# A chromosomal reference genome sequence for the malaria mosquito, Anopheles maculipalpis, Giles, 1902

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

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Nil Rahola, Diego Ayala, Lemonde B A Bouafou, Boris K Makanga, Harriet F Johnson, et al. A chromosomal reference genome sequence for the malaria mosquito, Anopheles maculipalpis, Giles, 1902. Wellcome Open Research, 2024, 9, pp.553. 10.12688/wellcomeopenres.22988.1. hal-05199084

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DATA NOTE

# A chromosomal reference genome sequence for the malaria mosquito, *Anopheles maculipalpis*, Giles, 1902

[version 1; peer review: 4 approved, 1 approved with reservations]

Nil Rahola<fnref n="1" />,<fnref n="2" />, Diego Ayala ID<fnref n="1" />,<fnref n="3" />, Lemonde B. A. Bouafou ID<fnref n="1" />,<fnref n="2" />, Boris K. Makanga<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" />, Daniel E. Neafsey<fnref n="7" />, Alex Makunin ID<fnref n="7" />, Mara K. N. Lawniczak ID<fnref n="7" />

**V1** First published: 26 Sep 2024, 9:553
https://doi.org/10.12688/wellcomeopenres.22988.1

Latest published: 26 Sep 2024, 9:553
https://doi.org/10.12688/wellcomeopenres.22988.1

## Abstract

We present a genome assembly from an individual female *Anopheles maculipalpis* (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae). The genome sequence is 224 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 maculipalpis, African malaria mosquito, genome sequence, chromosomal

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

## Open Peer Review

### Approval Status

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### version 1

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26 Sep 2024

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1. **Komal Kumar Bollepogu Raja** ID, Baylor College of Medicine, Houston, USA

2. **Daibin Zhong** ID, University of California at Irvine, Irvine, USA

3. **Craig Wilding** ID, University College Dublin School of Biology and Environmental Science (Ringgold ID: 123708), Dublin, Ireland

4. **Renee Ali** ID, Johns Hopkins University

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

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

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

<footnote n="4">⁴Département de Biologie et Écologie Animale, Institut de Recherche en Écologie Tropicale, Libreville, 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>
Bloomberg School of Public Health,
Baltimore, USA

5. **Din Syafruddin**, National Research and Innovation Agency (BRIN), Makassar, Indonesia

**Puji Budi Setia Asih Setia Asih**, National Research and Innovation Agency, Jakarta, 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:** Rahola N: Investigation, Resources; Ayala D: Investigation; Bouafou LBA: Investigation, Resources; Makanga BK: Data Curation, 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; 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, 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 Rahola N 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:** Rahola N, Ayala D, Bouafou LBA et al. A chromosomal reference genome sequence for the malaria mosquito, *Anopheles maculipalpis*, Giles, 1902 [version 1; peer review: 4 approved, 1 approved with reservations] Wellcome Open Research 2024, 9:553 https://doi.org/10.12688/wellcomeopenres.22988.1

**First published:** 26 Sep 2024, 9:553 https://doi.org/10.12688/wellcomeopenres.22988.1
## Species taxonomy

Animalia; Arthropoda; Insecta; Diptera; Culicidae; Anophelinae; Anopheles; *Anopheles maculipalpis*; Giles, 1902 (NCBI txid:1496333).

