Chromosome-scale genome assembly of the European common cuttlefish Sepia officinalis

Reviewer #1 (Public review):

Summary:

This manuscript presents a high-quality, chromosome-level genome assembly of the European cuttlefish (Sepia officinalis), a representative species of the cephalopod lineage. Using state-of-the-art sequencing and scaffolding technologies -including PacBio HiFi long reads and Hi-C chromatin conformation capture - the authors deliver a genome assembly with exceptional contiguity and completeness, as evidenced by high BUSCO scores. This genome resource fills a significant gap in cephalopod genomics and offers a valuable foundation for studies in neurobiology, behavior, and evolutionary biology. However, there are several major aspects that need to be strengthened.

Major Revisions Recommended:

(1) Single-individual genome limitation

The genome assembly is based on a single individual, which appears to be male. While this approach is common in genome projects, it does not capture the full genetic diversity of the species. As S. officinalis exhibits a wide geographical range and possible population structure, future efforts (or discussion in this manuscript) should consider re-sequencing multiple individuals - of both sexes and from diverse geographic origins - to characterize population-level variation, sex-linked features, and structural polymorphisms.

(2) Limited experimental validation of chromosomal inferences

The study reports chromosome-scale scaffolding using Hi-C data and proposes a revised karyotype for S. officinalis. However, these inferences would be significantly strengthened by orthogonal validation methods. In particular, fluorescence in situ hybridization (FISH) or karyotyping from cytogenetic preparations would provide direct confirmation of chromosome number and structural arrangements. The reliance solely on Hi-C contact maps for inferring chromosomal organization should be acknowledged as a limitation or supplemented with such validations.

(3) Shallow discussion of chromosomal evolution

The manuscript briefly mentions chromosomal number differences among cephalopods but does not explore their evolutionary or functional implications. A more thorough comparative analysis - linking chromosomal rearrangements (e.g., fusions, fissions) with ecological adaptation, life history, or neural complexity - would greatly enhance the impact of the findings. Referencing chromosomal dynamics in related taxa and possible links to behavioral innovations would contextualize these results more effectively.

(4) Underdeveloped gene family and pathway analysis

While the authors identify expansions in gene families such as protocadherins and C2H2 zinc finger transcription factors, the functional significance of these expansions remains speculative. The manuscript would benefit from:

a) Functional enrichment analyses (e.g., GO, KEGG) targeting these gene families.

b) Expression profiling across tissues or developmental stages to infer regulatory roles.

c) Comparison with expression or expansion patterns in other cephalopods with known behavioral complexity (e.g., Octopus bimaculoides, Euprymna scolopes).

d) Potential integration of transcriptomic or epigenomic data to support regulatory hypotheses.

Reviewer #2 (Public review):

Summary:

This paper concerns an interesting organism, Sepia officinalis. However, in the opinion of this reviewer, the paper reads somewhat like a genome report. The authors have used 23x PacBio HiFi in conjunction with relatively low coverage (11x) Hi-C to scaffold the genome into a karyotype of 47 chromosomes. They have used a combination of short and long read RNA seq to annotate the genome in what looks like a very good annotation. The paper offers basic analyses of the Busco evaluation, some descriptive analyses of gene family and repeat content, and a bit more focused analysis on synteny among sequenced squids. Generally, the data will be useful.

Strengths:

This is a high-quality annotation, and the data ultimately will be useful to other researchers. I appreciate trying to understand what's happening between assemblies of S. officinalis.

Weaknesses:

I don't believe the data at hand makes a strong case for the argument of 47 chromosomes. This is my biggest sticking point with the paper, and it is for a few reasons:

(1) The authors point to assembly differences between the DToL assembly and the one presented in the manuscript and seem to claim that DToL is incorrect. However, the DToL assembly (xcSepOffi3.1) is based on much deeper HiFi and HiC coverage than the one at hand (51x and 80+x respectively). There are many things to try here, including:

a) Downloading the DToL data and reassembling using a common pipeline.

b) Downsampling the DToL data to similar coverage as what the authors have achieved.

c) Combining your data and that of DToL for even deeper coverage (heterozygosity is low enough that I don't imagine this impeding things too badly).

(2) Looking at Figure 1, there appears to be a misjoin at chromosome 42. Looking carefully at Figure S1, that misjoin does not appear on any of the panels - this is confusing. Given the size of that chromosome and the authors' chromosome numbering, I'm guessing this is a manual merge (as it's larger than most of the chromosomes numerically close (40, 41, 43, etc). Further, staring closely at Figure 1, there appear to be cross-scaffold contacts between 42 and 43 and 42 and 44. Secondarily there are contacts between 43 and 44. This bit of the assembly seems potentially problematic.

Reviewer #3 (Public review):

Summary:

In this study, authors Simone Rencken and co-authors present and investigate the genome of the common cuttlefish Sepia officinalis.

Strengths:

The authors explain in a detailed yet concise manner the main steps for a genome assembly, with very robust methods for validation, and according to current best practices. In addition to the chromosomal assembly, the authors confirmed the presence of 47 chromosomes using Hi-C data and multiple species synteny. They also generated a comprehensive gene annotation, with assessments of gene completeness, providing a useful resource for the community of researchers interested in cuttlefish biology and comparative genomics.

Weaknesses:

While the study touches upon the subjects of gene content, TE activity, or species-level comparisons, the study does not provide in-depth investigations of these.