Jonathan Cooper

eLife assessment
This important study makes a bold step towards understanding what fraction of DNA that is liberated from different tissues in a healthy human is found in circulation as cell-free DNA. Unfortunately, the evidence for the conclusions is presently incomplete, but with additional controls, this could become a major achievement for reference in understanding changes in cell-free DNA in disease states.

anonymous

Reviewer #1 (Public Review):
Summary:
Sender et al describe a model to estimate what fraction of DNA becomes cell-free DNA in plasma. This is of great interest to the community, as the amount of DNA from a certain tissue (for example, a tumor) that becomes available for detection in the blood has important implications for disease detection.
However, the authors' methods do not consider important variables related to cell-free DNA shedding and storage, and their results may thus be inaccurate. At this stage of the paper, the methods section lacks important detail. Thus, it is difficult to fully assess the manuscript and its results.
Strengths:
The question asked by the authors has potentially important implications for disease diagnosis. Understanding how genomic DNA degrades in the human circulation can guide towards ways to enrich for DNA of interest or may lead to unexpected methods of conserving cell-free DNA. Thus, the question "how much genomic DNA becomes cfDNA" is of great interest to the scientific and medical community. Once the weaknesses of the manuscript are addressed, I believe this manuscript has the potential to be a widely used resource.
Weaknesses:
There are two major weaknesses in how the analysis is presented. First, the methods lack detail. Second, the analysis does not consider key variables in their model.
Issues pertaining to the methods section. 
The current manuscript builds a flux model, mostly taking values and results from three previous studies:
1- The amount of cellular turnover by cell type, taken from Sender &amp; Milo, 2021 
2- The fractions of various tissues that contribute DNA to the plasma, taken from Moss et al, 2018 and Loyfer et al, 2023
My expertise lies in cell-free DNA, and so I will limit my comments to the manuscripts in (2).
Paper by Loyfer et al (additional context):
Loyfer et al is a recent landmark paper that presents a computational method for deconvoluting tissues of origin based on methylation profiles of flow-sorted cell types. Thus, the manuscript provides a well-curated methylation dataset of sorted cell-types. The majority of this manuscript describes the methylation patterns and features of the reference methylomes (bulk, sorted cell types), with a smaller portion devoted to cell-free DNA tissue of origin deconvolution.
I believe the data the authors are retrieving from the Loyfer study are from the 23 healthy plasma cfDNA methylomes analyzed in the study, and not the re-analysis of the 52 COVID-19 samples from Cheng et al (MED 2021).
Paper by Moss et al (additional context):
Moss et al is another landmark paper that predates the Loyfer et al manuscript. The technology used in this study (methylation arrays) is outdated but is an incredible resource for the community. This paper evaluates cfDNA tissues of origin in health and different disease scenarios. Again, I assume the current manuscript only pulled data from healthy patients, although I cannot be sure as it is not described in the methods section.
This manuscript:
The current manuscript takes (I think) the total cfDNA concentration from males and females from the Moss et al manuscript (pooled cfDNA; 2 young male groups, 2 old male groups, 2 young female groups, 2 old female groups, Supplementary Dataset; "total_cfDNA_conc" tab). I believe this is the data used as total cfDNA concentration. It would be beneficial for all readers if the authors clarified this point.
The tissues of origin, in the supplemental dataset ("fraction" tab), presents the data from 8 cell types (erythrocytes, monocytes/macrophages, megakaryocytes, granulocytes, hepatocytes, endothelial cells, lymphocytes, other). The fractions in the spreadsheet do not match the Loyfer or Moss manuscripts for healthy individuals. Thus, I do not know what values the supplementary dataset represents. I also don't know what the deconvolution values are used for the flux model.
The integration of these two methods lack detail. Are the authors here using yields (ie, cfDNA concentrations) from Moss et al, and tissue fractions from Loyfer et al? If so, why? There are more samples in the Loyfer manuscript, so why are the samples from Moss et al. being used? The authors are also selectively ignoring cell-types that are present in healthy individuals (Neurons from Moss et al, 2018). Why?
Appraisal:
At this stage of the manuscript, I think additional evidence and analysis is required to confirm the results in the manuscript.
Impact:
Once the authors present additional analysis to substantiate their results, this manuscript will be highly impactful on the community. The field of liquid biopsies (non-invasive diagnostics) has the potential to revolutionize the medical field (and has already in certain areas, such as prenatal diagnostics). Yet, there is a lack of basic science questions in the field. This manuscript is an important step forward in asking more "basic science" questions that seek to answer a fundamental biological question.

