The overall and sequence-specific degradation of soil extracellular 16S rRNA genes across China: rates and influential factors

Reviewer #1 (Public review):

Summary:

This manuscript investigates the degradation dynamics of extracellular DNA in soils and its impact on estimates of microbial abundance and diversity. By combining a broad geographic sampling design with a primer-labeling strategy, qPCR quantification, amplicon sequencing, and PMA treatment, the authors aim to disentangle total versus intracellular DNA signals and explore sequence-specific degradation patterns. The topic is relevant, particularly given the increasing awareness of relic DNA as a confounding factor in microbial ecology. The experimental design is ambitious and potentially impactful. However, several conceptual inconsistencies, methodological ambiguities, and statistical limitations currently weaken the robustness of the conclusions. These issues need to be addressed.

Strengths:

The manuscript addresses a timely and important question in microbial ecology, particularly given the growing recognition that relic DNA can bias interpretations of community composition derived from amplicon sequencing. The study is ambitious in scope, incorporating a broad geographic sampling design across multiple soil types, which enhances the generalizability of the findings. The use of a controlled microcosm experiment combined with a primer-labeling strategy to track extracellular DNA dynamics is conceptually innovative and provides a structured framework to investigate degradation processes.

In addition, the integration of multiple approaches, including qPCR for absolute quantification, high-throughput sequencing for community profiling, and PMA treatment to differentiate extracellular from intracellular DNA, represents a comprehensive attempt to disentangle complex sources of bias in soil microbiome analyses. The effort to link degradation dynamics with environmental variables and to explore sequence-level patterns further demonstrates the authors' intent to move beyond descriptive analyses toward a mechanistic understanding.

Weaknesses:

Several conceptual and methodological issues currently limit confidence in the study's conclusions. Key terms such as "sequence-specific degradation" are not clearly defined or supported by a mechanistic or structural hypothesis, making it difficult to interpret the biological meaning of the results. In addition, the bioinformatic workflow presents inconsistencies, particularly the use of ASVs followed by clustering at 97% similarity, which undermines the resolution required to support sequence-level inferences. Statistical analyses are also insufficiently described, including unclear definitions of "T values," a lack of detail on pairing structure, and no indication of multiple testing correction.

Furthermore, important methodological details are missing or unclear, including primer design (e.g., GAPDH tag vs ACTF), Illumina library preparation (e.g., adapter and indexing strategy), and validation of PMA treatment efficiency. The interpretation of PMA-treated samples as representing "living communities" is likely overstated, given the known limitations of the method in soil systems. Finally, typographical errors, inconsistent terminology, and unclear phrasing throughout the manuscript reduce readability and further complicate interpretation.

Reviewer #2 (Public review):

Summary:

This manuscript describes the results of an interesting study examining the rate of degradation of extracellular DNA in soil ecosystems using a clever experimental approach. 16S ribosomal RNA genes were amplified from soil samples, and then purified PCR amplicons, containing a 5' linker sequence on the forward primer, were introduced to soils and monitored over time using real-time quantitative PCR and NGS amplicon sequencing. The study was able to measure rates of overall extracellular DNA degradation, but also sequence-specific degradation rates. I like the idea and execution of the study, and the results are interesting. The manuscript needs some help to improve the overall readability. Please see general and editorial comments below.

Strengths:

Innovative experimental design that is well deployed across a large number of soil types, revealing interesting variability in extracellular DNA degradation.

Weaknesses:

(1) The manuscript needs another review to improve the readability of the document.

(2) The authors have used 16S genes to look at sequence-specific degradation. But 16S rRNA genes are actually pretty well conserved, and there isn't as much genetic variation across this gene among organisms as there is for other genes. It might be more relevant to look at metagenomic DNA degradation from high AT, high GC organisms, etc. This would be more generalizable than 16S genes.

(3) Consideration of differential cell lysis during soil DNA extraction needs to be considered as well.

(4) It is not clear why the authors didn't put GAPDH linkers on the reverse primer as well. This would have given an easier amplicon to amplify (no degeneracies at all).