Comprehensive characterization of a transgene insertion in a highly repetitive, centromeric region of Anopheles mosquitoes

Abstract

The availability of the genomic sequence of the malaria mosquito Anopheles gambiae has in recent years sparked the development of transgenic technologies with the potential to be used as novel vector control tools. These technologies rely on genome editing that confer traits able to affect vectorial capacity. This can be achieved by either reducing the mosquito population or by making mosquitoes refractory to the parasite infection. For any genetically modified organism that is regarded for release, molecular characterization of the transgene and flanking sites are essential for their safety assessment and post-release monitoring. Despite great advancements, Whole-Genome Sequencing data are still subject to limitations due to the presence of repetitive and unannotated DNA sequences. Faced with this challenge, we describe a number of techniques that were used to identify the genomic location of a transgene in the male bias mosquito strain Ag(PMB)1 considered for potential field application. While the initial inverse PCR identified the most likely insertion site on Chromosome 3 R 36D, reassessment of the data showed a high repetitiveness in those sequences and multiple genomic locations as potential insertion sites of the transgene. Here we used a combination of DNA sequencing analysis and in-situ hybridization to clearly identify the integration of the transgene in a poorly annotated centromeric region of Chromosome 2 R 19D. This study emphasizes the need for accuracy in sequencing data for the genome of organisms of medical importance such as Anopheles mosquitoes and other tools available that can support genomic locations of transgenes.

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Comprehensive characterization of a transgene
insertion in a highly repetitive, centromeric region
of Anopheles mosquitoes
Matteo Vitale, Chiara Leo, Thomas Courty, Nace Kranjc, John B. Connolly,
Giulia Morselli, Christopher Bamikole, Roya Elaine Haghighat-Khah, Federica
Bernardini & Silke Fuchs
To cite this article: Matteo Vitale, Chiara Leo, Thomas Courty, Nace Kranjc, John B. Connolly,
Giulia Morselli, Christopher Bamikole, Roya Elaine Haghighat-Khah, Federica Bernardini
& Silke Fuchs (2022): Comprehensive characterization of a transgene insertion in a highly
repetitive, centromeric region of Anopheles mosquitoes, Pathogens and Global Health, DOI:
10.1080/20477724.2022.2100192
To link to this article: https://doi.org/10.1080/20477724.2022.2100192
© 2022 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
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ORIGINAL ARTICLE
Comprehensive characterization of a transgene insertion in a highly repetitive,
centromeric region of Anopheles mosquitoes
Matteo Vitale
a
, Chiara Leo
b
, Thomas Courty
c
, Nace Kranjc
a
, John B. Connolly
a
, Giulia Morselli
a
,
Christopher Bamikole
d
, Roya Elaine Haghighat-Khah
a
, Federica Bernardini
a
and Silke Fuchs
a
a
Department of Life Sciences, Imperial College London, London, UK;
b
Polo d’Innovazione di Genomica, Genetica, e Biologia, Siena, Italy;
c
Department of Infectious Diseases, King’s College London, London, UK;
d
Department of Metabolism, Digestion and Reproduction,
Imperial College London, London, UK
ABSTRACT
The availability of the genomic sequence of the malaria mosquito Anopheles gambiae has in
recent years sparked the development of transgenic technologies with the potential to be used
as novel vector control tools. These technologies rely on genome editing that confer traits able
to aect vectorial capacity. This can be achieved by either reducing the mosquito population or
by making mosquitoes refractory to the parasite infection. For any genetically modied
organism that is regarded for release, molecular characterization of the transgene and anking
sites are essential for their safety assessment and post-release monitoring. Despite great
advancements, Whole-Genome Sequencing data are still subject to limitations due to the
presence of repetitive and unannotated DNA sequences. Faced with this challenge, we
describe a number of techniques that were used to identify the genomic location of
a transgene in the male bias mosquito strain Ag(PMB)1 considered for potential eld applica-
tion. While the initial inverse PCR identied the most likely insertion site on Chromosome 3 R
36D, reassessment of the data showed a high repetitiveness in those sequences and multiple
genomic locations as potential insertion sites of the transgene. Here we used a combination of
DNA sequencing analysis and in-situ hybridization to clearly identify the integration of the
transgene in a poorly annotated centromeric region of Chromosome 2 R 19D. This study
emphasizes the need for accuracy in sequencing data for the genome of organisms of medical
importance such as Anopheles mosquitoes and other tools available that can support genomic
locations of transgenes.
KEYWORDS
Transgene; PiggyBac;
repetitive region;
centromere; whole genome
sequencing; fluorescence
in situ hybridization
Introduction
Advances in molecular technologies, as well as the avail-
ability of platforms for Next-Generation Sequencing, have
led to an unprecedented investigation of the genome of
human disease vector species such as Anopheles mosqui-
toes, vectors of malaria [1–4]. This knowledge has
enabled the development of techniques for genome
editing and the potential for vector control strategies
that aim at mosquito population replacement or suppres-
sion [5–7]. Genetic modication requires the integration
of a transgene into the genome of the host organism,
which confers phenotypes consistent with the control
strategy being pursued. Insertions of the transgene can
be achieved via a variety of molecular tools; PiggyBac
transposons, site-specic recombinases (like AttP/AttB)
and CRISPR/Cas9 systems are the most commonly used
for genomic engineering applications in insects [6–9].
Whilst AttB/AttP and CRISPR/Cas9 systems allow for
a site-directed insertion, PiggyBac-mediated transposon
integration occurs semi-randomly into the host genome
due to the simple requirement of a TTAA sequence motif.
