# Chromosomal reference genome sequences for the malaria mosquito, Anopheles coustani, Laveran, 1900

Lemonde B A Bouafou, Diego Ayala, Boris K Makanga, Nil Rahola, Harriet F Johnson, Haynes Heaton, Martin G Wagah, Joanna C Collins, Ksenia Krasheninnikova, Sarah E Pelan, et al.

► To cite this version:

Lemonde B A Bouafou, Diego Ayala, Boris K Makanga, Nil Rahola, Harriet F Johnson, et al. Chromosomal reference genome sequences for the malaria mosquito, Anopheles coustani, Laveran, 1900. Wellcome Open Research, 2024, 9, pp.551. 10.12688/wellcomeopenres.22983.1. hal-05199087

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DATA NOTE

# Chromosomal reference genome sequences for the malaria
mosquito, *Anopheles coustani*, Laveran, 1900

[version 1; peer review: 2 approved]

Lemonde B. A. Bouafou<sup>1,2</sup>, Diego Ayala<sup>1,3</sup>, Boris K. Makanga<sup>4</sup>, Nil Rahola<sup>1,2</sup>, Harriet F. Johnson<sup>5</sup>, Haynes Heaton<sup>6</sup>, Martin G. Wagah<sup>7</sup>, Joanna C. Collins<sup>7</sup>, Ksenia Krasheninnikova<sup>7</sup>, Sarah E. Pelan<sup>7</sup>, Damon-Lee B. Pointon<sup>7</sup>, Ying Sims<sup>7</sup>, James W. Torrance<sup>7</sup>, Alan Tracey<sup>7</sup>, Marcela Uliano-Silva<sup>7</sup>, Jonathan M.D. Wood<sup>7</sup>, Katharina von Wyschetzki<sup>7</sup>, Scientific Operations: DNA Pipelines collective, Shane A. McCarthy<sup>7,8</sup>, Daniel E. Neafsey<sup>9,10</sup>, Alex Makunin<sup>7</sup>, Mara K N Lawniczak<sup>7</sup>

**v1** First published: 26 Sep 2024, 9:551
https://doi.org/10.12688/wellcomeopenres.22983.1

Latest published: 26 Sep 2024, 9:551
https://doi.org/10.12688/wellcomeopenres.22983.1

## Abstract

We present genome assembly from individual female *An. coustani* (African malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae) from Lopé, Gabon. The genome sequence is 270 megabases in span. Most of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled for both species. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

## Keywords

Anopheles coustani, African malaria mosquito, genome sequence, chromosomal

## Open Peer Review

Approval Status

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1

2

version 1

26 Sep 2024

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1. **Qian Han**<sup>1,2</sup>, Hainan University, Haikou, China

2. **Daibin Zhong**<sup>1,2</sup>, University of California, California, USA

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

<footnote n="1"><sup>1</sup>MIVEGEC, Univ. Montpellier, CNRS, IRD, Montpellier, France</footnote>

<footnote n="2"><sup>2</sup>ESV, Centre Interdisciplinaire de Recherches Médicales de Franceville (CIRMF), Franceville, Gabon</footnote>

<footnote n="3"><sup>3</sup>Medical Entomology Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar</footnote>

<footnote n="4"><sup>4</sup>Département de Biologie et Écologie Animale, Institut de Recherche en Écologie Tropicale, Libreville, Gabon</footnote>

<footnote n="5"><sup>5</sup>Scientific Operations, Wellcome Sanger Institute, Hinxton, England, UK</footnote>

<footnote n="6"><sup>6</sup>CSSE, Auburn University, Auburn, Alabama, USA</footnote>

<footnote n="7"><sup>7</sup>Tree of Life, Wellcome Sanger Institute, Hinxton, England, UK</footnote>

<footnote n="8"><sup>8</sup>Department of Genetics, University of Cambridge, Cambridge, England, UK</footnote>

<footnote n="9"><sup>9</sup>Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA</footnote>

<footnote n="10"><sup>10</sup>Infectious Disease and Microbiome Program, Broad Institute, Cambridge, MA, USA</footnote>
<figure><img src="image_6.png" /></figure>

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

**Corresponding authors:** Alex Makunin (am60@sanger.ac.uk), Mara K N Lawniczak (mara@sanger.ac.uk)

**Author roles:** **Bouafou LBA:** Investigation, Resources; **Ayala D:** Investigation; **Makanga BK:** Investigation, Resources; **Rahola N:** Investigation, Resources; **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; **Sims Y:** 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; **Neafsey DE:** Conceptualization, Funding Acquisition;

**Makunin A:** Formal Analysis, Methodology, Project Administration, Visualization, Writing – Original Draft Preparation; **Lawniczak MKN:** Conceptualization, Funding Acquisition, Investigation, Supervision, Writing – Review & Editing

**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 (206194) [https://doi.org/10.35802/206194], which supports ML. SAM is supported by the Wellcome Trust Grant (WT207492). The research received funding from an ANR grant in France (ANR-18-CE35-0002-01-WILDING), which was awarded to D.A.

