Microhomology-Mediated Circular DNA Formation from Oligonucleosomal Fragments During Spermatogenesis

Reviewer #1 (Public Review):

This study aims to address the mechanism of eccDNA generation during spermatogenesis in mice. Previous efforts for cataloging eccDNA in mammalian germ cells have provided inconclusive results, particularly in the correlation between meiotic recombination and the generation of eccDNA. The authors employed an established approach (Circle-seq) to enrich and amplify eccDNA for sequencing analyses and reported that sperm eccDNA is not associated with miotic recombination hotspots. Rather, the authors reported that eccDNAs are widespread, and oligonucleosomal DNA fragments from sperm undergoing apoptosis, with the ligation of DNA ends by microhomology-mediated end-joining, would be a major source of eccDNA.

The strength of the study includes evaluating the eccDNA contents not only in sperm but also from earlier stages of cells in spermatogenesis. The differences in eccDNA size peaks between sperm and other progenitors, in particular, the unique peak in sperm around 360 bp, are intriguing. Results from sequencing data analysis were presented elegantly.

I also have critiques. First, the lack of eccDNA quality control step is a concern. Previous studies employed electron microscopy to ensure that DNA species are mostly circular before rolling-circle amplification. Phi29 polymerase is widely used for DNA amplification, including whole genome amplification of linear chromosomal DNA. Phi29 polymerase has a high processivity and strand displacement activity. When those activities occur within a molecule, it creates circular DNA from linear DNA in vitro. In vitro-created eccDNA from linear DNA would be randomly distributed in the genome, which may explain the low incidence of common eccDNA between replicates. Therefore, it will be crucial to show that DNA prior to amplification is dominantly circular. Electron microscopy would be challenging for the study because the relatively small number of cells were processed to enrich eccDNA. An alternative method for quality controls includes spiking samples with linear and circular exogenous DNA and measuring the ratios of circular/linear control DNA before and after column purification/exonuclease digestion. eccDNA isolation procedures can be validated by a very high circular/linear control DNA ratio.

Another critique is regarding the limitation of the study. It is important to remind the readers of the limitations of the study. As the authors mentioned, rolling circle amplification preferentially increases the copy numbers of smaller eccDNA. Therefore, the native composition of eccDNA is skewed. In addition, the candidate eccDNAs are identified by split reads or discordant read pairs. The details of the mapping process are unclear from the methods, but such a method would require reads with high mapping quality; the identification of eccDNA is expected to require sequencing reads that are mapped to genomic locations uniquely with high confidence, and reads mapped to more than one genomic location, such as highly similar repeat sequences or duplications, are eliminated. Such identification criteria would favor eccDNA formed by little or no homology at the junction sequences, and eliminate eccDNA formed by long homologies at the ends, such as eccDNA formed exclusively by satellite DNA. Therefore, it is not surprising that the authors found the dominance of microhomology-mediated eccDNA. It remains to be determined whether small eccDNA with microhomologies are the dominant species of eccDNA in the native composition. In this regard, it is noted that similar procedures of eccDNA enrichment (column purification, exonuclease digestion, and rolling circle amplification ) revealed variable sizes and characteristics of eccDNA in sperm (human from Henriksen et al. or mice from this study), dependent on the methods of sequencing (long-read or short-read sequencing). Considering these limitations, the last sentence of the introduction, "We conclude that germline eccDNAs are formed largely by microhomology mediated ligation of nucleosome protected fragments, and barely contribute to de novo genomic deletions at meiotic recombination hotspots" needs to be revised.

Small eccDNA (microDNA) data from various mouse tissues are available from the study by Dillion et al., (Cell Reports 2015). Authors are encouraged to examine whether the notable findings in this study (oligonucleosomal-sized eccDNA peaks and the association with apoptotic cell death) are unique to sperm or common in the eccDNA from other tissues.

