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DATA NOTE

# A chromosomal reference genome sequence for the malaria mosquito, *Anopheles (Nyssorhynchus) aquasalis*, Curry, 1932

[version 1; peer review: 1 approved, 1 not approved]

Mathilde Gendrin<sup>1</sup>, Katy Heu<sup>1</sup>, Harriet F Johnson<sup>2</sup>, Haynes Heaton<sup>3</sup>, Martin G Wagah<sup>4</sup>, Joanna C Collins<sup>4</sup>, Ksenia Krasheninnikova<sup>4</sup>, Sarah E Pelan<sup>4</sup>, Damon-Lee B Pointon<sup>4</sup>, James W Torrance<sup>4</sup>, Alan Tracey<sup>4</sup>, Marcela Uliano-Silva<sup>4</sup>, Jonathan M D Wood<sup>4</sup>, Katharina von Wyschetzki<sup>4</sup>, Shane A McCarthy<sup>4,5</sup>, Mara K N Lawniczak<sup>4</sup>, Daniel E Neafsey<sup>6,7</sup>, Alex Makunin<sup>4</sup>,

Wellcome Sanger Institute Scientific Operations: Sequencing Operations

**V1** First published: 25 Jul 2025, **10**:363
https://doi.org/10.12688/wellcomeopenres.24601.1

Latest published: 25 Jul 2025, **10**:363
https://doi.org/10.12688/wellcomeopenres.24601.1

## Abstract

We present a genome assembly from an individual female *Anopheles (Nyssorhynchus) aquasalis* (a malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), from a wild population in French Guiana. The genome sequence is 177 megabases in span. The majority of the assembly is scaffolded into four chromosomal pseudomolecules with the X sex chromosome assembled into two arms, XR and XL. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

## Keywords

Nyssorhynchus aquasalis, South American malaria mosquito, genome sequence, chromosomal

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This article is included in the **Tree of Life** gateway.

## Open Peer Review

Approval Status X ✓

1

version 1

25 Jul 2025

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2

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1. **Valerie Nguyen**<sup>1</sup>, University of Florida,
Vero Beach, USA

**Yoosook Lee**<sup>2</sup>, University of Florida,
Gainesville, USA

University of Florida, Vero Beach, USA

2. **Craig Wilding**<sup>3</sup>, University College Dublin
School of Biology and Environmental Science,
Dublin, Ireland

Any reports and responses or comments on the
article can be found at the end of the article.

<footnote n="1"><sup>1</sup>Microbiota of Insect Vectors Group, Institut Pasteur de la Guyane, Cayenne, Cayenne, French Guiana</footnote>

<footnote n="2"><sup>2</sup>Scientific Operations, Wellcome Sanger Institute, Hinxton, England, UK</footnote>

<footnote n="3"><sup>3</sup>CSSE, Auburn University, Auburn, Alabama, USA</footnote>

<footnote n="4"><sup>4</sup>Tree of Life, Wellcome Sanger Institute, Hinxton, England, UK</footnote>

<footnote n="5"><sup>5</sup>University of Cambridge Department of Genetics, Cambridge, England, UK</footnote>

<footnote n="6"><sup>6</sup>Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA</footnote>

<footnote n="7"><sup>7</sup>Infectious Disease and Microbiome Program, Broad Institute, Cambridge, Massachusetts, USA</footnote>
**Corresponding authors:** Mara K N Lawniczak (mara@sanger.ac.uk), Alex Makunin (am60@sanger.ac.uk)

**Author roles:** **Gendrin M:** Investigation; **Heu K:** Investigation; **Johnson HF:** Investigation, Methodology; **Heaton H:** Investigation, Methodology; **Wagah MG:** Visualization; **Collins JC:** Data Curation; **Krasheninnikova K:** Methodology; **Pelan SE:** Data Curation; **Pointon DLB:** Data Curation; **Torrance JW:** Data Curation; **Tracey A:** Data Curation; **Uliano-Silva M:** Investigation, Methodology; **Wood JMD:** Data Curation; **von Wyschetzki K:** Methodology; **McCarthy SA:** Investigation, Methodology; **Lawniczak MKN:** Conceptualization, Funding Acquisition, Investigation, Supervision, Writing - Review & Editing; **Neafsey DE:** Conceptualization, Funding Acquisition; **Makunin A:** Formal Analysis, Methodology, Project Administration, Visualization, Writing - Original Draft Preparation;

**Competing interests:** No competing interests were disclosed.

**Grant information:** This work was supported by a Bill & Melinda Gates Foundation Award (INV-009760) to ML. The Wellcome Sanger Institute is funded by the Wellcome Trust (220540), which supports ML. SAM is supported by the Wellcome Trust Grant (WT207492). This project has been funded in whole or in part with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Grant Number U19AI110818 to the Broad Institute (DEN). MG is supported by a grant from Collectivité Territoriale de la Guyane (2019/CTG/PEDNI).

