Pupal DNA was isolated from a fourth-generation inbred cohort that originated from a wild-caught female collected in Skåne, Sweden, using standard salt extraction (35). Illumina sequencing was used for all data generation used in genome construction. One paired end (PE) and the two MP libraries were constructed at the Science for Life Laboratory (SciLifeLab), the National Genomics Infrastructure, Sweden, using one polymerase chain reaction–free PE DNA library (180 bp) and two Nextera MP libraries (3 and 7 kb) all from a single individual. All sequencing was done on Illumina HiSeq 2500 High Output mode, PE 2 × 100-bp by SciLifeLab. Two additional 40-kb MP fosmid jumping libraries were constructed from a sibling used in the previous library construction. Genomic DNA, isolated as above, was shipped to Lucigen Co. (Middleton, WI, USA) for the fosmid jumping library construction, and sequencing was performed on an Illumina MiSeq using 2 × 250-bp reads (36). Last, a variable insert size library of 100 to 100,000 bp in length was generated using the Chicago and HiRise methods (37). Genomic DNA was again isolated from a sibling of those used in previous library construction. The genomic DNA was isolated as above and shipped to Dovetail Co. (Santa Cruz, CA, USA) for library construction, sequencing, and scaffolding. These library fragments were sequenced by Centrillion Biosciences Inc. (Palo Alto, CA, USA) using the Illumina HiSeq 2500 High Output mode, PE 2 × 100 bp. Nearly 500 million read pairs of data were generated, providing ~285× genomic coverage (table S1). The 3- and 7-kb MP pair libraries were filtered for high-confidence true MPs using NextClip v0.8 (38). All read sets were then quality-filtered, and the ends were trimmed of adapters and low-quality bases and screened of common contaminants using BBDuk v37.51 (39). Insert size distributions were plotted to assess library quality, which was high (fig. S6). The 180-bp, 3-kb, and 7-kb read datasets were used as input for AllpathsLG r50960 (40) for initial contig generation and scaffolding (note S1). AllpathsLG was run with haploidify = true to compensate for the high degree of heterozygosity. A further round of superscaffolding using the 40-kb library alongside the 3- and 7-kb libraries was done using SSPACE v2 (41). Last, the assembly was scaffolded a third time and error-corrected using the Chicago read libraries and the HiRise software pipeline. These steps produced a final assembly of 3005 scaffolds with an N50 length of 4.2 Mb and a total length of 350 Mb (note S1), and a 14,945-bp mitochondrial DNA (mtDNA) genome (note S1 and fig. S7). The final assembly’s complete and SCO content was 94% for P. napi as assessed by BUSCO v3.0.2 (42) (for more details, see note S1). Haploid status of the chromosome level assembly was further explored using self-alignments and HaploMerger2 (43). See note S1 for more details.

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