The genome sequence of the cinnamon sedge caddisfly, Limnephilus marmoratus (Curtis, 1834)

We present a genome assembly from an individual Limnephilus marmoratus (a caddisfly; Arthropoda; Insecta; Trichoptera; Limnephilidae). The genome sequence is 1,630 megabases in span. Most of the assembly (99.93%) is scaffolded into 30 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.4 kilobases in length.


Background
Limnephilus marmoratus (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 slowly flowing waters of all sizes.Sites may dry up over summer, but this is a species more associated with waters that do not dry up completely.Larvae are found amongst submerged vegetation or debris such as twigs.The larvae feed primarily on dead plant litter, but can eat a wide range of food.The life cycle is not clear, as those from permanent waters seem to emerge as adults in summer and lay then, but the laying site is not known, whilst those from temporary waters emerge as adults in spring and diapause until laying in late summer on the damp bottom, e.g.under logs, where water will flood later that year.A similar life cycle that may or may not have a diapause is found in some other Limnephilus species.
The adult is one of the most variable of the larger caddis but always has wings with large patches of brown and white.It can be distinguished from its relatives using a key (Barnard & Ross, 2012;Wallace et al., 2022).The larvae are of a group that makes their case by arranging the cut pieces of vegetation at a tangent to the long axis of the case.The larvae can only be identified to species when dead or anaesthetised using a key by Wallace, Wallace and Philipson, 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 first one reported for L. marmoratus, 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 a male L. marmoratus collected from Abbey Fields Lake Spring Source, UK (latitude 51.32, longitude 0.91).A total of 28-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 55-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 236 missing joins or misjoins and removed 48 haplotypic duplications, reducing the assembly length by 1.62% and the scaffold number by 65.82%, and increasing the scaffold N50 by 13.17%.
The final assembly has a total length of 1,630 Mb in 67 sequence scaffolds with a scaffold N50 of 56.2 Mb (Table 1).Most (99.93%) of the assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 29 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.Chromosomes 2 and 8 have a high level of shared Hi-C signal which seems likely to result from a high proportion of shared repeat.While not fully  RNA was extracted from whole body tissue of iiLimMarm3 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was eluted in 50 μL RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi), Illumina NovaSeq 6000 (RNA-Seq) and HiSeq X Ten (10X) instruments.Hi-C data were also generated from whole body tissue of iiLimMarm1 and iiLimMarm3 using the Arimav2 kit and sequenced on the Illumina NovaSeq 6000 instrument.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).Table 3 contains a list of all software tool versions used, where appropriate.In line with another reviewer's comment in the genome note of Glyphotaelius pellucidus, it would be good to make a comparison with other caddisfly genomes generated by Darwin Tree of Life.It would also be very helpful if the author could add some additional information about the challenges they have faced in the lab, such as DNA extraction, because sometimes it is difficult to get good-quality DNA of caddisflies.
Is the rationale for creating the dataset(s) clearly described?Jacqueline Heckenhauer LOEWE Centre for Translational Biodiversity Genomics (LOEWE-TBG), Frankfurt, Germany In their data note, the authors present the genome sequence of the cinnamon sedge caddisfly, Limnephilus marmoratus using a de novo assembly method following the approach used by the Darwin Tree of Life Project.The presented genome is of high quality and released openly for reuse.To my knowledge this is the first time the genome of this species has been assembled, therefore this high-quality reference genome is very beneficial to the field and I approve the article.
However, I have some comments/suggestions: Methods: No details are given with respect to the parameters of the bioinformatic tools used.Maybe it is possible to add the command used for assembly and quality control or refer to a data note which already includes this exact information since I imagine the pipeline for genome assembly is similar to other genomes assembled in the DToL Project. 1.
Other quality metrics besides BUSCO and N50 would be interesting to report in addition (for example assessing assembly continuity using LTR-RTs, and reporting back mapping rate of reads to the assembly...)

2.
Was the assembly length as long as the expected genome size? 3. I know that there is an alternate assembly for this specimen.This is really cool!I see the accession of the alternate haplotype is mentioned in the table but maybe also mention this in the text? 4.

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? Partly
Are the datasets clearly presented in a useable and accessible format?Yes

Figure 2 .
Figure 2. Genome assembly of Limnephilus marmoratus, iiLimMarm1.1:metrics.The BlobToolKit Snailplot 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 1,629,971,709 bp assembly.The distribution of chromosome lengths is shown in dark grey with the plot radius scaled to the longest chromosome present in the assembly (77,254,504 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 chromosome lengths (56,174,236 and 44,182,302 bp), respectively.The pale grey spiral shows the cumulative chromosome 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/iiLimMarm1.1/dataset/CAKJUN01.1/snail.

Figure 5 .
Figure 5. Genome assembly of Limnephilus marmoratus, iiLimMarm1.1:Hi-C contact map.Hi-C contact map of the iiLimMarm1.1 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=ZKeV_vHlTzyNCGL2cL5XEg.

Table 1 . Genome data for Limnephilus marmoratus, iiLimMarm1.1. Project accession data
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 90.4% using the endopterygota_odb10 reference set.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 spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

Table 3 . Software tools and versions used.
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the Darwin Tree of Life Project Sampling Code of Practice.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.

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.
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