*The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.*

**Copyright:** © 2025 Gendrin M et al. This is an open access article distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**How to cite this article:** Gendrin M, Heu K, Johnson HF et al. *A chromosomal reference genome sequence for the malaria mosquito, Anopheles (Nyssorhynchus) aquasalis, Curry, 1932 [version 1; peer review: 1 approved, 1 not approved]* Wellcome Open Research 2025, 10:363 https://doi.org/10.12688/wellcomeopenres.24601.1

**First published:** 25 Jul 2025, 10:363 https://doi.org/10.12688/wellcomeopenres.24601.1
## Species taxonomy

Animalia; Arthropoda; Insecta; Diptera; Culicidae; Anophelinae; *Anopheles*; *Anopheles aquasalis*; Curry, 1932 (NCBI txid: 42839).

## Background

*Anopheles* (*Nyssorhynchus aquasalis*) is a malaria vector endemic to the Caribbean and Central and South America, particularly in coastal regions. It is noted for its tolerance of brackish water as larval habitat². The first genome assembly for this species was released in 2017 (GCA_002846955.1). It was generated using Illumina (short) sequencing reads³, and it exhibits the gapped scaffolds and modest contig sizes typical of arthropod assemblies produced using short reads from heterozygous and/or pooled templates.

We report a new genome assembly of *Anopheles aquasalis*, which was sequenced as part of the Anopheles Reference Genomes Project (PRJEB51690). This chromosomally complete genome sequence was produced from one female from the brood of a wild collected mated female collected in Macouria,

French Guiana. The markedly improved contiguity of this assembly relative to the first one is attributable to the use of long read sequencing of DNA from a single specimen to minimize heterozygosity. This new assembly is presented as a high-quality reference genome for the species.

## Genome sequence report

The genome was sequenced from a single female *Anopheles aquasalis* offspring reared from a wild-caught female individual collected from Macouria, French Guiana (5.015, -52.528). A total of 94-fold coverage in Pacific Biosciences single-molecule HiFi long reads (N50 10.760 kb) and 132-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data from a male sibling. Manual assembly curation corrected 19 missing joins or misjoins, reducing the scaffold number by 14.3%.

The final assembly has a total length of 177 Mb in 91 sequence scaffolds with a scaffold N50 of 90.077 Mb (Table 1). 93.37% of the assembly sequence was assigned to four

<table><caption>Table 1. Genome data for *Anopheles aquasalis*, idAnoAquaMG_Q_19.</caption><thead><tr><th colspan="2">Project accession data</th></tr></thead><tbody><tr><td>Assembly identifier</td><td>idAnoAquaMG_Q_19</td></tr><tr><td>Species</td><td>Anopheles aquasalis</td></tr><tr><td>Specimen</td><td>idAnoAquaMG-Q_19</td></tr><tr><td>NCBI taxonomy ID</td><td>42839</td></tr><tr><td>BioProject</td><td>PRJEB53270</td></tr><tr><td>BioSample ID</td><td>ERS10527339</td></tr><tr><td>Isolate information</td><td>female, whole organism</td></tr><tr><td colspan="2"><strong>Raw data accessions</strong></td></tr><tr><td>PacificBiosciences SEQUEL II</td><td>ERR9439489</td></tr><tr><td>10X Genomics Illumina</td><td>ERR9356742, ERR9356743, ERR9356744, ERR9356745</td></tr><tr><td>Hi-C Illumina</td><td>ERR9356741</td></tr><tr><td>PolyA RNA-Seq Illumina</td><td>ERR9356750, ERR9356751</td></tr><tr><td colspan="2"><strong>Genome assembly</strong></td></tr><tr><td>Assembly accession</td><td>GCA_943734665</td></tr><tr><td>Accession of alternate haplotype</td><td>GCA_943734835</td></tr><tr><td>Span (Mb)</td><td>176.588</td></tr><tr><td>Number of contigs</td><td>118</td></tr><tr><td>Contig N50 length (Mb)</td><td>38.496</td></tr><tr><td>Number of scaffolds</td><td>91</td></tr><tr><td>Scaffold N50 length (Mb)</td><td>90.077</td></tr><tr><td>Longest scaffold (Mb)</td><td>90.077</td></tr><tr><td>BUSCO* genome score</td><td>C:97.5% [S:97.0%,D:0.5%],F:0.5%,M:2.0%,n:3285</td></tr></tbody></table>

<footnote>* BUSCO scores based on the diptera_odb10 BUSCO set using BUSCO 5.3.2. C=complete [S=single copy, D=duplicated], F=fragmented, M=missing, n=number of orthologues in comparison. A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/idAnoAquaMG_Q_19/dataset/CALSDQ01/busco.</footnote>
chromosomal-level scaffolds, representing two autosomes (numbered and oriented against the *An. albimanus* STECLA strain assembly (4; GCA_013758885.1)) with identical chromosome arms arrangement⁵, and the XR and XL sex chromosome arms (Figure 1–Figure 4; Table 2).

