A chromosomal reference genome sequence for the
malaria mosquito, Anopheles marshallii, Theobald, 1903

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

► To cite this version:

Boris K Makanga, Diego Ayala, Nil Rahola, Lemonde B A Bouafou, Harriet F Johnson, et al. A chromosomal reference genome sequence for the malaria mosquito, Anopheles marshallii, Theobald, 1903. Wellcome Open Research, 2024, 9, pp.554. 10.12688/wellcomeopenres.22989.1. hal-05199088

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DATA NOTE

# A chromosomal reference genome sequence for the malaria mosquito, *Anopheles marshallii*, Theobald, 1903

[version 1; peer review: 3 approved, 1 approved with reservations]

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

**V1** First published: 26 Sep 2024, **9:554**
https://doi.org/10.12688/wellcomeopenres.22989.1

**Latest published:** 26 Sep 2024, **9:554**
https://doi.org/10.12688/wellcomeopenres.22989.1

## Abstract

We present a genome assembly from an individual female *Anopheles marshallii* (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae) from Lopé, Gabon. The genome sequence is 225.7 megabases in span. Most of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

## Keywords

Anopheles marshallii, African malaria mosquito, genome sequence, chromosomal

## Open Peer Review

Approval Status

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26 Sep 2024

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1. Abdou Azaque Zouré, University of Liège, Institute of Health Sciences Research, (IRSS/CNRST), Gembloux, Belgium

2. Mary E. Gebhardt, Johns Hopkins University Bloomberg School of Public Health, Baltimore, USA

Renee Ali ID, Johns Hopkins University

<footnote n="1">¹Département de Biologie et Écologie Animale, Institut de Recherche en Écologie Tropicale, Libreville, Gabon</footnote>

<footnote n="2">²MIVEGEC, University of Montpellier, CNRS, IRD, Montpellier, France</footnote>

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

<footnote n="4">⁴ESV, Centre Interdisciplinaire de Recherches Médicales de Franceville (CIRMF), Franceville, Gabon</footnote>

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

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

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

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

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

<footnote n="10">¹⁰Infectious Disease and Microbiome Program, Broad Institute, Cambridge, Massachusetts, USA</footnote>
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This article is included in the **Tree of Life** gateway.

Bloomberg School of Public Health,
Baltimore, USA

3. **Craig Wilding** ID, Liverpool John Moores University, Liverpool, UK

4. **Din Syafruddin**, Hasanuddin University Medical Research Center (HUMRC), National Research and Innovation Agency (BRIN), Makassar, Indonesia

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

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

**Author roles:** Makanga BK: Investigation, Resources; Ayala D: Investigation; Rahola N: Investigation, Resources; Bouafou LBA: Investigation, Resources; Johnson H: Investigation, Methodology; Heaton H: Investigation, Methodology; Wagah M: Visualization; Collins J: Data Curation; Krasheninnikova K: Methodology; Pelan S: Data Curation; Pointon DL: Data Curation; Sims Y: Data Curation; Torrance J: Data Curation; Tracey A: Data Curation; Uliano-Silva M: Investigation, Methodology; Wood JMD: Data Curation; von Wyschetzki K: Methodology; McCarthy S: Investigation, Methodology; Neafsey D: 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 Makanga BK 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:** Makanga BK, Ayala D, Rahola N *et al*. A chromosomal reference genome sequence for the malaria mosquito, *Anopheles marshallii*, Theobald, 1903 [version 1; peer review: 3 approved, 1 approved with reservations] Wellcome Open Research 2024, 9:554 https://doi.org/10.12688/wellcomeopenres.22989.1

**First published:** 26 Sep 2024, 9:554 https://doi.org/10.12688/wellcomeopenres.22989.1
## Species taxonomy

Animalia; Arthropoda; Insecta; Diptera; Culicidae; Anopheliinae; Anopheles; *Anopheles marshallii*; Theobald, 1903 (NCBI txid:1521116).

