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PNAS January 21, 2020
We observed the presence of extrachromosomal circular DNA (eccDNA) in the plasma of pregnant women. We found that the plasma eccDNA molecules were longer than their linear counterparts. Among such eccDNA molecules, those of fetal origin were shorter than those of maternal origin
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 这些eccDNA分子显示出双峰大小分布,峰值分布在〜202和〜338 bp,在两个峰的整个大小范围内观察到明显的10 bp周期性,表明它们的核小体起源. Also, the predominance of the 338-bp peak of eccDNA indicated that eccDNA had a larger size distribution than linear DNA in human plasmaeccDNA的338bp峰占优势,表明eccDNA在人血浆中的分布比线性DNA大。. Moreover, eccDNA of fetal origin were shorter than the maternal eccDNA胎儿来源的eccDNA比母亲的eccDNA短. 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.
The fragmentation patterns of cell-free DNA (cfDNA) in human plasma is an area of intense research interest (–). Recent studies on the size distributions (), end locations (), and end motifs () revealed that these fragmentation patterns in cfDNA bore relationships with their tissues of origin. In pregnancy, fetal-derived plasma DNA (mainly of placental origin) were observed to be linear fragments of DNA that were shorter than the maternal-derived (mainly of hematopoietic origin) DNA (, , ). In cancer, tumor-derived cfDNA were detected with smaller sizes and preferred end coordinates from those derived from nonmalignant cells (–). Diagnostic applications had been demonstrated for using the fragmentation patterns of plasma DNA in noninvasive prenatal testing and cancer testing (, , –). However, the above-mentioned studies predominantly focused on linear DNA fragments in plasma. We have recently demonstrated that there were different topologic forms (i.e., linear as well as circular) of plasma mitochondrial DNA (mtDNA) (). Of interest, we observed that circular mtDNA molecules were predominantly of hematopoietic origin, whereas the liver-derived ones were predominantly linear.我们观察到圆形mtDNA分子主要来自造血,而肝脏衍生的分子主要是线性的。
In this work, we explored plasma DNA molecules originated from the genome that were of other topological forms. In particular, we focused on extrachromosomal circular DNA (eccDNA) molecules in the plasma of pregnant women. This special form of DNA molecules had previously been observed across different species of organisms from yeast to mouse (, ). The sizes of eccDNA varied widely, ranging from dozens of bases to hundreds of thousands of bases, with the majority of them being smaller than 1,000 bp (, ). These eccDNA molecules were found to be enriched from genomic regions with high gene densities and GC contents (, ). The presence of eccDNA in human and murine plasma had also been reported (, ). However, there are no published data on eccDNA in the plasma of pregnant women. Our first goal in this work was therefore to investigate whether a fetus might release eccDNA into the plasma of its pregnant mother, analogous to the presence of linear fetal DNA in the plasma of pregnant women (, ). Second, we compared the size profiles between maternal and fetal eccDNA. Last, we explored nucleotide motif signatures flanking the eccDNA junctional sites in the hope of gaining insights into eccDNA generation mechanisms.
We analyzed plasma DNA samples from five cases of third-trimester pregnancy by MspI digestion. Circular DNA molecules in plasma were first enriched by exonuclease V (exo V) digestion of the background linear DNA. MspI restriction enzyme was then used to linearize the remaining circular DNA, followed by library construction and next-generation sequencing. The workflow of eccDNA detection from plasma DNA is described in , from which we developed bioinformatics algorithms for eccDNA identification and downstream analyses (see details in ). The “junction” indicated the position where two ends of a genomic sequence were ligated, forming a DNA circle.
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 for details) were assigned as eccDNA fragment reads.
The plasma eccDNA counts of each sample was normalized as eccDNA per million mappable reads (EPM). The number of mappable reads of each sample used in this calculation was the total number of reads mapped to both chromosomal and mtDNA in that sample. We first confirmed the efficiency of exo V in enriching eccDNA molecules from plasma samples. We compared the EPM values of case 13007 with and without exo V treatment, followed by MspI digestion. We observed a 10,014-fold increase in EPM value after exo V treatment (EPM [exo V + MspI]: 6,409; EPM [MspI only]: 0.64). Thus, exo V treatment could significantly enrich eccDNA molecules. For the five pregnancy cases examined by the MspI approach (exo V + MspI), the median EPM value was 1,462 (range, 844 to 6,409). We further plotted the plasma eccDNA size distributions and compared them with their linear counterparts from the same subjects. Size profiles of these five cases showed that the linear DNA and eccDNA molecules in plasma had distinct size distributions (). The linear plasma DNA showed a predominant size peak at ∼166 bp with a 10-bp periodic pattern in molecules smaller than 166 bp. Such a size distribution is in concordance with previous reports on linear plasma DNA (, , ). On the other hand, eccDNA molecules detected from MspI-treated samples showed two major peaks at 202 and 338 bp. The 338-bp peak was around 10 to 30 times more pronounced than the 202-bp peak as indicated by their areas under the curves.按照曲线下的面积计算,338 bp的峰值比202 bp的峰值大10到30倍。 The predominant dinucleosomal size signature of plasma eccDNA observed here was in concordance with previous reports of plasma samples from nonpregnant subjects此处观察到的血浆eccDNA的主要二核小体大小特征与先前来自非妊娠受试者的血浆样品报道一致 (, , ). Moreover, there was a distinct 10-bp periodicity throughout the size ranges within each of the peaks. Notably, such small peaks at 10-bp intervals were of almost identical sizes among different cases. The eccDNA size profiles of individual cases and the sizes of each small peak are plotted in .
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.
It had previously been shown that fetal-derived linear plasma DNA molecules were generally shorter than the maternal-derived molecules (, ). To compare the maternal- and fetal-specific eccDNA in plasma, we classified eccDNA fragments carrying fetal-specific single-nucleotide polymorphism (SNP) alleles as fetal-derived eccDNA, and the ones that carried maternal-specific SNP alleles as maternal-derived eccDNA. show the size distributions of maternal- and fetal-derived eccDNA of the five pregnancy cases (MspI-treated), respectively. Both maternal and fetal eccDNA exhibited two major peaks at ∼202 and ∼338 bp, with both peaks being sharper for the fetal population 母体和胎儿的eccDNA都在〜202和〜338 bp处出现了两个主要峰,这两个峰对于胎儿群体而言都更为尖锐。 Furthermore, both maternal and fetal eccDNA plots showed a 10-bp periodicity in proximity to both peaks. shows that the cumulative frequency curve of fetal-specific eccDNA was located on the left of the maternal-specific curve. Hence, fetal-derived eccDNA molecules were generally shorter than the maternal-derived ones. Such size differences between maternal- and fetal-derived eccDNA were thus consistent with those of their linear counterparts (, ). Also, the fetal DNA fractions deduced from eccDNA showed a positive correlation with those deduced from linear DNA from the same samples (
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