Sepia officinalis assembly statistics and quality control.

A) Specimen of S. officinalis (credit: Stephan Junek, MPI Brain Research). B) Overview of the assembly workflow. Genome size was estimated using GenomeScope72,73. The primary assembly was generated from long DNA reads (PacBio Sequel II) and HiC reads (Dovetail OmniC) with hifiasm124. Assembly was scaffolded with YAHS77. C) Snail plot of chromosome-scale S. officinalis assembly generated using blobtools2135 showing scaffold statistics, base composition and BUSCO75 completeness against the metazoa_odb10 database. D) HiC heatmap showing 47 chromosome-scale scaffolds with very few sequences in unplaced scaffolds. Heatmap was generated using juicebox78, 0-7039 observed counts (balanced) are shown.

Syntenic comparison of three decapod species.

A) Taxonomy of selected cephalopod species showing their genome size and (haploid) chromosome numbers. Taxonomy information downloaded from NCBI taxonomy browser, divergence times from85,136. B) Synteny relationship between chromosomes of E. scolopes46 (top), D. pealeii46 (middle) and S. officinalis (bottom). Riparian plot was generated using orthogroups located on chromosomes of the species. Euprymna chromosomes 45 and 46 are not shown because they contain too few orthogroups. C) Synteny plot of Sepia chromosomes 40 (magenta) and 43 (darkblue), that are joined in the other species and cause the different haploid chromosome number in Sepia.

Genome annotation for Sepia officinalis.

A) Annotation of repeat landscape of the S. officinalis genome, annotated using RepeatModeler88. Full repeat landscape is shown on the left, annotated repeats (excluding unclassified or simple repeats) are shown on the right. B-C) Quality control of gene annotation and comparison to two other cuttlefish species using OMArk109. Results shown for Sepia lycidas (GCA_963932145.1, Ensembl Genebuild), Sepia officinalis (BRAKER, this study) and Sepia pharaonis137 (BRAKER). Lophotrochozoa was used as the ancestral clade. B) Completeness assessed by the presence of genes conserved in the clade, classified as single or multiple copies (duplicated), or missing. C) Consistency assessed by the proportion of proteins placed in the correct lineage (consistent); placement in incorrect lineages randomly (inconsistent) or to specific species (contamination), or no placement in known gene families (unknown).

HapHiC scaffolding for different numbers of expected chromosomes show 47 chromosomes as most supported.

Hi-C contact maps from HapHiC76 are shown for 46, 47, 48, 49 and 50 expected chromosome scaffolds. Assembled chromosomes are shown as blue boxes, HiC signal indicating a false (unsupported) merger is shown by cyan arrow, false splits are shown by black arrows.

Comparison of Sepia officinalis chromosome-scale assemblies.

A) HiC heatmap of GCA_964300435.1 scaffolded against Omni-C data collected from a different individual (this study). Assembled chromosomes are shown as blue boxes, HiC signal indicating a false merge is shown by cyan arrow, false splits are shown by black arrows. B) Whole-genome alignment of both S. officinalis assemblies (this study: rows, GCA_964300435.1: columns).

Syntenic relationship between S. officinalis and D. pealeii chromosomes.

Dot plot showing finer-resolution syntenic anchor hits (perfectly collinear blast hits within the same orthogroup). Genes are ordered along the chromosomes, gridlines are shown every 1000 genes.

Syntenic relationship between S. officinalis and E. scolopes chromosomes.

Dot plot showing finer-resolution syntenic anchor hits (perfectly collinear blast hits within the same orthogroup). Genes are ordered along the chromosomes, gridlines are shown every 1000 genes. E. scolopes chromosomes 45 and 46 are not shown because they contain too few orthogroups.

Syntenic comparison of four decapod species hints at a cephalopod sex chromosome.

A) Riparian plot showing synteny relationships of chromosomes from four decapod species, generated using orthogroups. Euprymna chromosomes 45 and 46 are not shown because they contain too few orthogroups. Chromosome split in S. officinalis compared to other species is shown in purple, putative sex chromosome as identified recently87 is shown in cyan. B) Normalized coverage of sequencing data in S. officinalis chromosomes. C) Normalized coverage of short reads to female S. esculenta genome, reproduced from87. Decrease in read coverage for chromosome 46 visible, the putative Z sex chromosome. Read depth was calculated from Illumina gDNA reads in windows of 500,000 bp and normalized to the median coverage of chromosome 1. Box plots showing median divergence (box dividing line), interquartile range (box), and 1.5 times the interquartile range (whiskers). Putative Z chromosome is highlighted in cyan.