## Background

*Anopheles marshallii* (Theobald, 1903) is a secondary malaria vector that inhabits across sub-Saharan Africa¹,². Together with twelve other species, *An. austenii*, *An. berghei*, *An. brohieri*, *An. hancocki*, *An. hargreavesi*, *An. mortiauxi*, *An. mousin-hoi*, *An. njombiensis*, *An. seydeli*, *An. hughi*, *An. kosiensis* and *An. letabensis*, it forms the Marshallii Group³. The last three species constitute the Marshallii complex with *An. marshallii*. This mosquito is present almost everywhere on the continent, from West Africa (Sierra Leone, Cote d'Ivoire, Ghana, etc) to Central Africa (Cameroon, Gabon, Congo, etc), East Africa (Ethiopia, Kenya, Tanzania, etc) and in a few Southern African countries such as Angola, Burundi and South Africa⁴⁻⁶.

*An. marshallii* larvae are usually present in streams and swampy areas, where the water is relatively clear and vegetation is abundant⁷⁻⁹. *An. marshallii* had been considered medically unimportant because of its zoophilic behaviour, but over time its generalist, including feeding on humans, behaviour and vectorial ability were demonstrated in the laboratory and the field¹,². It has been found in rural areas biting humans¹⁰, but also in the tropical forests of central Africa far from the presence of humans¹¹, where it is involved in the transmission of non-human primates malaria¹¹,¹². The generalist behaviour of this mosquito also makes it one of the potential bridge vectors in the transfer of malaria from non-human primates to humans¹². In addition, *An. marshallii* is capable of feeding on a wide range of hosts (rodents, wild ungulates, great apes, and bats) and is one of the vectors of the parasites described in this wildlife¹³,¹⁴.

Most of the genetic work done on *An. marshallii* has focused on the detection of cryptic species within the Marshallii complex¹⁵. To this end, chromosomal or isoenzymatic markers were used for the identification of four cryptic species¹⁶⁻¹⁸. In recent years, the use of molecular markers based on sequence variation within the ITS1 region or the mitochondrial genes (COI, COII) have made it possible to distinguish species within the group¹⁹⁻²¹. However, these genetic markers have not been involved in population genetic studies, neither to characterise the more or less zoophilic populations.

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

## Genome sequence report

The genome was sequenced from a single female *Anopheles marshallii* reared from a female mosquito collected in Lopé, Gabon (-0.187, 11.611) in April 2019. A total of 51-fold coverage in Pacific Biosciences single-molecule HiFi long reads

(N50 7.850 kb) and 86-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data from a sibling female individual.

The final assembly has a total length of 225.7 Mb in 289 sequence scaffolds with a scaffold N50 of 84.416 Mb (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. 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 using synteny to the AgamP3 assembly (22; GCF_000005575.2) based on cytogenetics data for other Myzomyia series species²³) (Figure 4). The assembly has a BUSCO 5.3.2²⁴ completeness of 97.5% 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 region were delineated based on the presence of characteristic tandem repeat arrays (Figure 5; Table 3). Candidate centromere regions in 2RL, 3RL and X comprised 189bp tandem repeat blocks. Predicted centromere locations agree well with synteny to *An. gambiae* (Figure 4) and Hi-C data (Figure 3). Clusters of rRNA genes were found on multiple unlocalised X-linked contigs.

Gene annotation was performed with the NCBI Eukaryotic Genome Annotation Pipeline and is available in RefSeq²⁵ under the accession GCF_943734725.1. A total of 13,358 genes were predicted, including 11,720 protein-coding genes and 1,204 non-coding RNAs. The genome assembly and gene annotations are hosted on VectorBase, www vectorbase.org²⁶ under the identifier AmarGA1.

## Methods

### Sample acquisition and nucleic acid extraction

*Anopheles marshallii* offspring were reared from a wild caught gravid female collected outdoors using human landing catch. A single female offspring idAnoMarsDA-429_01 was used for Pacific BioSciences and 10x genomics, a sibling female idAnoMarsDA-429_03 was used for Arima Hi-C.

