 977-85.Paulsen, T., P. Kumar, M. M. Koseoglu &amp; A. Dutta (2018) Discoveries of Extrachromosomal Circles of DNA in Normal and Tumor Cells. Trends Genet, 34, 270-278.Verhaak, R. G. W., V. Bafna &amp; P. S. Mischel (2019) Extrachromosomal oncogene amplification in tumour pathogenesis and evolution. Nat Rev Cancer, 19, 283-288.