Reviewer #2 (Public Review):
Summary:
Cell-free DNA (cfDNA) are short DNA fragments released into the circulation when cells die. Plasma cfDNA level is thought to reflect the degree of cell-death or tissue injury. Indeed, plasma cfDNA is a reliable diagnostic biomarker for multiple diseases, providing insights into disease severity and outcomes. In this manuscript, Dr. Sender and colleagues address a fundamental question: What fraction of DNA released from cell death is detectable as plasma cfDNA? The authors use public data to estimate the amount of DNA produced from dying cells. They also utilize public data to estimate plasma cfDNA levels. Their calculations showed that &lt;10% of DNA released is detectable as plasma cfDNA, the fraction of detectable cfDNA varying by tissue sources. The study demonstrates new and fundamental principles that could improve disease diagnosis and treatment via cfDNA.
Strengths:
1. The experimental approach is resource-mindful taking advantage of publicly available data to estimate the fraction of detectable cfDNA in physiological states. The authors did not assess if the fraction of detectable cfDNA changes in disease conditions. Nonetheless, their pioneering study lays the foundation and provides the methods needed for a similar assessment in disease states. 
2. The findings of this study potentially explain discrepancies in measured versus expected tissue-specific cfDNA from some tissues. For example, the gastrointestinal tract is subject to high cell turnover and release of DNA. Yet, only a small fraction of that DNA ends up in plasma as gastrointestinal cfDNA. 
3. The study proposes potential mechanisms that could account for the low fraction of detectable cfDNA in plasma relative to DNA released. This includes intracellular or tissue machinery that could "chew up" DNA released from dying cells, allowing only a small fraction to escape into plasma as cfDNA. Could this explain why the gastrointestinal track with an elaborate phagosome machinery contributes a small fraction of plasma cfDNA? Given the role of cfDNA as damage-associated molecular pattern in some diseases, targeting such a machinery may provide novel therapeutic opportunities.
Weaknesses: In vitro and in vivo studies are needed to validate these findings and define tissue machinery that contribute to cfDNA production. The validation studies should address the following limitations of the study design: -
1. Align the cohorts to estimate DNA production and plasma cfDNA levels. Cellular turnover rate and plasma cfDNA levels vary with age, sex, circadian clock, and other factors (Madsen AT et al, EBioMedicine, 2019). This study estimated DNA production using data abstracted from a homogenous group of healthy control males (Sender &amp; Milo, Nat Med 2021). On the other hand, plasma cfDNA levels were obtained from datasets of more diverse cohort of healthy males and females with a wide range of ages (Loyfer et al. Nature, 2023 and Moss et al., Nat Commun, 2018). 
2. "cfDNA fragments are not created equal". Recent studies demonstrate that cfDNA composition vary with disease state. For example, cfDNA GC content, fraction of short fragments, and composition of some genomic elements increase in heart transplant rejection compared to no-rejection state (Agbor-Enoh, Circulation, 2021). The genomic location and disease state may therefore be important factors to consider in these analyses. 
3. Alternative sources of DNA production should be considered. Aside from cell death, DNA can be released from cells via active secretion. This and other additional sources of DNA should be considered in future studies. The distinct characteristics of mitochondrial DNA to genomic DNA should also be considered.

What fraction of cellular DNA turnover becomes cfDNA?

Figures and data

cfDNA as a fraction of homeostatic cell turnover.

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