For high molecular weight (HMW) DNA extraction one whole insect (idAnoMarsDA-429_01) 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 modifications²⁷. 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
<table><caption>**Table 1.** Genome data for *An. marshallii*, idAnoMarsDA_429_01.</caption><thead><tr><th colspan="2">Project accession data</th></tr></thead><tbody><tr><td>Assembly identifier</td><td>idAnoMarsDA_429_01</td></tr><tr><td>Species</td><td>Anopheles marshallii</td></tr><tr><td>Specimen</td><td>idAnoMarsDA-429_01</td></tr><tr><td>NCBI taxonomy ID</td><td>1521116</td></tr><tr><td>BioProject</td><td>PRJEB53264</td></tr><tr><td>BioSample ID</td><td>ERS10527370</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>ERR9439506</td></tr><tr><td>10X Genomics Illumina</td><td>ERR9356824, ERR9356825, ERR9356826, ERR9356827</td></tr><tr><td>Hi-C Illumina</td><td>ERR9356819</td></tr><tr><td>PolyA RNA-Seq Illumina</td><td>ERR9356828, ERR9356829</td></tr></tbody><thead><tr><th colspan="2">Genome assembly</th></tr></thead><tbody><tr><td>Assembly accession</td><td>GCF_943734725</td></tr><tr><td>Accession of alternate haplotype</td><td>GCA_943734855</td></tr><tr><td>Span (Mb)</td><td>225.727</td></tr><tr><td>Number of contigs</td><td>305</td></tr><tr><td>Contig N50 length (Mb)</td><td>32.607</td></tr><tr><td>Number of scaffolds</td><td>289</td></tr><tr><td>Scaffold N50 length (Mb)</td><td>84.416</td></tr><tr><td>Longest scaffold (Mb)</td><td>92.345</td></tr><tr><td>BUSCO* genome score</td><td>C:97.5%[S:97.3%,D:0.2%],F:0.9%,M:1.6%,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/idAnoMarsDA_429_01/dataset/CALSDX01/busco.

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 mosquito specimen (idAnoMarsDA-429_03) 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 annotation, which will be made available through VectorBase²⁶ in due course, RNA was extracted from separate whole lab-reared offspring reared from an unrelated wild-caught female mosquito (female idAnoMarsDA-426_03 and male idAnoMarsDA-426_10) using TRIzol, according to the
<figure><img src="image_9.png" /><figcaption>**Figure 1.** Snail plot summary of assembly statistics for *Anopheles marshallii* assembly. The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 225,727,465 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 (92,345,055 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 sequence lengths (84,416,232 and 620,820 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/idAnoMarsDA_429_01/dataset/CALSDX01/snail.</figcaption></figure>

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
<figure><img src="image_10.png" /><figcaption>**Figure 2.** Blob plot of base coverage in a subset of idAnoMarsDA-429_01 10x linked reads against GC proportion for *An. marshallii* assembly idAnoMarsDA_429_01 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/idAnoMarsDA_429_01/dataset/CALSDX01/blob.</figcaption></figure>

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 Hifiasm²⁸; haplotypic duplications were identified and removed with purge_dups²⁹. One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with FreeBayes³⁰. The assembly was then scaffolded with Hi-C data³¹ using SALSA2³². The assembly was checked for contamination as described previously³³. Manual curation was performed using gEVAL³⁴, HiGlass³⁵ and Pretext³⁶.
<figure><img src="image_11.png" /><figcaption>**Figure 3.** Genome assembly of *An. marshallii*, *idAnoMarsDA_429_01: Hi-C contact map*. Visualised in HiGlass. Chromosomes order: 2RL, 3RL, X, followed by unlocalised scaffolds. The interactive Hi-C map can be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=PVHgSVbbST-xI-BNy6pPIA</figcaption></figure>

<table><caption>**Table 2.** Chromosomal pseudomolecules in the genome assembly of *An. marshallii*, *idAnoMarsDA_429_01*.</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>OX030903.1</td><td>2RL</td><td>92.345</td><td>1</td><td>6</td></tr><tr><td>OX030904.1</td><td>3RL</td><td>84.416</td><td>1</td><td>3</td></tr><tr><td>OX030905.1</td><td>X</td><td>19.747</td><td>1</td><td>1</td></tr><tr><td>OX030906.1</td><td>MT</td><td>0.015</td><td>1</td><td>0</td></tr><tr><td></td><td>X Unlocalised</td><td>29.203</td><td>285</td><td>6</td></tr></tbody></table>