Among *Anopheles* species, X chromosomes with two euchromatic arms are uncommon. In the *Nyssorhynchus* subgenus in particular, these were reported in *An. aquasalis*⁶, *An. nuneztovari*⁷, and possibly in *An. noroestensis*⁸, but not in *An. darlingi*⁷,⁹ or *An. albimanus*⁴,¹⁰,¹¹. Judging from the complete mitochondrial genome phylogeny¹², the biarmed X could be

associated with *An. oswaldoi* - *An. goeldii* branch roughly corresponding to *An. oswaldoi* group¹³. Synteny to *An. albimanus* (Figure 4) indicates that both X chromosome arms in *An. aquasalis* have orthologous segments in the X chromosome of *An. albimanus*. The segments from both arms seem to be distributed evenly across the *An. albimanus* X as a result of multiple rearrangements.

Chromosome arms, candidate centromere sequences, and the rDNA region were delineated based on the presence of characteristic tandem repeat arrays (Figure 5; Table 3). For candidate centromeres, most common tandem repeat units

<figure><img src="image_5.png" /><figcaption>**Figure 1.** Snail plot summary of assembly statistics for *Anopheles aquasalis* assembly idAnoAquaMG_Q_19. The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 176,588,384 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 (90,077,004 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 sequence lengths (90,077,004 and 8,489,435 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 diptera_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idAnoAquaMG_Q_19/dataset/CALSDQ01/snail.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Figure 2.** Blob plot of base coverage in a subset of idAnoAquaMG_Q_19 10x linked reads against GC proportion for *An. aquasalis* assembly idAnoAquaMG_Q_19. Chromosomes are coloured by phylum. Circles are sized in proportion to chromosome length. Histograms show the distribution of chromosome length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/idAnoAquaMG_Q_19/dataset/CALSDQ01/blob.</figcaption></figure>

were 97 bp long for chromosome 2, 107 bp long for chromosome 3 and chromosome arm XL, and 112 bp long for chromosome arm XR (Figure 6). Candidate centromeric repeat sequences were similar between chromosome 3 and arm XL, while other pericentric repeats lacked sequence similarity between chromosomes. In both XL and XR, pericentric tandem repeats were immediately followed by a short stretch of

rDNA at the very end of the chromosomal portion of assembly. This pericentric location of rDNA block in X chromosome agrees well with FISH results in a closely related species, *An. nuneztovari*¹⁴. The unassembled scaffolds comprised mostly rDNA with small arrays of 107 bp repeat, which suggests that all other repeat types were successfully incorporated in the chromosomal assembly. The two remaining major tandem
<figure><img src="image_7.png" /><figcaption>**Figure 3.** Hi-C contact map for genome assembly of *An. aquasalis*, idAnoAquaMG_Q_19. Visualised in HiGlass. Chromosomes order: 2RL, 3RL, XL, XR, then remaining scaffolds. XL and XR arms have lower contact density as individual Hi-C was a male. The interactive Hi-C map can be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=OC7yenNVRKWXH0x66BkBgQ.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 4.** Genome alignment dotplot between genome assemblies of *An. aquasalis*, idAnoAquaMG_Q_19 and *An. albimanus* (AalbS4). Visualised in DGenies. Identical autosomal chromosome arms arrangement suggested by 5.</figcaption></figure>
<table><caption>**Table 2.** Chromosomal pseudomolecules in the genome assembly of *An. aquasalis*, *idAnoAquaMG_Q_19*.</caption><thead><tr><th>INSDC accession</th><th>Chromosome</th><th>Size (Mb)</th><th>Count</th><th>Gaps</th></tr></thead><tbody><tr><td>OX030882.1</td><td>2RL</td><td>90.077</td><td>1</td><td>1</td></tr><tr><td>OX030883.1</td><td>3RL</td><td>68.231</td><td>1</td><td>3</td></tr><tr><td>OX030884.1</td><td>XL</td><td>8.489</td><td>1</td><td>13</td></tr><tr><td>OX030885.1</td><td>XR</td><td>6.560</td><td>1</td><td>10</td></tr><tr><td>OX030886.1</td><td>MT</td><td>0.015</td><td>1</td><td>0</td></tr><tr><td></td><td>X Unlocalised</td><td>3.216</td><td>86</td><td>0</td></tr></tbody></table>

repeats were found as multiple blocks in the euchromatic part of XL (202 bp unit) or 2R and XL (344 bp unit).