In 2014, the generation of an Anopheles gambiae auto-
somal sex distorter strain gfp124L-2 (renamed Ag(PMB)1
in [10]) was described [11]. This strain carries a modied
endonuclease variant, I-PpoIW124L, that cuts ribosomal
gene sequences that are highly repetitive and located on
the X chromosome of Anopheles gambiae mosquitoes
[11]. As a result, when expressed during the male sper-
matogenesis, this endonuclease causes damage to the
X-bearing gametes, leaving mostly Y-bearing sperm still
functional and generating approximately 95% male o-
spring. Since only female transmit malaria parasites, this
type of autosomal sex ratio distorter alone represents
a potential vector control that has been modeled to be
more ecient to eectively suppress mosquito popula-
tions than the sterile insect technique [12,13]. Moreover,
this type of genetic construct has the potential to be
developed into a gene drive if placed on the
Y chromosome, in this case, all males will inherit the
transgene. Therefore, this strain is of interest as
a potential vector control but also as an intermediate
step toward the development of a self-sustaining gene
CONTACT Federica Bernardini f.bernardini11@imperial.ac.uk Department of Life Sciences, Imperial College London, London, UK; CONTACT Silke
Fuchs silke.fuchs05@imperial.ac.uk
Supplemental data for this article can be accessed online at https://doi.org/10.1080/20477724.2022.2100192
PATHOGENS AND GLOBAL HEALTH
https://doi.org/10.1080/20477724.2022.2100192
© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

driven by Target Malaria, which is a not-for-prot research
consortium working on developing novel genetic tech-
nologies as a tool to ght malaria and to complement
existing vector control methods. However, for any strain
that is to be considered for release, including for small-
scale studies, there needs to be a full and exhaustive
characterization of the nature of the transgene insertion.
Ag(PMB)1 was generated via semi-random
PiggyBac mediated integration and this strain was
selected, based on fertility and male bias values, out
of 26 transgenic strains carrying transgenes with
various I-PpoI variants [11]. Some of the strains
with the same I-PpoI variants, but at dierent inser-
tion sites, showed a range of male bias and tness
costs, thus indicating a correlation between the
phenotype and the insertion site of the transgene.
The original insertion site of the Ag(PMB)1 trans-
gene was initially identied on Chromosome 3 R
36D by means of Inverse PCR [11]. Blast results of
the anking sequences at the time however indi-
cated a highly repetitive nature for these elements
with multiple hits in the Anopheles gambiae gen-
ome leaving the possibility of a potential dierent
insertion site. The occurrence of repetitive stretches
of DNA makes the process of sequencing, assembly
and annotation of genetic elements challenging
[14]. Similarly, the integration of a transgene in
a region of the genome with highly repetitive
sequences can lead to errors in the identication
of the integration site. Aware of this possibility, we
carried out an extensive analysis to characterize the
transgene integration in Ag(PMB)1 further. We com-
bined Whole-Genome Sequencing (WGS), Southern
blotting, Fluorescence in situ hybridization (FISH)
and Polymerase Chain Reaction (PCR) analysis and
showed a single integration of the PMB1 transgene
with the insertion on Chromosome 2 R 19D.
Materials and methods
Mosquito strains
Ag(PMB)1 is a transgenic Anopheles gambiae strain
where the beta 2 tubulin promoter mediated expres-
sion of the I-PpoI structural variant W124L fused to
eGFP during spermatogenesis leads to approximately
95% male ospring [11]. The strain was generated by
piggyBac transposon-mediated integration of the
pBac[3xP3-DsRed]b2eGFP::I-PpoI124L transgene into
germ cells of An. gambiae G3 strain embryos and
transgenic individuals can be identied via dsRed
marker expression in the eyes and nerve chord. The
G3 wild-type is an Anopheles gambiae/coluzzii hybrid
colony established initially from mosquitoes collected
in Gambia in 1975. BF_Ac(PMB)1 is a transgenic PMB1
strain that was introgressed for more than six back-
crosses into a local Anopheles coluzzii colony collected
from Bana, Burkina Faso in 2012. All mosquitoes were
maintained under standard mosquito rearing condi-
tions at 27°C ± 1 and 70% ± 5% relative humidity.
Inverse PCR
Primers were designed to amplify across the 5’ and 3’
genomic insertion boundaries of the Ag(PMB)1 trans-
gene. DNA was extracted from 4 individual pools (con-
taining 5 transgenic larvae each) of Ag(PMB)1 mosquitoes
using a Blood and Tissue Kit (Qiagen) following >100
generations of lab maintenance. PCR across the 5’ and 3’
transgene anking regions was performed using: pB-
5SEQ (5′-CGCGCTATTTAGAAAGAGAGA) with genomic
primer RH57 (5’-GAAAACCTTACAAGCGTCTTCAA) to
amplify the 5′ junction and pB-3SEQ (5′-CGATAAAACA
CATGCGTCAATT) with RH53 (5’-GATATTATCGCGCTC
GGTCC) to amplify the 3′ junction. Each PCR amplicon
was Sanger sequenced using both forward and reverse
primers and the resulting sequences were aligned to
compare genomic anking and transgenic sequences to
the published Inverse PCR data [11].
