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The genome sequence of the smoky wainscot, Mythimna impura (Hubner, 1808) [version 1; peer review: awaiting peer review]

We present a genome assembly from an individual female Mythimna impura (smoky wainscot; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 949 megabases in span. The majority of the assembly (98.39%) is scaffolded into 32 chromosomal pseudomolecules with the W and Z sex chromosomes assembled. The complete mitochondrial genome was also assembled and is 15.3 kilobases in length. Gene annotation of this assembly on Ensembl has identified 15,441 protein coding genes.


Background
The smoky wainscot, Mythimna impura (Hubner, 1808), is a common, nocturnal, non-pest, macro-moth species that occurs across the Palearctic. In Great Britain, M. impura has been categorised using the International Union for Conservation of Nature (IUCN) Red List criteria, as a resident species of Least Concern (Fox et al., 2021). Larvae feed on grasses (Gramineae; Robinson et al., 2010), and overwinter as small larvae. They can have one or two broods per year. Adults fly June to October. Noctuids are relatively mobile compared with other families; M. impura has a 'medium' dispersal ability (Jones et al., 2016).
Mythimna impura inhabit downland, sand dunes and rough grassy areas, including field margins. Moths are used as indicator species as they are sensitive to environmental change (Wagner et al., 2021); M. impura is a good indicator species for heavily grazed plots in the steppes of Mongolia (Enkhtur et al., 2017). Worldwide, the Noctuidae family contains many species considered agricultural pests. Integrated Pest Management programmes, including sex attractant trapping, have been developed for their control (Renou et al., 1991). The genome of M. impura, along with other species from this family, will provide valuable resources for comparative studies of these economically important insects.

Genome sequence report
The genome was sequenced from a single female M. impura collected from Ant Hills region, Wytham, Berkshire, UK (Figure 1). A total of 38-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 54-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 47 missing/misjoins and removed 9 haplotypic duplications, reducing the assembly size by 1.27% and the scaffold number by 30.83%, and increasing the scaffold N50 by 20.56%.  The final assembly has a total length of 949 Mb in 92 sequence scaffolds with a scaffold N50 of 30.6 Mb ( Table 1). The majority, 98.39%, of the assembly sequence was assigned to 32 chromosomal-level scaffolds, representing 30 autosomes (numbered by sequence length) and the W and Z sex chromosomes (Figure 2- Figure 5; Table 2).

Genome annotation report
The ilMytImpu1.2 genome has been annotated using the Ensembl rapid annotation pipeline (  Boyes (University of Oxford). The specimen was identified by Douglas Boyes and snap-frozen on dry ice.
DNA was extracted at the Scientific Operations Core, Wellcome Sanger Institute. The ilMytImpu1 sample was weighed and dissected on dry ice with head tissue set aside for Hi-C sequencing. Thorax and abdomen tissue was disrupted by manual grinding in lysis buffer with a disposable pestle. Fragment size analysis of 0.01-0.5 ng of DNA was then performed "using an Agilent FemtoPulse. High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. Low molecular weight DNA was removed from a 200-ng aliquot of extracted DNA using 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing. HMW DNA was sheared into an average fragment size between 12-20 kb in a Megaruptor 3 system with speed setting 30. Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample. The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
RNA was extracted from remaining thorax and abdomen tissue of ilMytImpu1 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions. RNA was then eluted in 50 μl RNAse-free water and  instruments. Hi-C data were generated in the Tree of Life laboratory from head tissue of ilMytImpu1 using the Qiagen kit and sequenced on an Illumina HiSeq (10X) instrument.

Genome assembly
Assembly was carried out with Hifiasm (Cheng et al., 2021); haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020). One round of polishing was performed by aligning 10X Genomics read data to the assembly with longranger align, calling variants with freebayes (Garrison & Marth, 2012). The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using SALSA2 (Ghurye et al., 2019). The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016) as described previously (Howe et al., 2021). Manual curation (Howe et al., 2021) was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and Pretext. The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2021), which performs annotation using MitoFinder (Allio et al., 2020). The genome was analysed and BUSCO scores generated within the BlobToolKit environment (Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the Mythimna impura assembly (GCA_905147345.2). Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).