Genome sequencing of Spiroplasma strain sHm

For genome sequencing of strain sHm, high molecular weight DNA was extracted from the egg masses of S+ line moths using Nanobind Tissue Big DNA Kit (Circulomics Inc., MD, USA) and used for library construction using Ultra-Long DNA Sequencing Kit (Oxford Nanopore Technologies, Oxford, UK) following the manufacture’s protocol. The constructed libraries were sequenced using ONT MinION flow cell (R 9.4.1) (Oxford Nanopore Technologies). The obtained reads were mapped to the H. magnanima reference genome (Jouraku et al., in preparation) with minimap2 (Li, 2018 (link)), and the non-mapped reads containing Spiroplasma reads were extracted with SAMtools v.1.9 (Li et al., 2009 (link)) and assembled using Canu 1.6 (Koren et al., 2017 (link)). The draft Spiroplasma genome (a circular main chromosome and plasmids) was annotated via BLASTn (NCBI nr database). The extracted DNA was also subjected to Illumina paired-end 150 bp sequencing (PE-150) at Novogene (Beijing, China). The Illumina data were used to polish the draft genome using minimap2 (Li, 2018 (link)) and Pilon v. 1.23 (Walker et al., 2014 (link)). Since no sequence changes were observed after the second polishing, the polished genome was considered as the complete genome of strain sHm. The circularity of the sHm genome was confirmed by BLASTn search, followed by manual deletion of overlapping sequence.