On telomeric ends of all chromosome arms except XL we found small (<10 kbp) blocks of a tandem repeat with a 35 bp unit (CTGTGGGGGTGGTAAGTGTCCTCTTCCCTTTCGAT; not visible in Figure 6 due small size), which we propose represents a species-specific telomere repeat candidate. The telomeric end of the XL arm was occupied by a tandem repeat with a 208 bp unit length and no sequence similarity to the 35 bp repeat. The same 208 bp repeat was immediately adjacent to a 35 bp telomeric repeat in arms 2R and 2L. While the 35 bp repeat could have been truncated in XL arm assembly, it is possible that this heterogeneity in telomeric and subtelomeric

repeat content reflects genuine biological variation characteristic of satellite derived telomeres in Diptera¹⁵.

The assembly has a BUSCO 5.3.2¹⁶ completeness of 97.5% using the diptera_odb10 reference set. 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 reconstructed without sequence gaps and was validated for reading frame accuracy and sequence similarity to public NCBI GenBank records.

Gene annotation was performed with the NCBI Eukaryotic Genome Annotation Pipeline and is available in the RefSeq¹⁷ under the accession GCF_943734665.1. A total of 13,026 genes were predicted, including 11,524 protein-coding genes and 1,790 non-coding RNAs. The genome assembly and gene annotations are hosted on VectorBase, www vectorbase.org¹⁸ under the identifier AaquGF1.

## Methods

### Sample acquisition and nucleic acid extraction

*Anopheles aquasalis* offspring were reared from a wild caught gravid female collected from Macouria, French Guiana (5.015, -52.528) by Katy Heu and Romuald Carinci in April 2019. A single female *idAnoAquaMG-Q_19* was used for Pacific BioSciences and 10x genomics, and its male sibling *idAnoAquaMG-Q_18* was used for Arima Hi-C.

For high molecular weight (HMW) DNA extraction, one whole insect (*idAnoAquaMG-Q_19*) was disrupted by manual grinding with a blue plastic pestle in Qiagen MagAttract lysis buffer and then extracted using the Qiagen MagAttract HMW DNA extraction kit with two minor modifications¹⁹. These

<figure><img src="image_9.png" /><figcaption>**Figure 5.** Sequence similarity heatmap for genome assembly of *An. aquasalis*, *idAnoAquaMG_Q_19*. Produced with StainedGlass, visualised in HiGlass. Chromosomes order: 2RL, 3RL, XR, XL - followed by the remaining scaffolds. Darker colours represent higher sequence similarity, notably at autosomal pericentric heterochromatin and sex chromosomes.</figcaption></figure>
<table><caption>**Table 3.** Chromosome features in the genome assembly of *An. aquasalis*, *idAnoAquaMG_Q_19*.</caption><thead><tr><td>Chromosome</td><td>Start</td><td>End</td><td>Feature</td></tr></thead><tbody><tr><td>2RL</td><td>1</td><td>51,547,091</td><td>Chromosome arm 2R</td></tr><tr><td>2RL</td><td>51,547,092</td><td>57,707,065</td><td>Chromosome 2 centromere</td></tr><tr><td>2RL</td><td>57,707,066</td><td>90,077,004</td><td>Chromosome arm 2L</td></tr><tr><td>3RL</td><td>1</td><td>34,075,603</td><td>Chromosome arm 3R</td></tr><tr><td>3RL</td><td>34,075,604</td><td>34,348,989</td><td>Chromosome 3 centromere</td></tr><tr><td>3RL</td><td>34,348,990</td><td>68,231,066</td><td>Chromosome arm 3L</td></tr><tr><td>XL</td><td>1</td><td>8,070,130</td><td>Chromosome arm XL</td></tr><tr><td>XR</td><td>1</td><td>6,404,761</td><td>Chromosome arm XR</td></tr></tbody></table>

<figure><img src="image_10.png" /><figcaption>**Figure 6.** Tandem repeats annotation for genome assembly of *An. aquasalis*, *idAnoAquaMG_Q_19*.</figcaption></figure>
modifications include halving volumes recommended by the manufacturer due to small sample size (*Anopheles* mosquitoes typically weigh 2–3 mg) and running two elutions to increase DNA yield. The quality of the DNA was evaluated using an Agilent FemtoPulse to ensure that most DNA molecules were larger than 30 kb, and preferably > 100 kb. In general, single mosquito extractions ranged in total estimated DNA yield from 200 ng to 800 ng, with an average yield of 500 ng. Low molecular weight DNA was removed using an 0.8X AMPure XP purification. A small aliquot (less than ~5% of the total volume) of HMW DNA was set aside for 10X Linked Read sequencing and the rest of the DNA was sheared to an average fragment size of 12–20 Kb using a Diagenode Megaruptor 3 at speeds ranging from 27 to 30. Sheared DNA was purified 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 and quality of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer with the Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sheared and cleaned sample on the FemtoPulse system once more. The median DNA fragment size for *Anopheles* mosquitoes was 15 kb and the median yield of sheared DNA was 200 ng, with samples typically losing about 50% of the original estimated DNA quantity through the process of shearing and purification.