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

**Copyright:** © 2024 Bouafou LBA 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:** Bouafou LBA, Ayala D, Makanga BK et al. Chromosomal reference genome sequences for the malaria mosquito, *Anopheles coustani*, Laveran, 1900 [version 1; peer review: 2 approved] Wellcome Open Research 2024, 9:551 https://doi.org/10.12688/wellcomeopenres.22983.1

**First published:** 26 Sep 2024, 9:551 https://doi.org/10.12688/wellcomeopenres.22983.1
## Species taxonomy

Animalia; Arthropoda; Insecta; Diptera; Culicidae; Anophelinae; Anopheles; *Anopheles coustani*; Laveran, 1900 (NCBI txid:139045).

## Background

*Anopheles coustani* (Laveran, 1900) belongs to the Coustani group together with the morphologically similar species *An. crypticus*, *An. fuscicolor*, *An. namibiensis*, *An. paludis*, *An. symesi*, *An. tenebrosus*, *An. caliginosus* and *An. ziemanni*¹. Although this mosquito was first described from Madagascar², it is widespread throughout the African continent. The larvae of *An. coustani* prefer to breed in natural clear water with aquatic vegetation while adults typically rest and feed outdoors³,⁴. The feeding preference of *An. coustani* is primarily zoophilic, including wild ungulates, but this zoophilic tendency greatly varies at a local scale from opportunistic to anthropophilic behaviour⁴⁻⁷. Regarding malaria transmission, *An. coustani* is considered a secondary vector, leading to the species being understudied. However, its epidemiological role in malaria transmission varies from minor importance to locally major vector, as in Madagascar⁸. The species has been found infected with various human *Plasmodium* species including *P. falciparum*, *P. vivax* and *P. malariae*⁵,⁹,¹⁰. In Madagascar and Cameroon, *An. coustani* was suspected to significantly contribute to malaria outbreaks and sustain malaria transmission⁸,¹⁰. Apart from human *Plasmodium* species, *An. coustani* has been involved in the transmission of other Haemosporidian parasites (including Hepatocystis) and a variety of arboviruses, including Rift Valley fever and Zika virus¹¹⁻¹³.

Early genetic works enabled distinguishing this species from its sister species, *An. crypticus*. This distinction was based mainly on a fixed chromosomal inversion of the X chromosome¹⁴. Very few studies have focused on the genetics of *An. coustani*, for example¹⁵ analysed the genetic diversity of *An. coustani*, using COI and ITS2 markers in 50 samples from several locations across Africa. The authors highlighted the existence of two genetic groups with a structure that was not geographically dependent. However, the authors could not rule out the possibility that *An. coustani* and *An. crypticus* are two separate species. One of the most important genomic studies carried out on *An. coustani* is the publication of its complete mitogenome, making available an interesting resource for phylogenetic analyses based on mitochondrial DNA¹⁶. Nonetheless, research on the nuclear DNA sequence is currently lacking and will be greatly facilitated by this new chromosomal reference genome.

The genome of the African malaria mosquito, *Anopheles coustani*, was sequenced as part of the Anopheles Reference Genomes Project (PRJEB51690). Here we present a chromosomally complete genome sequence for *Anopheles coustani*, based on a single wild-caught female.

## Genome sequence report

The genome was sequenced from a single female *Anopheles coustani* caught in Lopé, Gabon (-0.143, 11.610) in April 2019¹⁷. A total of 33-fold coverage in Pacific Biosciences single-molecule HiFi long reads (N50 11.273 kb) and 78-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data from an unrelated female individual. Manual assembly curation corrected 3 missing joins or misjoins, reducing the scaffold number by 0.7%.