## Background

The mosquito *Anopheles maculipalpis* (Giles, 1902) has a very large distribution throughout Africa and is also present in the islands of South-West Indian Ocean¹⁻⁴. It is a savannah species found up to 1000 metres above sea level in Central Africa¹,⁵. Its larvae develop in poorly oxygenated environments, typically among abundant aquatic vegetation. Their habitats are sunny with shallow, stagnant water, often muddy. They vary greatly in type: rice fields, stagnant ditches and canals, holes in the ground, hoofprints, rock hollows, shallow or drying ponds. Some larvae have been collected in rivers, which is exceptional. *An. maculipalpis* is often found in the larval stage in association with *An. gambiae*, *An. squamosus*, and *An. mascarensis*⁴. The pre-imaginal development period of this species is relatively long, at least one month. *An. maculipalpis* is generally considered zoophilic, exophagic, and exophilic⁴,⁶. In some localities, however, this species is reported to exhibit moderate anthropophily. This species is considered to have no medical importance, although a strain of the West Nile virus has been isolated in Madagascar, and experimental transmission of *Wuchereria bancrofti* has been achieved in the laboratory in Mauritius (Mascarene Archipelago, Indian Ocean)⁷. Few studies have generated genetic sequences such as cytochrome oxidase subunit II (COII) or internal transcribed spacer 2 (ITS2)⁸,⁹. However, no population genetic work has been published, because of its no proven medical importance as a malaria vector.

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

## Genome sequence report

The genome was sequenced from a single female *Anopheles maculipalpis* collected in Lopé, Gabon (-0.142, 11.610) in April 2019. A total of 52-fold coverage in Pacific Biosciences single-molecule HiFi long reads (N50 10.236 kb) and 79-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.

The final assembly has a total length of 224 Mb in 171 sequence scaffolds with a scaffold N50 of 92.549 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. 98.19% 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 using synteny to the AgamP3 assembly¹⁰ (accession GCF_000005575.2) (Figure 4) based on cytogenetics data¹¹ (Figure 4). The assembly has a BUSCO 5.3.2¹² completeness of 97.8% 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.

<table><caption>**Table 1.** Genome data for *An. maculipalpis*, idAnoMacuDA_375_x.</caption><thead><tr><th colspan="2">Project accession data</th></tr></thead><tbody><tr><td>Assembly identifier</td><td>idAnoMacuDA_375_x</td></tr><tr><td>Species</td><td>Anopheles maculipalpis</td></tr><tr><td>Specimen</td><td>idAnoMacuDA-375_x</td></tr><tr><td>NCBI taxonomy ID</td><td>1496333</td></tr><tr><td>BioProject</td><td>PRJEB53251</td></tr><tr><td>BioSample ID</td><td>ERS10527369</td></tr><tr><td>Isolate information</td><td>female, whole organism</td></tr><tr><th colspan="2">Raw data accessions</th></tr><tr><td>PacificBiosciences SEQUEL II</td><td>ERR9439503</td></tr><tr><td>10X Genomics Illumina</td><td>ERR9356813, ERR9356814, ERR9356815, ERR9356816</td></tr><tr><td>Hi-C Illumina</td><td>ERR9356811, ERR9356812</td></tr><tr><td>PolyA RNA-Seq Illumina</td><td>ERR9356817</td></tr></tbody></table>

<table><thead><tr><th colspan="2">Project accession data</th></tr><tr><th colspan="2">Genome assembly</th></tr></thead><tbody><tr><td>Assembly accession</td><td>GCA_943734695</td></tr><tr><td>Accession of alternate haplotype</td><td>GCA_943734695</td></tr><tr><td>Span (Mb)</td><td>224.075</td></tr><tr><td>Number of contigs</td><td>182</td></tr><tr><td>Contig N50 length (Mb)</td><td>22.618</td></tr><tr><td>Number of scaffolds</td><td>171</td></tr><tr><td>Scaffold N50 length (Mb)</td><td>92.549</td></tr><tr><td>Longest scaffold (Mb)</td><td>98.751</td></tr><tr><td>BUSCO* genome score</td><td>C:97.8%[S:97.5%,D:0.3%], F:0.4%,M:1,8%,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/idAnoMacuDA_375_x/dataset/CALSDU01/busco.</footnote>
<figure><img src="image_11.png" /><figcaption>**Figure 1.** Snail plot summary of assembly statistics for *Anopheles maculipalpis* assembly. The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 224,074,805 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 (98,751,411 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 sequence lengths (92,548,589 and 21,651,475 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/idAnoMacuDA_375_x/dataset/CALSDU01/snail.</figcaption></figure>