For Hi-C data generation, a separate sibling male mosquito specimen (idAnoAquaMG-Q_18) was used as input material for the Arima V2 Kit according to the manufacturer's instructions for animal tissue. This approach of using a sibling was taken in order to enable all material from a single specimen to contribute to the PacBio data generation given we were not always able to meet the minimum suggested guidance of starting with > 300 ng of HMW DNA from a specimen. Samples proceeded to the Illumina library prep stage even if they were suboptimal (too little tissue) going into the Arima reaction.

To assist with gene annotation, RNA was extracted from separate whole sibling male and female insect specimens (idAnoAquaMG-Q_16 and idAnoAquaMG-Q_21, respectively) using TRIzol, according to the manufacturer's instructions. RNA was then eluted in 50 µl RNAse-free water, and its concentration was 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. Samples were not always ideally preserved for RNA, so qualities varied but all were sequenced anyway.

## Sequencing

We prepared libraries as per the PacBio procedure and checklist for SMRTbell Libraries using Express TPK 2.0 with low DNA input. Every library was barcoded to support multiplexing. Final library yields ranged from 20 ng to 100 ng, representing only about 25% of the input sheared DNA. Libraries from two specimens were typically multiplexed on a single 8M SMRT Cell. Sequencing complexes were made using Sequencing Primer v4 and DNA Polymerase

v2.0. Sequencing was carried out on the Sequel II system with 24-hour run time and 2-hour pre-extension. A 10X Genomics Chromium read cloud sequencing library was also constructed according to the manufacturer's instructions (this product is no longer available). Only 0.5 ng of DNA was used and only 25–50% of the gel emulsion was put forward for library prep due to the small genome size. For Hi-C data generation, following the Arima HiC V2 reaction, samples were processed through Library Preparation using a NEB Next Ultra II DNA Library Prep Kit and sequenced aiming for 100x depth. RNA libraries were created using the directional NEB Ultra II stranded kit. Sequencing was performed by the Scientific Operations core at the Wellcome Sanger Institute on Pacific Biosciences SEQUEL II (HiFi), Illumina NovaSeq 6000 (10X and Hi-C), or Illumina HiSeq 4000 (RNAseq).

## Genome assembly

Assembly was carried out with Hifiasm²⁰; haplotypic duplications were identified and removed with purge_dups²¹. One round of polishing was performed by aligning 10X Genomics read data to the assembly with longranger align²², calling variants with freebayes²³. The assembly was then scaffolded with Hi-C data²⁴ using SALSA2²⁵. The assembly was checked for contamination as described previously²⁶. Manual curation was performed using gEVAL²⁷, HiGlass²⁸ and Pretext²⁹. The mitochondrial genome was assembled using MitoHiFi³⁰, which performs annotation using MitoFinder³¹. The genome was analysed and BUSCO scores were generated within the BlobToolKit environment³². Synteny analysis was performed with D-GENIES³³. Repetitive sequences were visualised with StainedGlass³⁴ and tandem repeats were annotated with TRASH³⁵. Table 4 contains a list of all software tool versions used, where appropriate.

<table><caption>**Table 4. Software tools used.**</caption><thead><tr><td>Software tool</td><td>Version</td><td>Source</td></tr></thead><tbody><tr><td>hifiasm</td><td>0.14</td><td>20</td></tr><tr><td>purge_dups</td><td>1.2.3</td><td>21</td></tr><tr><td>SALSA2</td><td>2.2-4c80ac1</td><td>25</td></tr><tr><td>longranger align</td><td>2.2.2</td><td>22</td></tr><tr><td>freebayes</td><td>1.3.1</td><td>23</td></tr><tr><td>MitoHiFi</td><td>2</td><td>30</td></tr><tr><td>gEVAL</td><td>N/A</td><td>27</td></tr><tr><td>HiGlass</td><td>1.11.6</td><td>28</td></tr><tr><td>PretextView</td><td>0.1.x</td><td>29</td></tr><tr><td>BlobToolKit</td><td>3.4.0</td><td>32</td></tr><tr><td>BUSCO</td><td>5.3.2</td><td>16</td></tr><tr><td>D-GENIES</td><td>1.4</td><td>33</td></tr><tr><td>StainedGlass</td><td>0.5</td><td>34</td></tr><tr><td>TRASH</td><td>1.2</td><td>35</td></tr></tbody></table>
## Ethics/compliance issues

The genetic resources accessed and utilised under this project were done so in accordance with the UK ABS legislation (Nagoya Protocol (Compliance) (Amendment) (EU Exit) Regulations 2018 (SI 2018/1393)) and the national ABS legislation within the country of origin, where applicable.

