Sampling, Nucleic Acid Extraction and Sequencing
For the whole genome assembly, a female individual of N. riversifrom Conness Lakes (N37.97224, W-119.30703), California, was used to conduct long-read sequencing, and a second female from the same population was used to conduct short-read sequencing. High molecular weight genomic DNA was extracted for the long-read library using the 10x Genomics protocol (DNA Extraction from Single Insects), followed by a purification and concentration step with SPRI magnetic beads. A PacBio library was prepared using the HS Large Fragment 50kb Kit by the UW-Madison Biotechnology Center, with an average library insert size of 35,640 base pairs. It was then sequenced on a PacBio Sequel II machine to approximately 24x coverage. The short-read, pair-end library (2 x 150 bp) was prepared using the Nextera DNA Flex kit and sequenced on the Illumina NovaSeq 6000 to approximately 100x coverage. Additional samples representing the egg stage, larva, adult male and adult female N. riversi were obtained for RNA sequencing. These samples were derived from Conness Lakes, California, with the exception that the larva was a progeny of a cross between Conness Lakes and a Central Sierra Nevada population. Total RNA was extracted using Trizol reagent (Life Technologies) and purified using the PureLink kit (Ambion). Pair-end libraries (2 x 150 bp) were then generated using the TruSeq Stranded Total RNA Library Prep kit by the UW-Madison Biotechnology Center, and samples were sequenced on the Illumina NovaSeq 6000 to generate approximately 30 million reads per sample. All raw sequencing data has been made publicly available on NCBI (SRA Accessions: SRR12967583- SRR12967588).