Reviewer #2 (Public Review):

This study presents a useful investigation of eccDNAs in spermatogenesis of mouse. It provides evidence about the biogenesis of eccDNAs and suggests that eccDNAs are derived from oligonucleosmal DNA fragmentation during apoptosis by MMEJ and may not be the direct products of germline deletions. However, the method of data analyses were not fully described and data analysis is incomplete. It provides additional observations about the eccDNA biogenesis and can be used as a starting point for functional studies of eccDNA in sperms. However, many aspects about data analyses and data interpretations need to be improved.

• Most of the conclusions made by the work are only based on the bioinformatics analyses, the validation of these foundlings using other method (biochemistry/molecular biology method) are missing. For example, no QC results presented for the eccDNA purification, which may show whether contaminates such as linear DNA or mitochondria DNA have been fully removed. Additionally, it is also helpful to use simple PCR to test the existence of identified eccDNAs in sperm or other samples to validate the specificity of the Circle-seq method.

• The reliability of the data analysis methods is uncertain, as the authors constructed and utilized their own pipeline to identify eccDNAs, despite the availability of established bioinformatics tools such as ECCsplorer, eccFinder, and Amplicon Architect. Moreover, the lack of validation of the pipeline using either ground truth datasets or simulation data raises concerns about its accuracy. Additionally, the methodology employed for identifying eccDNA that encompasses multiple gene loci remains unclear.

• Although the author stated that previous studies utilizing short-read sequencing technologies may have incorrectly annotated eccDNA breakpoints, this claim requires careful scrutiny and supporting evidence, which was not provided in the manuscript.

• The similarity between the eccDNA profiles of human and mouse sperm remains uncertain, and therefore, analyses of human eccDNA data and comparisons between the two are necessary if the authors claim that their findings of widespread eccDNA formation in mouse spermatogenesis extend to human sperms.

Author Response

eLife assessment

This study provides valuable information on the biogenesis of eccDNAs during spermatogenesis, i.e., eccDNAs in spermatogenic cells are not derived from miotic recombination hotspots but represent oligonucleosomal DNA fragments from apoptotic male germ cells, whose ends are ligated through microhomology-mediated end-joining. The study is currently incomplete because the method of bioinformatics needs more details and data interpretation should take the amplification bias into consideration.

We highly appreciate the positive assessment.

The negative assessment of our bioinformatics method is probably based on Reviewer #2’ comemnts. While Reviewer #1 considered that “Results from sequencing data analysis were presented elegantly”, Reviewer #2 overlooked some details and raised several critiques regarding our bioinformatics method. We respectfully disagree with many of his or her critiques: (I) Reviewer #2 considered that our method was not fully described. However, we have illustrated the principle and steps of our eccDNA detection method by Figure 4C and Figure 4-figure supplement 2, and submited our source codes to GitHub. (II) Reviewer #2 had concerns on the reliability of our method. However, we have revealed that it has comparible sensitivity and specificity with established bioinformatics tools (Figure 4—figure supplement 2C), and even higher accuracy on the assignment of eccDNA boundaries (Figure 4—figure supplement 2A). (III) Reviewer #2 also believed that “the similarity between the eccDNA profiles of human and mouse sperm remains uncertain”. However, we believe that our Fig. 5 have clearly shown that human sperm eccDNAs have exactly the same characteritics with mouse sperm eccDNAs. Nevertheless, in revised manuscript, we will add more description to help readers to better understand our method, and perform additional analyses to further back up our claims.

The amplification bias is indeed a problem of Circle-seq. Following editors’ and Reviewer #1’s insightful suggestions, we will analyze other datasets generated either by rolling circle amplification or not to see how our findings are affected. Additionally, we will consider to add one section to remind readers of the limitations of rolling-circle amplification-based Circle-seq and our data interpretation.

Reviewer #1 (Public Review):

This study aims to address the mechanism of eccDNA generation during spermatogenesis in mice. Previous efforts for cataloging eccDNA in mammalian germ cells have provided inconclusive results, particularly in the correlation between meiotic recombination and the generation of eccDNA. The authors employed an established approach (Circle-seq) to enrich and amplify eccDNA for sequencing analyses and reported that sperm eccDNA is not associated with miotic recombination hotspots. Rather, the authors reported that eccDNAs are widespread, and oligonucleosomal DNA fragments from sperm undergoing apoptosis, with the ligation of DNA ends by microhomology-mediated end-joining, would be a major source of eccDNA.