Southern blot analysis
A total of 60 individual adult mosquitoes were placed in 3
pools in 1.5 ml low adhesion microtubes and submerged
in dry ice until completely frozen, then crushed with
a micropestle and incubated at 37°C in a buer contain-
ing 200 µg/ml Proteinase K,200 mM Tris-CL, ph&.5,
30 mM EDTA and 2% SDS. The homogenate was
extracted twice in Phenol/Chloroform/Isoamyl alcohol
(25:24:1), then incubated with 50 U RNAase A at 37°C
for 15 minutes, before being extracted in Chloroform/
Isoamyl alcohol (24:1), then precipitated with 3 M Sodium
Acetate and Absolute ethanol and resuspended in water
overnight. A total of 2.5 µg genomic DNA was digested at
37°C with Fastdigest enzymes at 10 U/µl in reaction times
of typically 1 h. Digested genomic DNA was run on 0.8%
agarose gels, then transferred to positively charged nylon
blots overnight using capillary action. DIG Southern ana-
lysis was performed as per manufacturer’s instructions.
DIG labeled and unlabeled (control) I-PpoI probes of
499bp were generated with the primers PpoOR: 5’ CTT
TGT TGA GGA CCT GCC ACA GT 3’ and PpoIF: 5’ CGA CCT
AAG AAG AAG AGG AAG GTG 3’ [15] using the pBac
[3xP3-DsRed]b2eGFP::I-PpoI124L plasmid as template
DNA using PCR DIG Probe Synthesis Kit (Sigma-Aldrich
Catalog number11636090910). Probes were hybridized
at 50°C in DIG Easy Hyb buer overnight and detected
using DIG nucleic acid detection kit (Sigma Aldrich cat.
No. 11175041910).
2M. VITALE ET AL.

Sequence library preparation using illumina
TruSeq and in silico copy number analysis
DNA extractions were performed on mosquitoes
from two strains, namely Ag(PMB)1 and BF_Ac
(PMB)1, for each on 10 individuals (5 adult males,
5 non-fed adult females) using a Blood and Tissue
Kit (Qiagen). For each sample, 100 ng of input
gDNA were sheared using Covaris for a 350 bp
insert size. Library preparation was performed
using the Illumina TruSeq Nano kit. Each sample
was tagged with a unique barcode, followed by
three 2 × 150bp High Output V2.5 paired-end
sequencing runs on the Illumina NextSeq550 plat-
form, obtaining an average of 265 M reads per
sample. Raw data were checked with FastQC soft-
ware and aligned to Anopheles gambiae PEST
strain reference genome (AgamP4 from
VectorBase) using BWA mem software version
0.7.12. Unmapped reads were de novo assembled
using SPAdes aligner version 3.13.0 and contigs
that included eGFP::I-PpoI124L sequence were
aligned with ClustalO webserver and inspected
for consistency. To analyze the read coverage of
the transgene, sequencing reads were aligned,
using BWA mem [16], to AgamP4 reference gen-
ome with added expected insertion sequence. This
sequence contained the transgene sequences with
anking genomic regions [longer than expected
Illumina read of 250 bp) that allowed inspection
of reads spanning the putative integration site.
The genomic location of the predicted integration
site was selected based on AgamP4 PEST reference
genome and inverse PCR analysis performed
by [11].
A custom Python script was written and used to
extract informative reads from the alignments and
quantify them to obtain the read coverage and copy
number of the transgene. Genomic regions used for
mean coverage measurements were divided into three
separate categories (n-single copy, 2 n-two copies and
3 n-three copies] based on the expected number of
copies of a certain genomic feature. As 3 n-three copies
feature were selected beta2 tubulin (AGAP008622)
promoter and terminator sequences that are expected
to be in three copies (two copies from the endogenous
gene and a single copy from the transgene). DsRed
was selected as n-single copy feature, being present
only in the transgene. In addition, arbitrarily selected
15 random 20-bp-long genomic regions on autosomal
chromosomes were selected as 2 n-two copies geno-
mic features as these regions should correspond to the
coverage on two sets of chromosomes (2 n)
(Figure 1A). The random autosomal loci were selected
using BEDtools on AgamP4.12 genome annota-
tion [17].
Nanopore sequencing
The genomic DNA was extracted from a total of 5
BF_Ac(PMB)1 male and 5 female adult mosquitoes,
and 20 Ag(PMB)1 female adults using the Blood and
Tissue Kit (Qiagen) and prepared for long read
sequencing using MinION by Oxford Nanopore
Technology (ONT). BF_Ac(PMB)1 DNA was initially
fragmented using the Covaris G-tube to obtain frag-
ments of approximately 8kb in length and the ONT
adapters ligated (ONT 1D PCR adapters – SQK-
LSK109, PBGE12_9066_v109_revM_14Aug2019).
Library preparation for Ag(PMB)1 unfragmented
DNA was performed according to recommended
ONT protocol instructions for both Ligation
Sequencing Kit (SQK-LSK109) and Cas9 Sequencing
Kit (SQK-CS9109). All samples were puried with
AMPure XP magnetic beads. BF_Ac(PMB)1 samples
were barcoded and pooled for sequencing onto
seven ONT MinION R9.4.1 owcells. BF_Ac(PMB)1
samples from each library preparation kit were
loaded onto separate ONT MinION with R9.4.1 ow-
cell. The resulted long reads were rst processed
and quality checked before the alignment to the
AgamP4 reference genome concatenated to the
insertion sequence (this time without any putative
anking regions) using the Pomoxis tool (https://
github.com/nanoporetech/pomoxis) for BF_Ac(PMB)
1 samples and minimap2 [18] for Ag(PMB)1 sam-
ples. The reads that aligned to the expected inser-
tion region were extracted and de novo assembled
using the program Canu v1.8 [19].
