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. 2020 Jan 21;117(3):1658-1665.
doi: 10.1073/pnas.1914949117. Epub 2020 Jan 3.

Identification and characterization of extrachromosomal circular DNA in maternal plasma

Sarah T K Sin  1   2 , Peiyong Jiang  1   2 , Jiaen Deng  1   2 , Lu Ji  1   2 , Suk Hang Cheng  1   2 , Anindya Dutta  3 , Tak Y Leung  4 , K C Allen Chan  1   2 , Rossa W K Chiu  1   2 , Y M Dennis Lo  5   2
Affiliations

Identification and characterization of extrachromosomal circular DNA in maternal plasma

Sarah T K Sin et al. Proc Natl Acad Sci U S A. .

Abstract

We explored the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. Through sequencing following either restriction enzyme or Tn5 transposase treatment, we identified eccDNA molecules in the plasma of pregnant women. These eccDNA molecules showed bimodal size distributions peaking at ∼202 and ∼338 bp with distinct 10-bp periodicity observed throughout the size ranges within both peaks, suggestive of their nucleosomal origin. Also, the predominance of the 338-bp peak of eccDNA indicated that eccDNA had a larger size distribution than linear DNA in human plasma. Moreover, eccDNA of fetal origin were shorter than the maternal eccDNA. Genomic annotation of the overall population of eccDNA molecules revealed a preference of these molecules to be generated from 5'-untranslated regions (5'-UTRs), exonic regions, and CpG island regions. Two sets of trinucleotide repeat motifs flanking the junctional sites of eccDNA supported multiple possible models for eccDNA generation. This work highlights the topologic analysis of plasma DNA, which is an emerging direction for circulating nucleic acid research and applications.

Keywords: cell-free DNA; eccDNA; noninvasive prenatal testing; plasma DNA topologics.

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Conflict of interest statement

Competing interest statement: S.T.K.S., P.J., J.D., L.J., K.C.A.C., R.W.K.C. and Y.M.D.L. have filed a patent application based on the data generated from this work.

Figures

Fig. 1.
Fig. 1.
Workflow of eccDNA identification. eccDNA generated from the genome would possess a start (blue) and an end (red) position, which were ligated to form a junctional site. eccDNA molecules in the plasma were cleaved by MspI restriction enzyme, followed by library construction procedures. Paired-end sequencing of the DNA libraries were performed on the Illumina HiSeq 1500/2500 platforms. Sequencing reads were aligned to the reference genome, and algorithms were developed to identify eccDNA. Sequencing reads meeting the four criteria of eccDNA identification (see Materials and Methods for details) were assigned as eccDNA fragment reads.
Fig. 2.
Fig. 2.
eccDNA identification by the restriction enzyme (MspI) approach. Plasma samples of five pregnancy cases were analyzed. (A) Size distributions of linear (blue) and eccDNA (red) in the plasma. (B and C) Plots of size distributions of maternal- and fetal-derived plasma eccDNA, respectively. (D) Cumulative frequency plots of maternal- (blue) and fetal-derived (red) eccDNA in plasma.
Fig. 3.
Fig. 3.
Tagmentation-based approach for eccDNA analysis as a verification of the restriction enzyme method. Plasma DNA samples from five pregnancy cases were analyzed by tagmentation. (A) Plasma eccDNA counts detected in pregnancy cases by MspI digestion and tagmentation. P < 0.05, Wilcoxon rank-sum test. EPM, eccDNA per million mappable reads. (B) Size distributions of eccDNA detected by MspI digestion (blue) and tagmentation (red). (C and D) Plots of the size distributions of maternal- and fetal-derived eccDNA, respectively. (E) Cumulative frequency plots of maternal- (blue) and fetal-derived (red) plasma eccDNA.
Fig. 4.
Fig. 4.
Genomic distributions of (A) eccDNA and (B) linear DNA in maternal plasma. Plasma DNA samples from five pregnant subjects were analyzed for each groups. Data of eccDNA were obtained from the tagmentation approach. The “normalized genomic coverage” of linear/eccDNA for each class of genomic elements is the percentage of DNA molecules mapped to that class of genomic elements divided by the percentage of the genome covered by that class of elements. CpG2kbD, 2 kb downstream of CpG islands; CpG2kbU, 2 kb upstream of CpG islands; Gene2kbD, 2 kb downstream of genes; Gene2kbU, 2 kb upstream of genes; UTR, untranslated region.
Fig. 5.
Fig. 5.
Identification of junctional nucleotide motif patterns. (A) Trinucleotide motif sequences flanking the start and end positions of eccDNA molecules with 4-bp spacers in between. (B) The four trinucleotide segments flanking the start and end positions of eccDNA were labeled as I, II, III, and IV. The 4-bp spacers were termed S1 and S2. Trinucleotide motif sequences of I, II, III, and IV flanking eccDNA junctions with the top 20 frequencies were listed.
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