The genome sequence of a caddisfly, Limnephilus lunatus (Curtis, 1834)

We present a genome assembly from an individual Limnephilus lunatus (a caddisfly; Arthropoda; Insecta; Trichoptera; Limnephilidae). The genome sequence is 1,270 megabases in span. Most of the assembly is scaffolded into 13 chromosomal pseudomolecules, including the assembled Z chromosome. The mitochondrial genome has also been assembled and is 15.4 kilobases long.


Background
Limnephilus lunatus (Figure 1) is one of the most common British caddisflies, found from south-west England to Shetland and is one of the caddis that share the common name of 'cinnamon sedge'. Larvae can be found in still and flowing waters of all sizes, but they are almost all permanent and do not dry up over summer. Larvae are found amongst submerged vegetation or debris such as twigs. The life cycle can vary but eggs are laid in late summer, with the egg mass being laid close to the waterline, with the larvae hatching if the egg mass is wetted or eventually by liquefaction of the jelly. Unusual populations from waters that dry up over summer are also laid in late summer and, if the larvae hatch early, they burrow into the substratum awaiting return of the water to start active growth. While adults from temporary waters have to emerge early before the site dries up, adults from permanent waters emerge at different times, then enter an ovarian diapause, but are otherwise active, until late summer and autumn.
The adult is quite variable with a pale semi-lunar mark at the wing termen being a consistent feature but one that is not unique to the species. It can be distinguished from its relatives using (Barnard & Ross, 2012) and (Wallace et al., 2022). The larvae are of a group that makes their case by arranging the cut pieces of vegetation along the axis of the case; they have an irregular outline and are highly variable. The larvae can only be identified to species level when dead or anaesthetised using a key (Wallace et al., 2003), which only works for final and penultimate instars; there is no key to identify small larvae, pupae or eggs.
The high-quality genome sequence described here is, to our knowledge, the second reported for L. lunatus, with the first completed as part of the i5k initiative (assembly accession GCA_000648945.2). However, it is the first L. lunatus genome with chromosome-scale assembly, and has been generated as part of the Darwin Tree of Life project. It will aid in understanding the biology, physiology and ecology of the species, in addition to providing a mechanism to distinguish egg masses and early larval instars from those of its relatives.

Genome sequence report
The genome was sequenced from one female L. lunatus specimen, collected from Tewin Bury Farm, UK (latitude 51.81, longitude -0.16). A total of 36-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 44-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 59 missing/misjoins and removed 7 haplotypic duplications, reducing the assembly length by 0.96% and the scaffold number by 47.95%.
The final assembly has a total length of 1,270 Mb in 38 sequence scaffolds with a scaffold N50 of 95.4 Mb ( Table 1). Most (98.84%) of the assembly sequence was assigned to 13 chromosomal-level scaffolds, representing 12 autosomes and the Z sex chromosome (Figure 2- Figure 5; Table 2). Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size. Inversions between haplotypes were observed on chromosome 3 (104.64-110.84 Mb) and chromosome 11 . While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.
The assembly has a BUSCO v5.3. 2 (Manni et al., 2021) completeness of 89.8% using the OrthoDB-v10 endopterygota reference set. Although BUSCO coverage falls below the benchmark of 95%, the assembly is validated by high k-mer coverage and consensus quality QV scores (Table 1).

Sample acquisition and nucleic acid extraction
The L. lunatus specimen used for the genome assembly (iiLimLuna2) was collected and identified by Michael Austin (Environment Agency) from Tewin Bury Farm, Hertfordshire, UK (latitude 51.81, longitude -0.16). A second specimen (iiLimLuna7), which was used for RNA sequencing, was collected in Broadway Reen, Cardiff, Wales, UK (latitude 51.55, longitude -3.02) by Caleala Clifford (Natural Resources Wales). The specimen used for Hi-C analysis (iiLimLuna1) was collected from a pond in Gelli-hir Woods, Swansea, Wales, UK (latitude 51.62, longitude -4.08) by Graham Rutt (Natural Resources Wales). All specimens were collected from freshwater habitats using a kick-net. The specimens were preserved by snap-freezing in a dry shipper by Ben Price (Natural History Museum London).
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute. The iiLimLuna2 sample was weighed and    (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit. Low molecular weight DNA was removed from a 20-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 of 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    Hi-C data were also generated from iiLimLuna1 using the Arimav2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly
Assembly was carried out with Hifiasm (Cheng et al., 2021) and 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 Long Ranger 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 as described previously (Howe et al., 2021). Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022). The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2021), which performed annotation using MitoFinder (Allio et al., 2020). The genome was analysed and BUSCO scores generated within the BlobToolKit environment (Challis et al., 2020).  The genome sequence is released openly for reuse. The Limnephilus lunatus genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data and the assembly have been deposited in INSDC databases. The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute. Raw data and assembly accession identifiers are reported in Table 1. I am surprised the i5K initiative paper that previously sequenced this species is not cited 2 . 4. I presume the apparently poor Busco completeness is likely to reflect the Busco gene set rather than the assembly, It would be good to see some comment on this.

5.
provides standard assembly quality metrics. The genome quality is a tremendous improvement over the previous genome assembly by the i5k pilot project, and should be a welcome resource for arthropod genomics research.
My only suggestions are to: Include citations for the assertions about the species' biology in the introduction; 1.
When referring to the previous i5k assembly, cite the Thomas et al., paper 1 that first presented and used the original assembly. 2.