The assembly was then used to further investi-
gate the insertion site, blasting the regions, adja-
cent to the transgene, to the PEST reference
genome and on VectorBase (https://www.vector
base.org/). To identify the contig inclusive of the
inserted sequence, the eGFP and DsRed sequences
were used as query by BLASTn search [20]. Once
the transgenic sequence was detected, both BLAST
and tBLASTx searches were performed to better
dene the genomic region of the insertion. The
contig with the transgenic sequences was also rea-
ligned to the reference genome with Minimap2
[18] to identify sequence homology between the
contig and the reference genome. This latter tool
was further used to align the contig inclusive of
the transgene sequence to the high-quality assem-
bly obtained for Anopheles coluzzii by means of
PacBio sequencing [21]. This assembly of PacBio
long reads led to a considerably improved resolu-
tion of readings in repetitive and unassembled
regions in the An. gambiae PEST reference. In par-
ticular, Kingan et al. were able to assign 40% of
sequences that so far were misplaced in the arti-
cial AgamP4 PEST chromosome UNKN to specic
PATHOGENS AND GLOBAL HEALTH 3

chromosomes. Since many of the BLAST hits were
reported in the UNKN chromosome, the PacBio An.
coluzzii assembly was used in the present analysis
to assign the potential insertion site (Figure 1B).
A dot plot was produced to compare the PacBio
An. coluzzii assembly and the AgamP4 PEST refer-
ence genome and visualize the putative position of
the inserted sequence. The dot plot was generated
using the online tool D-Geneies v1.2.0 (run ID:
PNqgs_20210713201144) [22].
In Situ Hybridization of polytene chromosomes
Ovaries at Christopher stage III from 18/33-h half-
gravid females were dissected from 7 wild-type and 8
Ag(PMB1) individuals and xed for 24-h at Room
Temperature (RT) in Carnoy’s solution. Polytene chro-
mosome preparations were obtained according to Xia
et al. [23]. Chromosome spread quality check was per-
formed using a phase-contrast microscope and then
immersed in Liquid Nitrogen. Subsequently, they were
Figure 1. Overview of the methods to determine A Ag(PMB1) copy number and B Ag(PMB1) transgene integration site.
4M. VITALE ET AL.

dehydrated in a series of ethanol (50%, 70%, 90%,
100%) for 5 minutes each and then allowed to air dry
at RT and stored at – 20°C. To perform Fluorescent In
Situ Hybridization (FISH) on each chromosome pre-
paration, 800 ng of pBac [3xP3-DsRed]b2eGFP::
I-PpoI124L plasmid DNA [11] was labeled with dUTP-
Cy5 (GE Healthcare) by nick translation reaction
(ROCHE). The reaction mixture was incubated at 15°C
for 90 minutes and stopped by heating at 65°C for
10 minutes. PCR reaction (F-TATCGGCTGCAACATC
AAAC, R-ACAGAGGTTGTTGAGGAACCA) was used to
generate probes from the gene AGAP004670 [24].
DNA probes were precipitated by adding 1/10 volume
of 3 M NaAC and 2.5 volume of 100% ethanol and
centrifuged at 14,000 g for 20 minutes at 4°C. The
Probe was then resuspended in hybridization buer
that was pre-warmed at 37°C. FISH was performed as
previously described by Xia et al. [23].
After denaturation, hybridization, and washing
steps, the microscope slides were mounted with
Prolong Antifade + DAPI, sealed with Cytobond and
stored at 4°C until usage. Microscope slides were ana-
lyzed using a Leica SP8 inverted confocal microscope.
Pictures were observed with a 40x oil immersion objec-
tive and analyzed using Fiji and Photoshop.
Results
Characterization of the PMB1 integration anking
sites by inverse PCR
In order to characterize the integration site of the
transgene in the Ag(PMB)1 strain we rst performed
an inverse PCR using primers previously used by Galizi
et al [11]. Our results showed the same anking regions
(~400 bp on each side) as originally described for this
strain (Supplementary Figure 1). A subsequent BLAST
search of the combined 5’ and 3’ anking sequences
found the best match with over 97% sequence identity
over a 754-bp alignment length at the previously iden-
tied Chromosome 3 R 36D location. However, we also
obtained over 500 hits on dierent chromosomal loca-
tions, suggesting that the integration occurred in
a very repetitive region in the genome and that the
integration potentially occurred at a dierent site than
previously described. In fact, 12 of these autosomal hits
showed over 90% sequence identity of an alignment
length of over 700 bp and they also contained the
TTAA recognition sites required for piggyBac mediated
insertion of the transformation plasmid (Figure 2A).
Conrmation of a single integration event by
southern blot and illumina sequencing
In order to determine whether the integration could
have occurred at multiple repetitive sites or whether
there may have been copy number or structural
alterations in the transgene, we performed
a Southern blot analysis. Our results revealed only
single bands produced by two dierent enzyme diges-
tions of Ag(PMB)1 genomic DNA, demonstrating that
Ag(PMB)1 indeed contains a single insertion of the
transgene (Figure 2B). Similar to our previous BLAST
search of sequences obtained by Inverse PCR, the size
of the DNA fragment spanning part of the transgene
and its anking sites could not be matched to a specic
insertion site. This was due to multiple possible sites
with similar fragment sizes and the fact that some
possible sites were not assigned to chromosomes in
the Anopheles gambiae PEST reference genome
(Figure 2C). In addition, we investigated whether
Illumina sequencing could be used instead of
Southern blot analysis to conrm transgene integra-
tion events. We aligned WGS reads to a sequence of
the transgene anked by genomic regions upstream
and downstream from the putative integration site.
