Sample collection and DNA extraction
An adult Nucella lapillus individual was collected in May 2024 from Nahant, MA (42.419732, −70.902171). The foot tissue was used for DNA extraction and isolation.

High molecular weight (HMW) DNA was extracted from the foot via the CTAB method (1.4 M NaCl and 2% CTAB), followed by three chloroform reactions. The complete extraction protocol is available on the project GitHub repository (https://github.com/meghanclownfish/Nucella-lapillus-genome/tree/main/1_extraction). The HMW DNA was precipitated in ethanol (EtOH) and resuspended in Tris-EDTA buffer. The sample was further purified using a Genomic DNA Clean and Concentrator kit (gDCC-10, ZYMO Research, Irvine, CA, USA) per manufacturer’s instructions. Sample quality and concentration were assessed by running 2 μL of the sample on NanoDrop (Thermo Fisher Scientific, Singapore). A sample was deemed ready for sequencing if the 260/280 ratio was ∼1.9 and the 260/230 ratio fell between 2.0 and 2.2, following Sun et al. 2020.

Library prep and sequencing
N. lapillus long reads were sequenced using ONT platforms and libraries were prepared with the ONT Ligation sequencing kit (SQK-LSK114, ONT, Oxford, UK) and NEBNext Companion Module (E7180S NEB). Standard manufacturer's protocol was implemented with a few exceptions (see Ford et al., 2025 for details). Sequencing was done on a PromethION. Six flow cells in total were used to generate 103,553,219,099 bp raw data. PromethION flow cells (FLO-PRO114M) were primed and loaded per the standard manufacturer's protocol. To increase the yield of each flow cell, runs were paused and flushed using the EXP-WSH004 (ONT) kit and reloaded. High quality base calling was performed with Dorado 0.7.1 (ONT).

Genome size and heterozygosity
Genome size was estimated using JELLYFISH v2.2.10 to count canonical 41-mers from high quality ONT reads (min quality: 5) and computed a histogram of k-mer occurrence (Marcais and Kingsford 2011). The histogram was used to estimate heterozygosity with GenomeScope (Ranallo-Benavidez et al. 2020).