## Data availability

European Nucleotide Archive: *Anopheles aquasalis* genome assembly, idAnoAquaMG_Q_19. Accession number PRJEB53270; https://identifiers.org/bioproject/PRJEB53270.

The genome sequence is released openly for reuse. The *Anopheles aquasalis* genome sequencing initiative is part of the Anopheles Reference Genomes project PRJEB51690. All raw sequence data and the assembly have been deposited in INSDC databases. Raw data and assembly accession identifiers are reported in Table 1.

## Author information

Members of the Sanger Scientific Operations: Sequencing Operations team are listed here: https://doi.org/10.5281/zenodo.4790456.

## Acknowledgments

We thank Romuald Carinci (Institut Pasteur de la Guyane) for assistance with field collection.

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# Open Peer Review

Current Peer Review Status: [X] [✓]

## Version 1

Reviewer Report 04 September 2025

https://doi.org/10.21956/wellcomeopenres.27110.r130202

© 2025 Wilding C. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_11.png" /></figure>

Craig Wilding

<figure><img src="image_12.png" /></figure>

University College Dublin School of Biology and Environmental Science, Dublin, Leinster, Ireland

Overall: this is a valuable additional high-quality *Anopheles* genome sequence that contributes both to the *Anopheles* Reference Genomes Project and more widely. Below I provide some comments on the report that perhaps could be considered for improvements and to make it more widely accessible.

Author list. Methods section states that specimens were collected by Katy Heu and Romuald Carinci. Heu is a named author but not Carinci. Why is this given that collectors are supposedly first authors on Genome Notes? (I note the Acknowledgement so perhaps this is personal choice.)

Background. This is quite basic and lacking in detail - it really only mentions the larval habitat and some broad information on distribution. It would be good to have further details on e.g. full distribution, whether it is a major/minor vector, how it is reliably identified.

**Genome Sequence Report:**

It is not clear why synteny is being mapped to *An. albimanus*. It would be good to have this clarified if only in the legend to Figure 4.

Figure 3 - contact map. There are 5 main blocks on here then the paler areas (which I assume are the XL and XR which are paler due to lower contact density). So the 5 darker 'blocks' then are 2R, 2L, 3R, 3L and ..what? It would be good if this figure were annotated to show which chromosomal arms are being depicted.

I found this sentence unclear "the biarmed X could be associated with *An. oswaldoi* - *An. goeldii* branch roughly corresponding to *An. oswaldoi* group". Perhaps it could be amended for clarity. The legend to Figure 6 is inadequate and does not explain this figure. If nothing else, the internal boxed legend (containing coloured lines and numbers - repeat unit sizes) needs explaining.

Will the information from the first genome assembly (GCA_002846955.1) be available for variant mapping and depicted on the Ensembl pipeline? This could be mentioned in the Genome
Sequence Report.

Methods:

Whilst these are fully explanatory, they seem quite generic and not necessarily about this particular experiment, but more about how *Anopheles* samples are dealt with in the project as a whole. e.g. "In general, single mosquito extractions ranged in total estimated DNA yield from 200 ng to 800 ng". Well, you only sequenced 1 mosquito so provide the information for that specimen, and "The median DNA fragment size for *Anopheles* mosquitoes was 15 kb and the median yield of sheared DNA was 200 ng, with samples typically losing about 50% of the original estimated DNA quantity through the process of shearing and purification." You have just one sample and one species so is it necessary to talk about median sizes for all *Anopheles* here?

**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:** Evolutionary biology, genetics

**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.**

Reviewer Report 30 August 2025

https://doi.org/10.21956/wellcomeopenres.27110.r128987

© 2025 Lee Y et al. This is an open access peer review report distributed under the terms of the [Creative Commons Attribution License](https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

<figure><img src="image_13.png" /></figure>

Valerie Nguyen

<figure><img src="image_14.png" /></figure>

Florida Medical Entomology Laboratory, University of Florida, Vero Beach, Florida, USA

Yoosook Lee

<figure><img src="image_15.png" /></figure>

**Overall:** The report is written as if it is reporting to *Anopheles* genome specialist and omitted a lot

<footnote n="1">¹ Department of Entomology and Nematology, University of Florida, Gainesville, USA</footnote>

<footnote n="2">² Florida Medical Entomology Laboratory, University of Florida, Vero Beach, Florida, USA</footnote>
of information that is needed for college-level students or mosquito biologists who are not specialized in *Anopheles* genus. Some information like 'XR and XL' are foreign even to people knowledgeable about *Anopheles* genome in general. The report needs to explain a lot better what this study is about to make the content a lot easier to absorb.

Method section did not include manufacturer information typically included in peer-review papers. It would be also useful to include how many chromosome-level assembly has been done for mosquito species and how this assembly compare with others, highlighting contribution of this study to more broad audience.