The mitochondrial genome was assembled using MitoHiFi³⁷, which performs annotation using MitoFinder³⁸. The genome was analysed and BUSCO scores were generated within the Blob-ToolKit environment³⁹. Synteny analysis was performed with D-GENIES⁴⁰ and minimap2⁴¹. Repetitive sequences were visualised with StainedGlass⁴² and tandem repeats were annotated with ULTRA⁴³. 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
<figure><img src="image_12.png" /><figcaption>**Figure 4.** Alignment dotplot between genome assemblies of *An. marshallii*, *idAnoMarsDA_429_01* and *An. gambiae*, *AgamP3* (PEST). Chromosome arms correspondence (marshallii-gambiae): 2R-2R, 2L-3L, 3R-3R, 3L-2L in agreement with 23.</figcaption></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 5.** Sequence similarity heatmap for genome assembly of *An. marshallii*, *idAnoMarsDA_429_01*. Produced with StainedGlass, visualised in HiGlass. Chromosomes order: 2RL, 3RL, X - followed by unlocalised scaffolds. Darker colours represent higher sequence similarity, notably at pericentric and intercalary heterochromatin as well as in unassembled scaffolds.</figcaption></figure>
<table><caption>**Table 3.** Chromosome arms in the genome assembly of *An. marshallii*, idAnoMarsDA_429_01.</caption><thead><tr><th>Chromosome</th><th>Start</th><th>End</th><th>Chromosome arm</th></tr></thead><tbody><tr><td>2RL</td><td>1</td><td>54,056,034</td><td>2R</td></tr><tr><td>2RL</td><td>54,238,357</td><td>92,345,055</td><td>2L</td></tr><tr><td>3RL</td><td>1</td><td>42,547,211</td><td>3R</td></tr><tr><td>3RL</td><td>42,764,533</td><td>84,416,232</td><td>3L</td></tr><tr><td>X</td><td>1</td><td>19,545,021</td><td>X</td></tr></tbody></table>

<table><caption>**Table 4.** Software tools used.</caption><thead><tr><th>Software tool</th><th>Version</th><th>Source</th></tr></thead><tbody><tr><td>hifiasm</td><td>0.14</td><td>28</td></tr><tr><td>purge_dups</td><td>1.2.3</td><td>29</td></tr><tr><td>SALSA2</td><td>2.2-4c80ac1</td><td>32</td></tr><tr><td>Long Ranger align</td><td>2.2.2</td><td>44</td></tr><tr><td>FreeBayes</td><td>1.3.1</td><td>30</td></tr><tr><td>MitoHiFi</td><td>2</td><td>37</td></tr><tr><td>gEVAL</td><td>N/A</td><td>34</td></tr><tr><td>HiGlass</td><td>1.11.6</td><td>35</td></tr><tr><td>PretextView</td><td>0.1.x</td><td>36</td></tr><tr><td>BlobToolKit</td><td>3.4.0</td><td>39</td></tr><tr><td>BUSCO</td><td>5.3.2</td><td>24</td></tr><tr><td>D-Genies</td><td>1.4</td><td>40</td></tr><tr><td>minimap2</td><td>2.24</td><td>41</td></tr><tr><td>StainedGlass</td><td>0.5</td><td>42</td></tr><tr><td>ULTRA</td><td>1.0.0 beta</td><td>43</td></tr></tbody></table>

(Nagoya Protocol (Compliance) (Amendment) (EU Exit) Regulations 2018 (SI 2018/1393)) and the national ABS legislation within the country of origin, where applicable.

The genome sequence is released openly for reuse. The *Anopheles marshallii* 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.

## Data availability

European Nucleotide Archive: *Anopheles marshallii* genome assembly, idAnoMarsDA_429_01. Accession number PRJEB53264; https://identifiers.org/bioproject/PRJEB53264.

## 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 08 November 2024

https://doi.org/10.21956/wellcomeopenres.25314.r106640

© 2024 Syafruddin 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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Din Syafruddin

Hasanuddin University Medical Research Center (HUMRC), National Research and Innovation Agency (BRIN), Makassar, Indonesia

**General comment:**

The authors presented a complete genome sequence of *Anopheles marshalli*, including both chromosomal and extrachromosomal (mitochondrial) genomes. Therefore, the title should represent the entire results and should be changed accordingly. Complete genome sequence of many major malaria vectors such as *Anopheles gambiae* and *An. dirus* have been available in the GenBank and the authors could compare their results with the 2 malaria mosquitoes.

