Comment from Amirhossein Daneshpajouh

This comment supersedes my earlier submission on this docket (comment tracking number mrl-e7yr-j4u0). It is identical in substance; only internal source-file names in the citations were replaced with descriptive names. Please treat this version as authoritative, and I have separately requested withdrawal of the earlier submission. Executive summary. This comment asks the Agency to require four additive, quantitative reporting elements layered onto the existing draft guidance, each anchored to a specific guidance section and each computable today on public data alone: 1. (Sections VI-VII, off-target nomination completeness.) A sponsor relying on an in silico off-target candidate list should disclose the completeness of that list against three explicit, computable channels - reference-only, patient short variants, and patient structural-variant junctions - rather than leaving the list's completeness unstated. 2. (Section VIII, chromosomal-integrity / loss-of-genome-integrity analysis.) A sponsor relying on a short-read structural-variant caller for large-indel or translocation detection should report a distribution-free, caller-specific certified miss-rate floor against an orthogonal high-fidelity (e.g., long-read) truth set, because short-read SV callers carry large, caller-specific blind spots that a "sensitive and quantitative" claim (guidance's own language) does not, by itself, rule out. 3. (Sections IV-VII, sequencing-depth / sample-size adequacy.) Any zero-observed-failure calibration or validation claim should distinguish an existence floor (the sample size at which a bound is merely defined) from the much larger certification floor (the sample size actually needed to certify the stated miss-rate), because the two are numerically far apart and are conflated in current practice. 4. (Submission of study reports, bioinformatics reproducibility.) Every certified or reported number should be traceable to a hash-bound, independently re-derivable artifact and pipeline, as a concrete operationalization of the guidance's reproducibility expectations. Two worked examples (an approved ex vivo product and an investigational in vivo product) show the off-target completeness gap is measurable today on public reference data, and that for at least one documented site the ancestry-conditional coverage gap spans two orders of magnitude (8.9% vs 0.029% carrier probability, AFR vs NFE, at a single created candidate). An independent public-data benchmark shows that a widely used short-read structural-variant caller, evaluated against a long-read gold-standard truth set, has a certified deletion miss-rate of at least 70.7% (one-sided 95% lower bound) on the benchmarked class - a concrete instance of the detection-floor gap that Section VIII's "sensitive and quantitative" language does not itself quantify. Full 23-page comment is attached as FDA_COMMENT_FINAL_CLEAN.pdf. Affiliation: Simon Fraser University's Computational Biology Lab, under the supervision of Dr. Kay C. Wiese. Conflict of interest: The author declares no competing interests.