The analysis of the aligned reads showed that, as
expected for a single integration, the coverage on the
loci specic to eGFP::I-PpoI124L transgene and
piggyBac anking arms was consistent with single
copy coverage (n) compared to the coverage from
autosomal (2 n) loci as the heterozygous individuals
only contained one copy of the transgene (1 n). In
addition, the coverage at the promoter and terminator
regions of endogenous and transgenic beta-tubulin
gene was higher and consistent with 3 n (expected
single copy of the sequence from the transgene and
two copies from the endogenous sequences)
(Figure 2D). The analysis also showed inconsistently
high coverage in the immediate upstream and down-
stream transgene-anking regions. However, this
observation conrms the repetitive nature of the
sequences anking the integration site, which was
also conrmed with BLAST search that resulted in
more than 500 hits of homologous sequences from
other sites in the Anopheles genome. Overall, this
type of coverage analysis could be a useful alternative
to Southern blot analysis to conrm copy number
when characterizing transgene insertions.
Limitations of whole genome sequencing
approaches to pinpoint integration sites in
insuciently annotated regions in the reference
genome
Because the transgene anking regions occur in
a highly repetitive region, which are usually collapsed
into a single sequence in de novo assemblies using
Illumina reads, we performed Nanopore sequencing
analysis to obtain longer reads that would span this
region. Despite low coverage (<10x) we obtained 3’
end sequences that signicantly aligned to the PMB1
transgene and to a 563bp region about 1.5 kb
upstream of the repetitive sequence corresponding
PATHOGENS AND GLOBAL HEALTH 5

to a locus on chromosome arm 2 R (2 R | 58,941,996–
58,942,559). This places Ag(PMB)1 in band 19D of
chromosome arm 2 R, a region previously identied
as heterochromatic and rich in retroelements and
tandem repeats [25]. Some reads also aligned the
transgene anking regions on the 3’ end to the 3 R
(3 R | 47,762,128–47,762,483) which corresponds to the
published insertion site at 36D of chromosome 3 R [11]
Figure 2. Potential chromosomal insertion sites of the Ag(PMB)1 transgene based on Inverse PCR and subsequent blast search in
Vectorbase database. A A blast search of the sequence obtained by Inverse PCR revealed 12 autosomal locations with high
sequence similarity (>90%) over long Inverse PCR sequence stretches (~700/788bp), including the Chr 3 R 36D location (3 R|
47761729-47762482) previously proposed as the insertion site [11]. B Southern blot probed with I-PpoI specific transgene
sequences. As expected, a 8.7kb band was detected in ‘Plasmid’ positive control consisiting of pBac [3xP3-DsRed]b2eGFP::
I-PpoI124L linearized with AfeI. No signals were detected in DraI-cut and ScaI-cut wild type genomic DNA. Single postive bands of
the expected sizes, around 3 and 7kb, were detected in Ag(PMB)1 genomic DNA cut with DraI and ScaI. respectively. All numbers
refer to DNA fragement sizes in kb. C Schematic maps of transgene insertion in Ag(PMB)1 at the Chromosome 3 R 36D and two
newly identified potential integration sites on Chromosome 2 R in the Anopheles gambiae PEST reference genome (source
Vectorbase: AgamPEST). Digestion of Ag(PMB)1 genomic DNA with DraI and ScaI is expected to produce 3.2 and 7–8.2kb
fragments respectively, both of which can be detected using a probe against I-PpoI (in green). D Illumina sequencing reads
aligned to the transgenic sequences and integration site flanking regions.
6M. VITALE ET AL.

although this alignment was much more tenuous with
lower mapping quality compared to the alignment on
the 2 R band 19D region. This suggests the previously
published insertion site of chromosome 3 R band 36D
was prematurely attributed due to an inability to char-
acterize a sucient length of the anking regions.
We also investigated the transgene location in
a dierent genetic background. A de novo genome
assembly was performed using nanopore sequencing
on the introgressed BF_Ac(PMB)1 strain that contains
the same transgene but is located in an Anopheles
coluzzii instead of an An. gambiae background.
Assembly of reads with transgenic sequences (DsRed
and eGFP::PpoI124L) resulted in a 27 kb long contig,
which showed high sequence identity (> 90%) when
compared to the sequences obtained with inverse PCR
and Illumina sequencing from Ag(PMB)1 and BF_Ac
(PMB)1 (Figure 3A). This result supports correct assem-
bly of the reads obtained from nanopore sequencing.
Additionally, the comparison of the transgene-anking
regions from the transgene bearing contig with high-
quality de novo assembly of An. coluzzii [21] showed
high sequence identity with two An. coluzzii contigs
(000049 F and 000035 F). A 17.6 kb DNA sequence from
5’ transgene-anking region and a 2.6 kb sequence
from the 3’ transgene-anking region matched An.
coluzzii contigs 000049 F and 000035 F, respectively,
with relatively high sequence identity (> 70%).
According to the dot plot, both An. coluzzii contigs
(000049 F and 000035 F) correspond to the centro-
meric region of the 2 L chromosome in proximity to
the 2 R centromeric chromosome region predicted by
Figure 3. WGS sequencing analysis of the PMB1 insertion site. A Alignment of the PMB1 transgene and flanking sequences from
Inverse PCR (iPCR) from Ag(PMB)1 (New and [11]), Illumina sequencing from Ag(PMB)1 and BF_Ac(PMB)1, Nanopore sequencing
LSK* from BF_Ac(PMB)1), Nanopore Cas9 and Nanopore LSK from Ag(PMB)1. B Dot plot of the alignment between the de novo
assembly of An. coluzzii genome (contigs on the y axis) and the AgamP4 PEST reference genome (on the x axis). The box shows the
genomic position of the PacBio contigs (000035 F and 000049 F), where there was an alignment of the transgene flanking regions.