Chromosome arms, candidate centromere and the rDNA regions were delineated based on the presence of characteristic tandem repeat arrays (Figure 5; Table 3). Putative centromeres of autosomes comprised long stretches of irregular tandem repeats with varying unit lengths and arrangements, but with significant sequence homology between chromosomes 2 and 3. These locations were in agreement with the Hi-C contact map (Figure 3) and synteny to *An. gambiae* (Figure 4). Terminal part of chromosome X, starting at 17,790,992, also featured

variable tandem repeats, which could be grouped into at least six different types based on sequence similarity. None of those could be reliably identified as centromeric. Arrays of rRNA genes were located in X-linked unlocalised scaffolds.

Gene annotation was performed with NCBI Eukaryotic Genome Annotation Pipeline and is available in the RefSeq¹³ under the accession GCF_943734695.1. A total of 12,929 genes were predicted, including 11,533 protein-coding genes and
<figure><img src="image_12.png" /><figcaption>**Figure 2.** Blob plot of base coverage in a subset of *idAnoMacuDA-375_x* 10x linked reads against GC proportion for *An. maculipalpis* assembly *idAnoMacuDA_375_x*. 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/idAnoMacuDA_375_x/dataset/CALSDU01/blob.</figcaption></figure>

1,092 non-coding RNAs. The genome assembly and gene annotations are hosted on VectorBase, www vectorbase.org¹⁴ under the identifier AmacGA1.

## Methods

### Sample acquisition and nucleic acid extraction

*Anopheles maculipalpis* individuals were caught using a human landing catch. A single female *idAnoMacuDA-375_x* was used for Pacific BioSciences and 10x genomics, an unrelated female *idAnoMacuDA-405_x* was used for Arima Hi-C.

For high molecular weight (HMW) DNA extraction one whole insect (*idAnoMacuDA-375_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 modifications¹⁵. 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
<figure><img src="image_13.png" /><figcaption>**Figure 3.** Genome assembly of *An. maculipalpis*, *idAnoMacuDA_375_x*: Hi-C contact map. Visualised in HiGlass. Chromosomes are ordered as 2RL, 3RL, X, then remaining scaffolds. The interactive Hi-C map can be viewed at https://genome-note-higlass.tol.sanger.ac.uk/ I/?d=LR0ErKCBRiyLbpoq-qAnnw.</figcaption></figure>

<table><caption>**Table 2.** Chromosomal pseudomolecules in the genome assembly of *An. maculipalpis*, *idAnoMacuDA_375_x*.</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>OX030895.1</td><td>2RL</td><td>98.751</td><td>1</td><td>3</td></tr><tr><td>OX030896.1</td><td>3RL</td><td>92.549</td><td>1</td><td>3</td></tr><tr><td>OX030897.1</td><td>X</td><td>21.651</td><td>1</td><td>1</td></tr><tr><td>OX030898.1</td><td>MT</td><td>0.015</td><td>1</td><td>0</td></tr><tr><td></td><td>X Unlocalised</td><td>7.073</td><td>155</td><td>3</td></tr><tr><td></td><td>Unplaced</td><td>4.035</td><td>12</td><td>1</td></tr></tbody></table>
<figure><img src="image_14.png" /><figcaption>**Figure 4.** Alignment dotplot between genome assemblies of *An. maculipalpis* idAnoMacuDA_375_x and *An. gambiae*, AgamP4 (PEST). Chromosome arms correspondence (maculipalpus-gambiae): 2R-2R, 2L-3L, 3R-3R, 3L-2L in agreement with¹¹.</figcaption></figure>