The final assembly has a total length of 270 Mb in 420 sequence scaffolds with a scaffold N50 of 94.852 Mb (Figure 1–Figure 2; Table 1). The snail plot in Figure 1 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 2. 89.87% of the assembly sequence was assigned to three chromosomal-level scaffolds, representing two autosomes and the X sex chromosome (Figure 3; Table 2). Chromosomes were numbered and oriented against the *An. atroparvus* assembly AatrE4¹⁸ (accession GCA_015501955.1) (Figure 4) and double checked by polytene chromosome arms lengths, where 2L and 3R arms are the longest, 2R has intermediate length, followed by 3L and, finally, X¹⁴. The assembled portion of chromosome 3RL is about 3Mbp longer than 2RL, which means the naming convention here of naming the longer chromosome as 2 is not precisely followed. The assembly has a BUSCO 5.3.2¹⁹ completeness of 97.4% using the diptera_odb10 reference set. While not fully phased, the assembly deposited is of one haplotype, and also includes the circular mitochondrial genome. Contigs corresponding to the second haplotype have also been deposited.

Chromosome arms, candidate centromere sequences, and the rDNA regions were delineated based on the presence of characteristic tandem repeat arrays (Figure 5; Table 3). Candidate centromere regions of autosomes 2RL and 3RL comprised 52-53bp tandem repeat blocks with questionable sequence homology between chromosomes. On 3RL, a more pronounced tandem repeat region was found. Predicted centromere locations agree well with Hi-C signal (Figure 3) and synteny to *An. atroparvus* (Figure 4). In X chromosome assembly, no plausible centromere region was found. rDNA clusters were scattered across unlocalised X-linked scaffolds; they were often associated with tandem repeat blocks with unit length of 737 bp.

Gene annotation was performed with NCBI Eukaryotic Genome Annotation Pipeline and is available in the RefSeq²⁰ under the accession GCF_943734705.1. A total of 14,493 genes were predicted, including 12,032 protein-coding genes and 2,426 non-coding RNAs. The genome assembly and gene annotations are hosted on VectorBase, www vectorbase.org²¹ under the identifier AcouGA1.
<figure><img src="image_7.png" /></figure>

**Figure 1.** Snail plot summary of assembly statistics for *Anopheles coustani* assembly idAnoCousDA_361_x.2. The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 269,999,061 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 (97,602,170 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 sequence lengths (94,852,749 and 274,232 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/Anopheles%20coustani/dataset/CALSDV01/snail.

# Methods

## Sample acquisition and nucleic acid extraction

*Anopheles coustani* female individuals were caught in Lopé, Gabon using an animal-bait catch²². A single female idAnoCousDA-361_x was used for Pacific BioSciences and 10x genomics, an unrelated female idAnoCousDA-364_x was used for Arima Hi-C.

For high molecular weight (HMW) DNA extraction one whole insect (idAnoCousDA-361_x) 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 modifications²³. The quality of the DNA was evaluated using an Agilent FemtoPulse to ensure that most DNA molecules were
<figure><img src="image_8.png" /><figcaption>**Figure 2.** Blob plot of base coverage in a subset of *idAnoCousDA_361_x* 10x linked reads against GC proportion for *An. coustani* assembly *idAnoCousDA_361_x.2*. 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/Anopheles%20coustani/dataset/CALSDV01/blob.</figcaption></figure>

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 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 to 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
<table><caption>**Table 1.** Genome data for *An. coustani*, *idAnoCousDA_361_x*.</caption><thead><tr><th colspan="2">Project accession data</th></tr></thead><tbody><tr><td>Assembly identifier</td><td>idAnoCousDA_361_x.2</td></tr><tr><td>Species</td><td>Anopheles coustani</td></tr><tr><td>Specimen</td><td>idAnoCousDA-361_x</td></tr><tr><td>NCBI taxonomy ID</td><td>139045</td></tr><tr><td>BioProject</td><td>PRJEB53256</td></tr><tr><td>BioSample ID</td><td>ERS10527346</td></tr><tr><td>Isolate information</td><td>female, whole organism</td></tr></tbody><thead><tr><th colspan="2">Raw data accessions</th></tr></thead><tbody><tr><td>PacificBiosciences SEQUEL II</td><td>ERR9439496</td></tr><tr><td>10X Genomics Illumina</td><td>ERR9356773, ERR9356774, ERR9356775, ERR9356776</td></tr><tr><td>Hi-C Illumina</td><td>ERR9356772</td></tr><tr><td>PolyA RNA-Seq Illumina</td><td>ERR9356777, ERR9356778</td></tr></tbody><thead><tr><th colspan="2">Genome assembly</th></tr></thead><tbody><tr><td>Assembly accession</td><td>GCA_943734705</td></tr><tr><td>Accession of alternate haplotype</td><td>GCA_943734715</td></tr><tr><td>Span (Mb)</td><td>269.999</td></tr><tr><td>Number of contigs</td><td>448</td></tr><tr><td>Contig N50 length (Mb)</td><td>27.992</td></tr><tr><td>Number of scaffolds</td><td>420</td></tr><tr><td>Scaffold N50 length (Mb)</td><td>94.852</td></tr><tr><td>Longest scaffold (Mb)</td><td>97.602</td></tr><tr><td>BUSCO* genome score</td><td>C:97.4%[S:96.3%,D:1.1%], F:0.8%,M:1.8%,n:3,285</td></tr></tbody></table>

* 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/Anopheles%20coustani/dataset/CALSDV01/busco.