PATHOGENS AND GLOBAL HEALTH 7

the nanopore sequencing results of Ag(PMB)1
(Figure 3B). Despite lower sequence identity of the
immediate repetitive sequences neighboring the
transgene, the long anking regions around the trans-
gene on the 27 kb contig allowed us to locate the
transgene more precisely compared to the results
from other sequencing methods. Importantly, the 27
kb contig provided an accurate consensus sequence of
the inserted transgene and its genomic context.
In situ hybridization as a tool to locate transgenes
in highly repetitive non-annotated regions
In order to overcome the limitations of the various
sequencing approaches and existing reference gen-
omes we then applied FISH to validate the potential
centromeric 2 L/2 R chromosomal insertion site of the
transgene. This technique has been widely used on
polytene chromosomes for the creation of cytogenetic
maps and genome annotation in Anopheles mosqui-
toes [26,27]. A probe consisting of the original trans-
formation plasmid was generated to bind the
transgene in Ag(PMB)1 individuals as well as the endo-
genous beta2 tubulin promoter (AGAP008622) on
chromosome 3 R:13,725,120.13,726,724 as endogen-
ous positive control for Ag(PMB)1 and wild-type (WT)
individuals. In total, we examined 54 polytene chromo-
somes from 7 WT An. gambiae individuals and 63
polytene chromosome from 8 Ag(PMB)1 individuals.
As expected for a single integration of the transgene,
94% of PMB1 individuals (59 out of 63) showed two
FISH signals, one corresponding to the endogenous
beta2-tubulin promoter, located on the chromosome
3 R, band 31 division A and the other one to the
transgene in the chromocenter of chromosome 2 R
band 19, division D-E (Figure 4A). A few samples
(n = 4) showed only one signal in the chromocenter
of chromosome 2 R but none in chromosome 3 R. As
expected, 70% of wild-type samples (38 of 54) showed
only one single uorescent band that corresponded to
the endogenous beta2 tubulin promoter on chromo-
some 3 R (Figure 4B and Figure 4C), the rest (n = 16)
showed no uorescent signal. The failure of labeling of
the endogenous site on chromosome 3 R for WT and
PMB1 samples, was possibly due to insucient amount
of the probe in these samples. Importantly none of the
54 WT samples showed a signal on chromosome 2 R.
To conrm the transgene site integration on
Figure 4. FISH analysis on polytene chromosomes of Ag(PMB)1 and wild-type (WT) mosquitoes. FISH was performed with a probe
consisting of the original transformation plasmid specific to the transgene in Ag(PMB)1 individuals as well as the endogenous
beta2 tubulin promoter (AGAP008622) on Chromosome 3 R:13,725,120 . . . 13,726,724 in WT and Ag(PMB)1 individuals A PMB1
samples showed two red signals labeling the endogenous beta2-tubulin promoter on chromosome 3 R and the PMB1 transgene
insertion site in the proximity of the centromere of chromosome 2 R. B and C WT samples only showed one probe signal for the
endogenous beta2 tubulin promoter in the distal region of chromosome 3 R D Signals from the PMB1 transgene (in red) and the
flag gene AGAP004670 with known integration site (in white). E Chromosomal map of the proposed location of the PMB1
transgene and the reference gene (AGAP004670) with known chromosomal location at Chromosome 2 R: 60845862 . . . 60895998.
8M. VITALE ET AL.

chromosome 2 R, band 19D, a second probe targeting
exon 1 of a gene, AGAP004670, with known location
on chromosome 2 R, band 19, division E (position
60845862–60895998) was used for FISH analysis. As
expected, the probe (in white) gave a signal in
between the chromocenter of chromosome 2 R and
the signal from the PMB1 transgene (in red)
(Figure 4D).
Together, these results conrmed that the insertion
of the PMB1 transgene is on chromosome 2 R 19D
(Figure 4E).
Discussion
In the last 20 years, progress in the development of
genetic strategies for the control of vector-borne dis-
eases such as malaria has grown in parallel with the
exponential availability of whole-genome sequencing
data [1–4,21,28,29]. Although Next-Generation
Sequencing technologies have provided reliable
sequence information for most of the genome, het-
erochromatic and repetitive regions can often lead to
uncertainty or errors in DNA assemblies. It has been
estimated that 33% (about 86 Mbp) of the An. gam-
biae genome is composed of repetitive elements,
which are mostly found in the pericentromeric
regions of the autosomes and make up almost the
entirety of the Y chromosome [28,30]. A substantial
eort has been made by the scientic community to
improve data on genome assembly and annotation
over the last years [1–4,21,28,29,31]. In a study by
Sharakhova et al. [31] cDNA, BAC clones and PCR
amplied gene-fragments were used as probes for
in situ hybridization to place about 33% of previously
unmapped sequences that in total, accounted for
around 15 Mbp. These sequences fell mostly within
pericentromeric regions of the chromosomes; in addi-
tion, around 1.32 Mbp covered the physical gaps
between scaolds on euchromatic parts of the chro-
mosomes. Further, in 2019, a new low-input PacBio-
based approach led to a de novo assembly from
a single An. coluzzii Ngousso; this study succeeded in
placing 667 (>90%) of previously unplaced genes into
their appropriate chromosomal contexts in the
AgamP4 PEST reference genome [21]. AgamP4 still
includes a number of unplaced contigs (27.3 Mb
excluding unidentied nucleotides -Ns-) that are cur-
rently labeled as the ‘UNKN’ (unknown) chromosome
as well as misassembled haplotype scaolds. Further,
over 6302 gaps of Ns (ranging from 20bp-36kb) in the
primary chromosome scaolds makes the areas of
genome assembly still highly fragmented [21].