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 unrelated mosquito specimen (idAnoMacuDA-405_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 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 VectorBase¹⁴ in due course, RNA was extracted
<figure><img src="image_15.png" /><figcaption>**Figure 5.** Sequence similarity heatmap for genome assembly of *An. maculipalpis*, *idAnoMacuDA_375_x*. Produced with StainedGlass, visualised in HiGlass. CChromosomes are ordered as 2RL, 3RL, X, then remaining scaffolds. Darker colours represent higher sequence similarity, notably at putative centromeres and terminal part of X.</figcaption></figure>

from a separate whole unrelated female mosquito specimen (*idAnoMacuDA-401_x*) 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 check-list 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 Hifiasm¹⁶; haplotypic duplications were identified and removed with purge_dups¹⁷. One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger

ALIGN, calling variants with FreeBayes¹⁸. The assembly was then scaffolded with Hi-C data¹⁹ using SALSA2²⁰. The assembly was checked for contamination as described previously²¹. Manual curation was performed using gEVAL²², HiGlass²³ and Pretext²⁴. The mitochondrial genome was assembled using MitoHiFi²⁵, which performs annotation using MitoFinder²⁶. The genome was analysed and BUSCO scores were generated within the BlobToolKit environment²⁷. Synteny analysis was performed with D-GENIES²⁸. Repetitive sequences were visualised with StainedGlass²⁹ and tandem repeats were annotated with ULTRA³⁰. Table 4 contains a list of all software tool versions used, where appropriate.

<table><caption>**Table 3.** Chromosome arms in the genome assembly of *An. maculipalpis*, idAnoMacuDA_375_x.</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>57,070,844</td><td>2R</td></tr><tr><td>2RL</td><td>60,243,198</td><td>98,751,411</td><td>2L</td></tr><tr><td>3RL</td><td>1</td><td>46,889,622</td><td>3R</td></tr><tr><td>3RL</td><td>47,935,636</td><td>92,548,589</td><td>3L</td></tr><tr><td>X</td><td>1</td><td>21,651,475</td><td>X</td></tr></tbody></table>

<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>16</td></tr><tr><td>purge_dups</td><td>1.2.3</td><td>17</td></tr><tr><td>SALSA2</td><td>2.2-4c80ac1</td><td>20</td></tr><tr><td>longranger align</td><td>2.2.2</td><td>31</td></tr><tr><td>freebayes</td><td>1.3.1</td><td>18</td></tr><tr><td>MitoHiFi</td><td>2</td><td>25</td></tr><tr><td>gEVAL</td><td>N/A</td><td>22</td></tr><tr><td>HiGlass</td><td>1.11.6</td><td>23</td></tr><tr><td>PretextView</td><td>0.1.x</td><td>24</td></tr><tr><td>BlobToolKit</td><td>3.4.0</td><td>27</td></tr><tr><td>BUSCO</td><td>5.3.2</td><td>12</td></tr><tr><td>D-GENIES</td><td>1.4</td><td>28</td></tr><tr><td>StainedGlass</td><td>0.5</td><td>29</td></tr><tr><td>ULTRA</td><td>1.0.0 beta</td><td>30</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 maculipalpis* genome assembly, idAnoMacuDA_375_x. Accession number PRJEB53251; https://identifiers.org/bioproject/PRJEB53251.

The genome sequence is released openly for reuse. The *Anopheles maculipalpis* 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 30 December 2024

https://doi.org/10.21956/wellcomeopenres.25313.r114741

© 2024 Syafruddin D 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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Din Syafruddin

National Research and Innovation Agency (BRIN), Makassar, Indonesia

Puji Budi Setia Asih Setia Asih

Eijkman Research Center for Molecular Biology, National Research and Innovation Agency, Jakarta, Indonesia

**General comment:**

The manuscript reports the complete genome sequence of *Anopheles maculipalpis*, one of the malaria vectors in Africa and in the islands of South-West Indian Ocean. While the manuscript contains novel and Indexing data, the complete genome sequence of many species of Anopheles, such as *An. gambiae* and others have been published previously and are available in the database. Therefore, rather than providing a genome sequence analysis, data on RNAomics, particularly related to vector status, blood meal preference and other aspect related to disease transmission should be explored.