The strength of the study includes evaluating the eccDNA contents not only in sperm but also from earlier stages of cells in spermatogenesis. The differences in eccDNA size peaks between sperm and other progenitors, in particular, the unique peak in sperm around 360 bp, are intriguing. Results from sequencing data analysis were presented elegantly.

We are grateful to Reviewer #1 for his or her recognition of the strength of this study.

I also have critiques. First, the lack of eccDNA quality control step is a concern. Previous studies employed electron microscopy to ensure that DNA species are mostly circular before rolling-circle amplification. Phi29 polymerase is widely used for DNA amplification, including whole genome amplification of linear chromosomal DNA. Phi29 polymerase has a high processivity and strand displacement activity. When those activities occur within a molecule, it creates circular DNA from linear DNA in vitro. In vitro-created eccDNA from linear DNA would be randomly distributed in the genome, which may explain the low incidence of common eccDNA between replicates. Therefore, it will be crucial to show that DNA prior to amplification is dominantly circular. Electron microscopy would be challenging for the study because the relatively small number of cells were processed to enrich eccDNA. An alternative method for quality controls includes spiking samples with linear and circular exogenous DNA and measuring the ratios of circular/linear control DNA before and after column purification/exonuclease digestion. eccDNA isolation procedures can be validated by a very high circular/linear control DNA ratio.

We highly appreciate Reviewer #1’s insightful suggestions. We would like to perform eccDNA quality control by introducing circular exogenous DNA into our samples and measuring its ratio to endogenous linear DNA before and after eccDNA isolation procedures.

Another critique is regarding the limitation of the study. It is important to remind the readers of the limitations of the study. As the authors mentioned, rolling circle amplification preferentially increases the copy numbers of smaller eccDNA. Therefore, the native composition of eccDNA is skewed. In addition, the candidate eccDNAs are identified by split reads or discordant read pairs. The details of the mapping process are unclear from the methods, but such a method would require reads with high mapping quality; the identification of eccDNA is expected to require sequencing reads that are mapped to genomic locations uniquely with high confidence, and reads mapped to more than one genomic location, such as highly similar repeat sequences or duplications, are eliminated. Such identification criteria would favor eccDNA formed by little or no homology at the junction sequences, and eliminate eccDNA formed by long homologies at the ends, such as eccDNA formed exclusively by satellite DNA. Therefore, it is not surprising that the authors found the dominance of microhomology-mediated eccDNA. It remains to be determined whether small eccDNA with microhomologies are the dominant species of eccDNA in the native composition. In this regard, it is noted that similar procedures of eccDNA enrichment (column purification, exonuclease digestion, and rolling circle amplification ) revealed variable sizes and characteristics of eccDNA in sperm (human from Henriksen et al. or mice from this study), dependent on the methods of sequencing (long-read or short-read sequencing). Considering these limitations, the last sentence of the introduction, "We conclude that germline eccDNAs are formed largely by microhomology mediated ligation of nucleosome protected fragments, and barely contribute to de novo genomic deletions at meiotic recombination hotspots" needs to be revised.

We thank Reviewer #1 for pointing out limitations of the study. We will take into account and integrate the perspectives of Reviewer #1 in our revised manuscript. We will also try to analyze eccDNA datasets generated by long-read sequencing to see how our conclusions might be affected. However, we envision that it might be challenging to examine the contribution of microhomology-mediated ligation to eccDNA biogenesis using long-read sequencing data as the sequencing error rate of nanopore long-read sequencing data is very high.

Small eccDNA (microDNA) data from various mouse tissues are available from the study by Dillion et al., (Cell Reports 2015). Authors are encouraged to examine whether the notable findings in this study (oligonucleosomal-sized eccDNA peaks and the association with apoptotic cell death) are unique to sperm or common in the eccDNA from other tissues.

We are thankful to Reviewer #1 for this suggestion. We would like to analyze additional eccDNA sequencing datasets to see whether our findings are unique to sperm or common for other tissues.