In this manuscript, we report a number of strate-
gies based on sequencing data as well as Southern
blotting and FISH analysis that were used to charac-
terize transgene site integration in the An. gambiae
strain Ag(PMB)1. This strain produces a male bias
phenotype that is desirable for potential vector con-
trol. As part of the strain assessment a molecular
characterization of its transgene and insertion site
was performed [32].
Here, we show that the preliminary assignment
of the transgene location to Chromosome 3 R 36D
[11] was incorrect due the presence of highly repe-
titive sequences at the insertion site. Inverse PCR is
a standard method that allows the isolation and
sequencing of the regions anking the transgene.
However, in our case, a BLAST analysis provided
500 hits of which 12 showed 90% sequence iden-
tity over almost all of the anking sequence and
they also contained the TTAA recognition sites
required for PiggyBac mediated insertion. Based
on these results, we concluded that Inverse PCR,
which produces relatively short anking sequences,
is less suitable for highly repetitive long sequences
(>700 bp) to ascertain correct identication of
transgene landing sites. We then ruled out the
possibility of multiple insertions of the transgene
in the genome by performing Southern blotting
and coverage analysis based on Illumina sequen-
cing data generated using samples of the Ag(PMB)
1 strain. Both approaches conrmed a single inser-
tion of the transgene and coverage analysis could
be used as an alternative to classical Southern blot
analysis to determine copy number of transgenes.
However, neither provided denite evidence of the
location of the transgene. Nanopore sequencing
was then used to generate long reads of genomic
sequence with the aim of extending the sequence
obtained in the regions anking the transgene.
These data were used for a BLAST analysis that
suggested band 19D of chromosome 2 R as the
most likely location of the transgene, however
some reads also aligned to band 36D on chromo-
some 3 R, leading to persistent ambiguity of the
precise genomic location of the transgene.
Alignment of Nanopore sequencing data gener-
ated from the introgressed An. coluzziii BF_Ac
(PMB)1 strain to the de novo An. coluzziii Ngousso
assembly [21] was also unsuccessful in determining
the exact insertion site of the transgene.
Finally, we examined the presence of the transgene
in the genome of the Ag(PMB)1 strain by FISH analysis.
A uorescent probe consisting of the original transfor-
mation plasmid was generated that was complemen-
tary to and could therefore hybridize with the
sequences of the PMB1 transgene on polytene chro-
mosomes preparations. The result was consistent with
one of the integration sites identied by sequencing-
based approaches and show that FISH can be used as
a complementary tool to conrm insertion sites in
highly repetitive regions.
PATHOGENS AND GLOBAL HEALTH 9

However, genome assembly for species with
highly polymorphic genomes, such as mosquitoes,
can be challenging, novel techniques in sequencing
and scaolding methods promise to greatly
improve current data that could support the devel-
opment of novel targets for vector control, o-
target analysis, and characterization of insertion
sites of transgenic strains. In combination with phe-
notypic data, this will support the prediction of
dynamics of a transgene in potential target eld
populations.
Acknowledgments
We thank Barbara Fasulo for her guidance on the develop-
ment of FISH and the Facility for Imaging by Light
Microscopy (FILM) at Imperial College London. We are also
grateful for the helpful feedback on the manuscript by Tony
Nolan and John Mumford.
Data availability
The data that support the ndings of this study will be
openly available at https://zenodo.org/record/6010699
(DOI: 10.5281/zenodo.6010699) upon publication.
Disclosure statement
No potential conict of interest was reported by the author(s).
Funding
The authors are members of the Target Malaria not-for-prot
research consortium, which receives core funding from the
Bill & Melinda Gates Foundation (Grant INV006610 Target
Malaria Phase II) and from the Open Philanthropy Project
Fund [Grant O-77157]. The Facility for Imaging by Light
Microscopy (FILM) at Imperial College London is part-
supported by funding from the Welcome Trust [grant
104931/Z/14/Z] and BBSRC [grant BB/L015129/1].
ORCID
Chiara Leo http://orcid.org/0000-0003-0510-2535
Thomas Courty http://orcid.org/0000-0002-3424-886X
Nace Kranjc http://orcid.org/0000-0001-7720-8430
John B. Connolly http://orcid.org/0000-0002-7033-3463
Roya Elaine Haghighat-Khah http://orcid.org/0000-0002-
9741-5282
Federica Bernardini http://orcid.org/0000-0001-7526-
0155
Silke Fuchs http://orcid.org/0000-0001-8921-7422
References
[1] Anopheles Gambiae Genomes C. Genome variation and
population structure among 1142 mosquitoes of the
African malaria vector species Anopheles gambiae and
Anopheles coluzzii. Genome Res. 2020;30:1533–1546.
[2] The Anopheles gambiae 1000 Genomes Consortium.
Genetic diversity of the African malaria vector
Anopheles gambiae. Nature 2017;552:96–100.
[3] Holt RA, Subramanian GM, Halpern A, et al. The gen-
ome sequence of the malaria mosquito Anopheles
gambiae. Science. 2002;298:129–149.
[4] Neafsey DE, Waterhouse RM, Abai MR, et al. Mosquito
genomics. Highly evolvable malaria vectors: the genomes
of 16 Anopheles mosquitoes. Science. 2015;347:1258522.
