The genome sequence of a cased caddisfly, Mystacides longicornis (Linnaeus, 1758)

We present a genome assembly from an individual male Mystacides longicornis (cased caddisfly; Arthropoda; Insecta; Trichoptera; Leptoceridae). The genome sequence is 665.1 megabases in span. Most of the assembly is scaffolded into 20 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 15.75 kilobases in length.


Background
Mystacides longicornis is a cased caddis in the family Leptoceridae, which are easily recognised by their very long antennae.The commonest form of the adult is also easily recognised, having wings with dark bands on a fawn background; the other form has plain brown wings.Other features making identification easy are the red eyes and black hairy maxillary palps that protrude outwards (Barnard & Ross, 2012).The larval case is straight or slightly curved, composed of sand grains and plant fragments.Often fragments of the case project beyond the case, presumably to make them less easy for predators to swallow.The larva is omnivorous and can be found in both still and flowing water with the adults flying from May to September (Wallace et al., 2003).It is widespread and very common In England and Wales but less so in Scotland (NBN Atlas Partnership, 2024).It is widespread in Europe (O'Connor, 2015).
The genome of Mystacides longicornis was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.Here we present a chromosomal-level whole genome sequence for Mystacides longicornis, based on one male specimen from Lea Broad, England, UK.

Genome sequence report
The genome was sequenced from a male Mystacides longicornis (Figure 1) collected from Lea Broad, England, UK (52.62,1.23).A total of 43-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 13 missing joins or mis-joins and removed 2 haplotypic duplications, reducing the scaffold number by 13.64%, and increasing the scaffold N50 by 0.34%.
The final assembly has a total length of 665.1 Mb in 37 sequence scaffolds with a scaffold N50 of 33.6 Mb (Table 1).The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.88%) of the assembly sequence was assigned to 20 chromosomal-level scaffolds, representing 19 autosomes and the Z sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).Chromosome Z was assigned based on synteny to Athripsodes cinereus (GCA_947579605.1)(Wallace et al., 2023).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition and nucleic acid extraction
A male Mystacides longicornis (specimen ID NHMUK014438422, ToLID iiMysLong1) was hand-picked from Lea Broad, England, UK (latitude 52.62, longitude 1.23) on 2022-04-07.The specimen was hand-picked by Derek Coleman (Dipterists Forum), who also formally identified the species.It was preserved by dry-freezing at -80°C.
The workflow for high molecular weight (HMW) DNA extraction at the Wellcome Sanger Institute (WSI) Tree of Life Core Laboratory includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up.The sample was prepared for DNA extraction at the WSI Tree of Life Core Laboratory: the iiMysLong1 sample was weighed and dissected on dry ice (Jay et al., 2023).Tissue of the whole organism was homogenised using a PowerMasher II tissue disruptor (Denton et al., 2023a), setting aside tissue for Hi-C sequencing.
HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol   Protocols developed by the WSI Tree of Life laboratory are publicly available on protocols.io(Denton et al., 2023b).

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences Sequel IIe (HiFi) instrument.Hi-C data were also generated from remaining tissue of iiMysLong1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly and curation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with  et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.

Final assembly evaluation
The final assembly was post-processed and evaluated with the three Nextflow (Di Tommaso et al., 2017) DSL2 The sanger-tol/blobtoolkit pipeline is a Nextflow port of the previous Snakemake Blobtoolkit pipeline (Challis et al., 2020).It aligns the PacBio reads with SAMtools and mini-map2 (Li, 2018) and generates coverage tracks for regions of fixed size.In parallel, it queries the GoaT database (Challis et al., 2023) to identify all matching BUSCO   et al., 2021) blastp.The genome is also split into chunks according to the density of the BUSCO genes from the closest taxonomically lineage, and each chunk is aligned to the Uniprot Reference Proteomes database with DIAMOND blastx.Genome sequences that have no hit are then chunked with seqtk and aligned to the NT database with blastn (Altschul et al., 1990).All those outputs are combined with the blobtools suite into a blobdir for visualisation.

Gurinder Kaur Walia
Punjabi University, Patiala, Punjab, India The genome of Mystacides longicornis should be compares to those of other related species.This can provide insights into evolutionary changes and genomic adaptations. 1.
The article should also add the outlining of future research directions or potential applications of the genome data which provide a clearer picture of how the data can be utilized.

2.
Is the rationale for creating the dataset(s) clearly described?

Doga Cedden
University of Göttingen, Göttingen, Germany The data note by Derek Coleman presents a genome assembly from Mystacides longicornis of sufficient quality.The insect is adequately described, and the species taxonomy is accurate.The author presents a sufficient quality assessment of the assembly.The assembly was made publicly accessible.My only concern regarding the data note is that the antennae are not present in Figure 1 and should be one of the defining features of this species (longicornis).Please consider replacing Figure 1. with a more appropriate photograph where the antennae are present and visible.
Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: I work on beetle pests using RNAi and transcriptomics.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Genomics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Genome assembly of Mystacides longicornis, iiMysLong1.1:metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 665,109,236 bp assembly.The distribution of sequence lengths is shown in dark grey with the plot radius scaled to the longest sequence present in the assembly (41,430,793 bp, shown in red). .Orange and pale-orange arcs show the N50 and N90 sequence lengths (33,624,892 and 27,270,352 bp), respectively.The pale grey spiral shows the cumulative sequence count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the endopterygota_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Mystacides_longicornis/dataset/GCA_963576905.1/snail.

Figure 3 .
Figure 3. Genome assembly of Mystacides longicornis, iiMysLong1.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Mystacides_longicornis/dataset/GCA_963576905.1/blob.

Figure 4 .
Figure 4. Genome assembly of Mystacides longicornis, iiMysLong1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Mystacides_longicornis/dataset/GCA_963576905.1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Mystacides longicornis, iiMysLong1.1:Hi-C contact map of the iiMysLong1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=Ndau4nLsQF2mSl7UaJ5hCA.

Table 1 :
Proposed standards and metrics for defining genome assembly quality" from Rhie et al. (2021).
(Oatley et al., 2023).The DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 31(Bates et al., 2023).Sheared DNA was purified by solid-phase reversible immobilisation(Strickland  et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA.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.

Darwin Tree of Life Project Sampling nature
, the Biocontainers infrastructure(da Veiga Leprevost et al., 2017), and the Docker(Merkel,  2014)andSingularity (Kurtzer et al., 2017)containerisation solutions.of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project,

Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.