Reviewer #2 (Public Review):

This study presents a useful investigation of eccDNAs in spermatogenesis of mouse. It provides evidence about the biogenesis of eccDNAs and suggests that eccDNAs are derived from oligonucleosmal DNA fragmentation during apoptosis by MMEJ and may not be the direct products of germline deletions. However, the method of data analyses were not fully described and data analysis is incomplete. It provides additional observations about the eccDNA biogenesis and can be used as a starting point for functional studies of eccDNA in sperms. However, many aspects about data analyses and data interpretations need to be improved.

We thank Reviewer #2 for his or her critical reading. However, we respectfully disagree with some critiques on our data analyses (see below). Anyway, we will provide more method details in addition to Fig. 4C and Figure 4-figure supplement 2 that have illustrated the principle and steps of our method, as well as the performance in comparison with established methods. We will also perform additional analyses and make some clarifications in revised manuscript (see below).

• Most of the conclusions made by the work are only based on the bioinformatics analyses, the validation of these foundlings using other method (biochemistry/molecular biology method) are missing. For example, no QC results presented for the eccDNA purification, which may show whether contaminates such as linear DNA or mitochondria DNA have been fully removed. Additionally, it is also helpful to use simple PCR to test the existence of identified eccDNAs in sperm or other samples to validate the specificity of the Circle-seq method.

Following both this Reviewer’s and Reviewer #1’s suggestions, we will introduce circular exogenous DNA into our samples and measure its ratio to endogenous linear DNA and to mitochondria DNA before and after eccDNA isolation procedures. We will also try to perform PCR to test the existence of identified eccDNAs.

• The reliability of the data analysis methods is uncertain, as the authors constructed and utilized their own pipeline to identify eccDNAs, despite the availability of established bioinformatics tools such as ECCsplorer, eccFinder, and Amplicon Architect. Moreover, the lack of validation of the pipeline using either ground truth datasets or simulation data raises concerns about its accuracy. Additionally, the methodology employed for identifying eccDNA that encompasses multiple gene loci remains unclear.

In fact, we have compared the performance between our method and established methods for identification of eccDNA regions, such as Circle_finder, Circle_Map and ecc_finder. Our method has comparable sensitivity and specificity with existing methods, especially Circle_finder and Circle_Map (Figure 4—figure supplement 2C). We also used one specific genomic region to show that existing methods identified the same eccDNA regions but misassigned the eccDNA boundaries (Figure 4—figure supplement 2A). These results have been shown in Figure 4—figure supplement 2. We will highlight the information to make it more clear in our revised manuscript. We will further detect eccDNAs by ECCsplorer for comparison. Since Amplicon Architect is more specifically designed for detection of ecDNAs, it will not be included in our comparison. We will also try to perform PCR to validate the identified eccDNAs.

As pointed out by Reviewer #2, similar to ECCsplorer, Circle_finder, Circle_Map and ecc_finder, our method fails to identity ecDNAs that encompass multiple gene loci. We will remind readers of this limitation in our revised manuscript.

• Although the author stated that previous studies utilizing short-read sequencing technologies may have incorrectly annotated eccDNA breakpoints, this claim requires careful scrutiny and supporting evidence, which was not provided in the manuscript.

As abovementioned, we used one specific genomic region to show that existing methods all misassigned the eccDNA boundaries (Figure 4—figure supplement 2A). In revised manuscript, we will provide necessary statistics to support this claim.

• The similarity between the eccDNA profiles of human and mouse sperm remains uncertain, and therefore, analyses of human eccDNA data and comparisons between the two are necessary if the authors claim that their findings of widespread eccDNA formation in mouse spermatogenesis extend to human sperms.

We believe that our Fig. 5 have clearly shown that human sperm eccDNAs are also originated from oligonucleosomal fragmentation (Fig. 5A-C), not associated with meiotic recombination hotspots (Fig. 5D and E) but formed by microhomology directed ligation (Fig. 5F and G). These findings are consistent with what we observed in mouse sperm eccDNAs. Nevertheless, we will analyze additional public datasets to further back up our claim in revised manuscript.