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 unrelated mosquito specimen (idAnoCousDA-364_x) 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 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 unrelated insect specimens (idAnoCousDA-54_x and idAnoCousDA-63_x) using TRIzol, according to the
<figure><img src="image_9.png" /><figcaption>**Figure 3.** Genome assembly of *An. coustani*, *idAnoCousDA_361_x.2: Hi-C contact map*. Visualised in HiGlass. Chromosomes order: 3RL, 2RL, X, then remaining samples. Off-diagonal signal in 2L indicates a heterozygous inversion in the individual *idAnoCousDA-364_x* used for Hi-C. The interactive Hi-C map can be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=TOv9LjXMTYKBy8dC3rTKgQ.</figcaption></figure>

<table><caption>**Table 2.** Chromosomal pseudomolecules in the genome assembly of *An. coustani*, *idAnoCousDA_361_x.2*.</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>OX030900.2</td><td>2RL</td><td>94.853</td><td>1</td><td>3</td></tr><tr><td>OX030901.1</td><td>3RL</td><td>97.602</td><td>1</td><td>5</td></tr><tr><td>OX030902.1</td><td>X</td><td>19.034</td><td>1</td><td>4</td></tr><tr><td>OX030903.1</td><td>MT</td><td>0.015</td><td>1</td><td>0</td></tr><tr><td></td><td>X Unlocalised</td><td>31.162</td><td>166</td><td>3</td></tr><tr><td></td><td>Unplaced</td><td>27.333</td><td>250</td><td>13</td></tr></tbody></table>

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
<figure><img src="image_10.png" /><figcaption>**Figure 4.** Alignment dotplot between genome assemblies of *An. coustani*, *idAnoCousDA_361_x.2* and *An. atroparvus*, *AatrE4*. Visualised in D-Genies. Chromosome arms arrangement is the same for these representatives of *Anopheles* subgenus.</figcaption></figure>

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,
<figure><img src="image_11.png" /><figcaption>**Figure 5.** Sequence similarity heatmap for genome assembly of *An. coustani*, *idAnoCousDA_361_x.2*. Produced with StainedGlass, visualised in HiGlass. Chromosomes order: 2RL, 3RL, X - followed by the remaining scaffolds. Darker colours represent higher sequence similarity, notably at pericentric and intercalary heterochromatin as well as in unassembled X-linked scaffolds.</figcaption></figure>

<table><caption>**Table 3.** Chromosome arms in the genome assembly of *An. coustani*, *idAnoCousDA_361_x.2*.</caption><thead><tr><td>Chromosome</td><td>Start</td><td>End</td><td>Chromosome arm</td></tr></thead><tbody><tr><td>2RL</td><td>1</td><td>48,615,516</td><td>2R</td></tr><tr><td>2RL</td><td>49,081,485</td><td>94,852,749</td><td>2L</td></tr><tr><td>3RL</td><td>1</td><td>57,704,850</td><td>3R</td></tr><tr><td>3RL</td><td>57,761,701</td><td>97,602,170</td><td>3L</td></tr><tr><td>X</td><td>1</td><td>19,033,788</td><td>X</td></tr></tbody></table>

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) instruments.

## Genome assembly

Assembly was carried out with Hifiasm²⁴; haplotypic duplications were identified and removed with purge_dups²⁵. One round of polishing was performed by aligning 10X
Genomics read data to the assembly with LongRanger align, calling variants with FreeNayes²⁶. The assembly was then scaffolded with Hi-C data²⁷ using SALSA2²⁸. The assembly was checked for contamination as described previously²⁹. Manual curation was performed using gEVAL³⁰, HiGlass³¹ and Pretext³². The mitochondrial genome was assembled using MitoHiFi³³, which performs annotation using MitoFinder³⁴. The genome was analysed and BUSCO scores were generated within the BlobToolKit environment³⁵. Synteny analysis was performed with D-GENIES³⁶. Repetitive sequences were visualised with StainedGlass³⁷ and tandem repeats were annotated with ULTRA³⁸. Table 4 contains a list of all software tool versions used, where appropriate.