### Abstract:

1. remove 'sex' in 'X sex chromosome'

2. While title indicates that this is chromosome level assembly, autosome information is not provided in the abstract and only X and mitogenome information is provided.

### Keywords

1. change 'chromosomal' to 'chromosome'

### Background:

1. Revise the sentence "It is noted for its tolerance..." to "The initial description of this species by Curry (1932) noted for its tolerance..."

2. provided the number of contig size and scaffold numbers of the 2017 release in the first paragraph. In addition, there needs to be a space after 2017 and before parenthesis begins.

3. provide assembly name or version that people can reference easily (e.g. idAnoAquaMG_Q_19 or AaquGF1?) in the 2nd paragraph of the background section.

4. "Anopheles" should be in italic in the 1st sentence of the 2nd paragraph of the Background section.

5. Rephrase the 2nd sentence of the 2nd paragraph in the Background section. "collected" are repeated in the sentence.

6. Background needs to include the core long read sequencing technology and other technology (Hi-C) used to generate the chromosome-level assembly.

### Genome sequence report

### Valerie's notes:

1. May need to add information on rearing conditions.

2. Explain why Hi-C data from a male sibling was used for the chromosome confirmation. Was the sequencing performed in the same way as the female?

3. Explain the data within Figure 2. The font of the legend should be larger.

4. For figure 3, explaining where the low and high contact densities are will help readers understand the figure.

5. If "(5.015, -52.528)" is for indicating GPS coordinate, to avoid confusion, I suggest revising to "(5.015 N, 52.528 W)".

6. "(N50 10.760kb)" needs to be removed as alternative number is provided in the 2nd sentence of the 1st paragraph of the Genome sequence report section.

7. In the 1st paragraph of the Genome sequence report section, it provides the genome coverage but for people working on the genome assembly, it would be more useful to have read numbers when dealing with uncertain genome size.

8. It is not explained why Hi-C data from male sibling was used rather than the same female or sibling female. The reason may be obvious to writer but not obvious to majority readers.

9. It is not explained why manual assembly curation is needed and what researchers look for to identify missing joins or misjoins. The term missing joins and misjoins are also not
explained.

10. The rationale to orient the assembly using *An. albimanus* is not explained.

11. Figure 1 is not useful for chromosome level assembly. It is not clear what Dataset 'CALSDQ01' is from what database. I expect a number more akin to each chromosome length, GC content over some window for each chromosome, and perhaps table of contig size distribution (mean, se or std) for each chromosome in table.

12. Figure 2. This figure is not explained at all. The legend should be self-explanatory and can stand on its own irrespective of what's in the text. It is not explained what 'ERR9356742_cov' or 'gc' is and the unit of the numbers on x and y axis. It is indicated that 'Chromosomes are coloured by phylum' but I only see blue circle and blue bar so not sure this is supposed to be the right caption.

13. Figure 3. This is first time in the paper that shows the autosome label like 2RL and 2RL. I am familiar with *An. gambiae* chromosome structure like chromosome 2 and 2L and 2R for indicating left and right arm of the chromosome 2 with R (right arm) being the longer arm. It is not explained what 2RL and 3RL are in the paper leading up to Figure 3.

14. Traditionally, the chromosome with longer arm is called 'right arm' in the past but this assembly has left arm longer than right arm. Author need to explain how this came about so that readers can reconcile the information provided here with the traditional chromosome naming convention.

15. Table 2 caption need to include what 'INSCD' stands for and what each chromosome acronym means.

16. Figure 4 is hard to see for X chromosome section and verify the following statement: "Synteny to *An. albimanus* (Figure 4) indicates that both X chromosome arms in *An. aquasalis* have orthologous segments in the X chromosome of *An. albimanus*."

17. Figure 5 do not have any axis label or color legend.

18. Figure 6 – inner circle for left over scaffold numbers along the outside of the circle are not legible. It is not explained what the number outside of the circle means in the Figure caption.

19. "not visible in Figure 6 due small size" -> you can create zoomed in version to make it visible if this is important to mention in the main text.

20. Need introductory paragraph before explaining 'BUSCO' results what authors trying to explain here.

21. The accession number of mitogenome should be provided in the text not just in a table. It is not clear what is INSCD in Table 2 and the data link should be provided in the text or reference section.

22. There are two numbers for genome identifier: idAnoAquaMG_Q_19 or AaquGF1 and perhaps need to be clearer to readers in the text and abstract.

23. Results section doesn't include DNA amount that the extraction method yielded, size range of the fragment while method section explained the process and indicated what is expected 'in general' for single mosquito extraction.

## Methods

### Valerie's notes:

1. An explanation of why different genome sequencing methods were used for each sample would be helpful to readers. Explaining the mechanics of why PacBio was used for the female and Arima Hi-C was used for the male.