[5] Gantz VM, Jasinskiene N, Tatarenkova O, et al. Highly
ecient Cas9-mediated gene drive for population
modication of the malaria vector mosquito
Anopheles stephensi. Proc Natl Acad Sci U S A.
2015;112:E6736–43.
[6] Grossman GL, Raerty CS, Clayton JR, et al. Germline
transformation of the malaria vector, Anopheles gam-
biae, with the piggyBac transposable element. Insect
Mol Biol. 2001;10:597–604.
[7] Kyrou K, Hammond AM, Galizi R, et al. A CRISPR-Cas9
gene drive targeting doublesex causes complete
population suppression in caged Anopheles gambiae
mosquitoes. Nat Biotechnol. 2018;36:1062–1066.
[8] Handler AM. Use of the piggyBac transposon for
germ-line transformation of insects. Insect Biochem
Mol Biol. 2002;32:1211–1220.
[9] Nimmo DD, Alphey L, Meredith JM, et al. High e-
ciency site-specic genetic engineering of the mos-
quito genome. Insect Mol Biol. 2006;15:129–136.
[10] Pollegioni P, North AR, Persampieri T, et al. Detecting
the population dynamics of an autosomal sex ratio
distorter transgene in malaria vector mosquitoes.
J Appl Ecol. 2020;57:2086–2096.
[11] Galizi R, Doyle LA, Menichelli M, et al. A synthetic sex
ratio distortion system for the control of the human
malaria mosquito. Nat Commun. 2014;5:3977.
[12] Schliekelman P, Ellner S, Gould F. Pest control by
genetic manipulation of sex ratio. J Econ Entomol.
2005;98:18–34.
[13] Deredec A, Burt A, Godfray HCJ. The population genet-
ics of using homing endonuclease genes in vector and
pest management. Genetics. 2008;179:2013–2026.
[14] Torresen OK, Star B, Mier P, et al. Tandem repeats lead
to sequence assembly errors and impose multi-level
challenges for genome and protein databases. Nucleic
Acids Res. 2019;47:10994–11006.
[15] Windbichler N, Papathanos PA, Crisanti A. Targeting
the X chromosome during spermatogenesis induces
Y chromosome transmission ratio distortion and early
dominant embryo lethality in Anopheles gambiae.
PLoS Genet. 2008;4:e1000291.
[16] Li H, and Durbin R. Fast and accurate short read align-
ment with Burrows-Wheeler transform. Bioinformatics.
2009;25(14):1754–1760.
[17] Quinlan AR, Hall IM. BEDTools: a exible suite of utili-
ties for comparing genomic features. Bioinformatics.
2010;26:841–842.
[18] Li H. Minimap2: pairwise alignment for nucleotide
sequences. Bioinformatics. 2018;34:3094–3100.
[19] Koren S, Walenz BP, Berlin K, et al. Canu: scalable and
accurate long-read assembly via adaptive k-mer
weighting and repeat separation. Genome Res.
2017;27:722–736.
[20] Altschul SF, Gish W, Miller W, et al. Basic local align-
ment search tool. J Mol Biol. 1990;215:403–410.
[21] Kingan SB, Heaton H, Cudini J, et al. A high-quality de
novo genome assembly from a single mosquito using
pacbio sequencing. Genes (Basel). 2019;10:62.
10 M. VITALE ET AL.

[22] Cabanettes F, Klopp C. D-GENIES: dot plot large genomes
in an interactive, ecient and simple way. PeerJ. 2018;6:
e4958.
[23] Xia A, Peery A, Kamali M, et al. Fluorescence in situ
hybridization to the polytene chromosomes of
Anopheles mosquitoes. Bio-protocol. 2013;3:e860.
[24] Timoshevskiy VA, Sharma A, Sharakhov IV, et al.
Fluorescent in situ hybridization on mitotic chromo-
somes of mosquitoes. J Vis Exp. 2012;17:e4215.
[25] Sharakhova MV, George P, Brusentsova IV, et al. Genome
mapping and characterization of the Anopheles gambiae
heterochromatin. BMC Genomics. 2010;11:459.
[26] Coluzzi M, Sabatini A, Della Torre A, et al. A polytene
chromosome analysis of the Anopheles gambiae spe-
cies complex. Science. 2002;298:1415–1418.
[27] George P, Sharma A, Sharakhov IV. 2D and 3D chromo-
some painting in malaria mosquitoes. J Vis Exp. 2014;6:
e51173.
[28] Hall AB, Papathanos PA, sharma A, et al. Radical remo-
deling of the Y chromosome in a recent radiation of
malaria mosquitoes. Proc Natl Acad Sci U S A.
2016;113:E2114–23.
[29] Zamyatin A, Avdeyev P, Liang J, et al. Chromosome-
level genome assemblies of the malaria vectors
Anopheles coluzzii and Anopheles arabiensis.
Gigascience. 2021;10:giab017.
[30] Besansky NJ, Powell JR. Reassociation kinetics of
Anopheles gambiae (DIPTERA: culicidae) DNA. J Med
Entomol. 1992;29:125–128.
[31] Sharakhova MV, Hammond MP, Lobo NF, et al. Update
of the Anopheles gambiae PEST genome assembly.
Genome Biol. 2007;8:R5.
[32] World Health Organization. Guidance framework for test-
ing of genetically modied mosquitoes. 2nded. Geneva
Switzerland: Licence: CC BY-NC-SA 3.0 IGO.2021. https://
www.who.int/publications/i/item/9789240025233
PATHOGENS AND GLOBAL HEALTH 11