## 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.

<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>24</td></tr><tr><td>purge_dups</td><td>1.2.3</td><td>25</td></tr><tr><td>SALSA2</td><td>2.2-4c80ac1</td><td>28</td></tr><tr><td>longranger align</td><td>2.2.2</td><td>39</td></tr><tr><td>freebayes</td><td>1.3.1</td><td>26</td></tr></tbody></table>

<table><thead><tr><td>Software tool</td><td>Version</td><td>Source</td></tr></thead><tbody><tr><td>MitoHiFi</td><td>2</td><td>33</td></tr><tr><td>gEVAL</td><td>N/A</td><td>30</td></tr><tr><td>HiGlass</td><td>1.11.6</td><td>31</td></tr><tr><td>PretextView</td><td>0.1.x</td><td>32</td></tr><tr><td>BlobToolKit</td><td>3.4.0</td><td>35</td></tr><tr><td>BUSCO</td><td>5.3.2</td><td>19</td></tr><tr><td>D-GENIES</td><td>1.4</td><td>36</td></tr><tr><td>StainedGlass</td><td>0.5</td><td>37</td></tr><tr><td>ULTRA</td><td>1.0.0 beta</td><td>38</td></tr></tbody></table>

## Data availability

European Nucleotide Archive: *Anopheles coustani* genome assembly, idAnoCousDA_361_x.2. Accession number PRJEB53256; https://identifiers.org/bioproject/PRJEB53256.

The genome sequence is released openly for reuse. The *Anopheles coustani* 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 Wellcome Sanger Institute Scientific Operations: Sequencing Operations are listed here: https://doi.org/10.5281/zenodo.12165051.

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# Open Peer Review

Current Peer Review Status: ✓ ✓

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## Version 1

Reviewer Report 19 October 2024

https://doi.org/10.21956/wellcomeopenres.25308.r103180

© 2024 Zhong D. 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.

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Daibin Zhong

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University of California, California, USA

The study by Bouafou et al., titled "Chromosomal Reference Genome Sequences for the Malaria Mosquito, *Anopheles coustani*, Laveran, 1900," presents a genome assembly from a single wild-caught female *An. coustani* collected in Lopé, Gabon. The mosquito's DNA and RNA were sequenced using Pacific Biosciences and Illumina technologies (10X Genomics, Hi-C, and RNAseq). Chromosome conformation Hi-C data from an unrelated female were utilized to scaffold the primary assembly contigs. The resulting genome sequence spans 270 megabases, with most of the assembly organized into three chromosomal pseudomolecules, and also includes the complete mitochondrial genome. The paper is clearly written, with a solid experimental design and appropriate statistical methods for genome assembly. It provides important new insights into the reference genome of this species, which will serve as a valuable resource for further research into the genetics and biology of *Anopheles coustani*, supporting the development of effective malaria control strategies. I have no further comments.

**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:** population 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.

Reviewer Report 17 October 2024

https://doi.org/10.21956/wellcomeopenres.25308.r103182

© 2024 **Han Q.** 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.

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Qian Han

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Hainan University, Haikou, Hainan, China

The report titled "Chromosomal reference genome sequences for the malaria mosquito, *Anopheles coustani*, Laveran, 1900" authored by Lemonde B. A. Bouafou et al., presents a genome assembly of the *An. coustani* mosquito, a species known to transmit malaria. The study is significant as it aims to understand the genetic makeup and evolutionary history of this mosquito, which may be useful for developing effective control strategies against malaria.

In this report, the team successfully generated a genome assembly using Pacific Biosciences SEQUEL II and Illumina sequencing technologies. The genome assembly statistics are provided, including the chromosome sizes and counts, and the BUSCO scores, which assess the completeness of the assembly. The article also includes a list of references, providing additional context and supporting information for the study.

Overall, this report contributes valuable genomic data for *An. coustani*, which could aid in the development of targeted interventions to control malaria transmission. The comprehensive approach taken by the authors, including the use of multiple sequencing technologies and bioinformatics tools, ensures the reliability and accuracy of the genome assembly.

**Minor:**

I would like to know some interesting aspects, but not sure if they could be done further in this report or in somewhere else. For example,

(1) The similarities and differences between the genomes of *An. coustani* and other known major malaria vectors, such as *An. gambiae*. Do these differences explain the differences in their ability to transmit malaria?

(2) Are there any specific genes found in this genome that are involved in Plasmodium infection or transmission compared with other mosquito species?

(3) The article mentions that the feeding preference of this species varies from animal to human preference, are there genes or regulatory regions in the genome that are associated with this behavioral plasticity?
**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:** vector biology, vector borne diseases, parasitology, structural biology.

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.