**Suggested Title:**

A reference genome sequence for the malaria mosquito, *Anopheles marshallii*, Theobald, 1903

**Specific comment:**

1. The authors reported the complete sequence of mitochondrial DNA. "*The complete mitochondrial genome was also assembled and is 15.4 kilobases in length*". The finding should also be discussed and compared with the previously reported mitochondrial genomes of other *Anopheles* species, such as An. gambiae etc. Otherwise, it may be published separately.

2. The authors may also discussed differences of *Anopheles marshalli* with the major malaria vector in Africa, *Anopheles gambiae*

**Recommendation:** Revise

**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.

**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, however I have significant reservations, as outlined above.**

Reviewer Report 06 November 2024

https://doi.org/10.21956/wellcomeopenres.25314.r103927

© 2024 **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.

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**Craig Wilding**

Liverpool John Moores University, Liverpool, UK

This Genome Note provides a useful review of the genome assembly and annotation efforts for *Anopheles marshallii*, the first genome from the Marshallii group and therefore an important reference genome for further research in this group.

**Background:**

It would be good to see some wider taxonomic context – approximately when did marshallii and the gambie complex last share a common ancestor (especially given the later dot plot). But this would be also useful to know given they are likely to be found sympatrically.

These are minor pedantic points, but I would suggest two changes:

Rather than "that inhabits across sub-Saharan Africa" I suggest " is found across sub-Saharan Africa"

I would suggest changing "its generalist, including feeding on humans, behaviour" to "its generalist behaviour, including feeding on humans,"

**Methods:**

Where the specimen is referred to as idAnoMarsDA-429_01 should this be ID AnoMarsDA-429_01?

Given the projected closure of Vectorbase (https://www.nature.com/articles/d41586-024-03196-6) albeit with a temporary reprieve, it seems advisable to not suggest that the annotation will be made available there. All DToL assemblies/annotations are made available on Ensembl! so I would
argue that this sentence is unnecessary.

**Results:**

Figure 2 shows a blob plot. However, there is no mention of the significance of the small blobs outside the main area. What are these? From symbionts? This should at least be discussed.

Figure 4. I am really unsure what this is showing me and what the point of it is. It seems to have been done because it could be done, rather than because it needed doing. Whilst there is mention of it showing synteny of predicted centromere locations with those of *An. gambiae* it is really not clear to me how it shows this. It could be better explained or the figure annotated to indicate this. It would also be useful to have some context about how closely related *An. marshallii* and *A. gambiae* are.

There is no mention of the size and completeness of the mitochondrial genome.

**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.

**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 05 November 2024

https://doi.org/10.21956/wellcomeopenres.25314.r106646

© 2024 Gebhardt M 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.

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**Mary E. Gebhardt**

Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA

**Renee Ali**

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Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School of Public
Health, Baltimore, Maryland, USA

In this article, the authors present a whole genome assembly of *Anopheles marshallii*, which is a secondary vector involved in malaria transmission. They state the known bionomic and behavioral knowledge of this species and outline the importance of contributing comprehensive genetic data to help further our knowledge of population genetics and phylogenetic studies among secondary vector species. The authors then summarize their methods for extraction, sequencing, and annotation of a single female *An. marshallii* from Gabon.

The protocols used in this article are sound and easy to follow and the data is publicly available and easy to access. There are a few small clarifications that could be made to enhance the article:

1. How was this species verified as *An. marshallii*? Were the specimens used morphologically verified by an expert? Some publicly available genetic data can be mislabeled without correct and verified morphology.

2. For the annotation, why were separate non-related mosquitoes used instead of the parent or another sibling?

**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:** Anopheles ecology and genetics

**We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.**

Reviewer Report 02 November 2024

https://doi.org/10.21956/wellcomeopenres.25314.r106648

© 2024 Zouré A. 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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Abdou Azaque Zouré
University of Liège, Institute of Health Sciences Research, (IRSS/CNRST), Gembloux, Belgium

* I propose to make the link with the *An.gambiae* complex, which is the main malaria vector.

* I will then propose a phylogenetic tree that puts together the sequences of *An gambiae*?
your *Anopheles marshallii* and if possible other *Anopheles marshallii* sequences.

* In the methods, you need to give a lot of information about the collection site.

**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:** Genetic, molecular biology, genomic

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.