2. Rearing information for the specimens needs to be stated.

3. Manufacturing information needs to be listed for the reagents.
1. PacBio

2. Arima

3. Qiagen

4. Agilent

5. AMpure

6. Qubit

7. TRIzol

8. NanoDrop

4. It seems as if the Hi-C data generation was not as in-depth as the previous step. What was done to ensure the quality of the sample? Was it checked on Qubit or Nanodrop?

5. "Samples were not always ideally preserved for RNA, so qualities varied but all were sequenced anyway." - Needs to be explained how samples were preserved.

6. Include manufacturer information (name, city, country) for typical peer-review publications throughout the method section where a reagent or equipment is mentioned.

7. If "(5.015, -52.528)" is for indicating GPS coordinate, to avoid confusion, I suggest revising to "(5.015 N, 52.528 W)".

8. Remove 'by Katy Heu and Romuald Carinci'. One is listed as co-author and the other is acknowledged in acknowledgement section.

9. "Low molecular weight DNA was removed using 0.8X AMpure XP purification". - the expected fragment size cutoff of 0.8X AMpure XP purification is not included. 'Low molecular weight' is subjective to interpretation. AMpure XP reagent manufacturer and city, country information is not provided in the method section.

10. "A small aliquot (less than ~5% of the total volume) of HMW DNA ": include the total volume of DNA.

11. "Sheared DNA was purified using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample." - typically 1X cleanup removes 80-90bp long fragments. The author already clean up using 0.8X ratio to remove shorter fragments and you want to use the same ratio 0.8X or lower if you want to retain longer fragments. So the choice of 1.8X clean up doesn't make sense given the intent of the step explained in the text.

12. "The median DNA fragment size for *Anopheles* mosquitoes was 15 kb and the median yield of sheared DNA was 200 ng, with samples typically losing about 50% of the original estimated DNA quantity through the process of shearing and purification." This seems like results. But only one sample is used for genome assembly. This may indicate multiple samples are used to find the right sample and condition for the library prep and pick one for the actual sequencing? Perhaps author want to revise the manuscript to be consistent with the actual number of mosquitoes used for the experiment. And better separate what belongs to method or result sections.

13. "This approach of using a sibling was taken in order to enable all material from a single specimen to contribute to the PacBio data generation": In the previous paragraph, the single specimen was used for the PacBio and 10X sequencing. Revise this sentence to be consistent with the rest of the manuscript.

14. "Samples proceeded to the Illumina library prep stage even if they were suboptimal (too little tissue) going into the Arima reaction.": It is not clear the connection between Hi-C method and Illumina library prep. In order for this to make sense, author many need to explain what entails Hi-C data generation briefly so this sentence makes sense to people.
15. Need to specify what life stage of sibling male and female were at as well as rearing conditions or time of the day when RNA extraction occurred as transcriptome composition is varies at different life stage, feeding status, time of the day, etc.

16. "Samples were not always ideally preserved for RNA, so qualities varied" - Needed to be provided in the results section on the qualities of samples used for RNA sequencing and discuss it by providing the ideal range of RNA quality in comparison.

17. "We prepared libraries as per the PacBio procedure and checklist for SMRTbell Libraries using Express TPK 2.0 with low DNA input." : Add the what is required for 'low DNA input' in ng amount in parenthesis after 'low DNA input'.

18. "Every library was barcoded to support multiplexing.", "Libraries from two specimens were typically multiplexed on a single 8M SMRT Cell." - there are multiple languages here to indicate multiple libraries were created for PacBio sequencing. Are these all from a single individual? Or different individuals are used per library and only one was sequenced? Clarification is needed.

19. Version numbers are missing for all programs used for the genome assembly. For reproducibility, it is important to include version numbers.

**Ethics/compliance issues**

1. "the national ABS legislation within the country of origin, where applicable." : There is only one country of origin for the samples used in this study. They should provide any permit or authorization used to collect samples in French Guiana. If not needed, should state as such.

**Data availability**

1. Should spell it out INSDC.

**Acknowledgement**

1. Any funding acknowledgement of funding agency and the role they played in this study is missing.

**References**

1. Harbach RE 2013 Mosquito Taxonomic Inventory was last accessed on 2019. I suggest to make sure the information is consistent with more current dates of 2025.

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

Partly

**Are the protocols appropriate and is the work technically sound?**

Partly

**Are sufficient details of methods and materials provided to allow replication by others?**

No

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

Partly

**Competing Interests:** No competing interests were disclosed.

**Reviewer Expertise:** vector biology, genomics, bioinformatics, Anopheles, Aedes, mosquito control research

**We confirm that we have read this submission and believe that we have an appropriate level**
of expertise to state that we do not consider it to be of an acceptable scientific standard, for
reasons outlined above.