**Specific comments:**

1. The title should not mention "chromosomal" only as the manuscript reports full chromosomal genome sequence of *Anopheles maculipalpis* as well as the mitochondrial genome.

2. The phylogenetic tree of the *Anopheles maculipalpis* and other major malaria vector worldwide might be reconstructed, based on mitochondrial genome or internal transcribed spacer 2 (ITS2) gene.

**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:** Molecular entomology and parasitology

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, however we have significant reservations, as outlined above.

Reviewer Report 23 December 2024

https://doi.org/10.21956/wellcomeopenres.25313.r115140

© 2024 Ali R. 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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Renee Ali

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

The rationale for this study is sound and would be useful as reference material for future comparisons with specimens from other endemic regions. The BUSCO score of 97.8% indicates a high-quality gene content and the assembly is reasonably correct. The methods presented are clearly described and can be reproducible if implemented on other *Anopheles* mosquito species.

**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:** entomology, mosquito population genomics, molecular 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.

Reviewer Report 17 December 2024

https://doi.org/10.21956/wellcomeopenres.25313.r114735

© 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

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University College Dublin School of Biology and Environmental Science (Ringgold ID: 123708),
Dublin, Leinster, Ireland

Rahola et al. describe the sequencing of a single *Anopheles maculipalpis* individual, reporting a high quality (in terms of scaffold-length and BUSCO completeness) genome generated using a combination of PacBio and Hi-C scaffolding for genome sequencing, and Illumina for RNAseq. The methods utilized are reported clearly although there is one exception. The sample collection section of the method says "A single female idAnoMacuDA-375_x was used for Pacific BioSciences and 10x genomics, an unrelated female idAnoMacuDA-405_x was used for Arima Hi-C." Then the Hi-C methods section states "For Hi-C data generation, a separate unrelated mosquito specimen (idAnoMacuDA-405_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 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." My question is, how is this using a sibling if this was, as stated twice previously, an unrelated female?

All data are deposited and freely available.

The manuscript is clearly written and the background provides useful information on this species and the utility of the reported resource which should be of value for comparative genomics work and further research on this species.

**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 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 16 December 2024

https://doi.org/10.21956/wellcomeopenres.25313.r114738

© 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 ID

University of California at Irvine, Irvine, USA

The study by Rahola et al. presents a high-quality chromosome-level genome assembly for Anopheles maculipalpis. Utilizing a combination of long-read and short-read sequencing technologies, including PacBio and Illumina, along with Hi-C scaffolding, the authors generated a robust genome assembly with a scaffold N50 of 92.549 Mb. A high BUSCO completeness score (97.8%) indicates a highly accurate and complete assembly. The paper is well-written and presents the results clearly and concisely. The experimental design and methodology are sound. This valuable genomic resource will significantly contribute to our understanding of An. maculipalpis biology and facilitate future research on 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 genetics, molecular biology, genomics, and the interplay between parasites and their vectors.
**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 15 October 2024

https://doi.org/10.21956/wellcomeopenres.25313.r104027

© 2024 Bollepogu Raja K. 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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**Komal Kumar Bollepogu Raja**

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Department of Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA

The manuscript by Rahola et al. provides a comprehensive overview of the genome sequencing and assembly of *Anopheles maculipalpis*. The authors have provided specific details about the ecological background of this species. The methods section is thorough and provides clear descriptions of the techniques used and allow reproducibility. The genome assembly results including mitochondrial genome are well presented with specific statistics and references to figures and tables. The BUSCO completeness score of 97.8% indicates a very high-quality assembly. The authors have deposited their data and made it available to public. Overall, the manuscript is well written with the genomic data useful for research on comparative genomics and broad mosquito research community.

**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:** Genomics, Single cell omics, Bioinformatics and Neurobiology

**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.**