Jun 02, 2026
Mosquitoes DNA extraction
- Jimena Belen Raez Pereyra1
- 1Universidad Peruana Cayetano Heredia
- Mosquitoes mitogenome
Protocol Citation: Jimena Belen Raez Pereyra 2026. Mosquitoes DNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6jq21vqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 06, 2026
Last Modified: June 02, 2026
Protocol Integer ID: 316396
Keywords: mosquitoes dna extraction mosquito, individual mosquito specimen, studies in mosquito taxonomy, mosquito taxonomy, genomic dna extraction, standardized workflow for genomic dna extraction, dna extraction, mitochondrial genome sequencing, important vectors of numerous pathogen, genomic characterization essential for surveillance, weight dna suitable for downstream molecular application, genome sequencing, maximizing dna yield, evolutionary genomic, pathogen detection, dna yield, weight dna, numerous pathogen, other genomic analysis, genomic characterization, resulting dna, making accurate molecular identification, accurate molecular identification, dna
Abstract
Mosquitoes are important vectors of numerous pathogens affecting human and animal health, making accurate molecular identification and genomic characterization essential for surveillance and research. This protocol describes a standardized workflow for genomic DNA extraction from individual mosquito specimens. The procedure is designed to obtain high-quality and high-molecular-weight DNA suitable for downstream molecular applications, including PCR amplification, mitochondrial genome sequencing, whole-genome sequencing, and other genomic analyses. The protocol includes specimen preparation, tissue disruption, DNA extraction, purification, and quality assessment steps. Particular attention is given to maximizing DNA yield while minimizing degradation and contamination. The resulting DNA is compatible with both short-read and long-read sequencing platforms and can support studies in mosquito taxonomy, population genetics, vector biology, pathogen detection, and evolutionary genomics.
Materials
- BG-Sentinel traps
- Prokopack aspirators
- DNA/RNA Shield
- Stereomicroscope
- Taxonomic keys
- PBS
- Proteinase K
- Zymo Research squisher-singles
- Ethanol
- DNA Clean 26 Concentrator™-5 kit
- Qubit
- NanoDrop
- Agilent TapeStation
- Oxford Nanopore Technologies (ONT) Ligation Sequencing Kit (SQK-LSK114)
- PromethION flow cells
- Ubuntu 22.04.4 LTS
- Intel Core i9-14900K CPU
- NVIDIA GeForce RTX 4090 GPUs
- Dorado v1.0.2
- Samtools v1.22
- Minimap2 v2.28-r1209
- Flye v2.9.6
- MitoFinder v1.4.1
- DNAapler v1.2.0
- NanoPlot v1.46.2
- ModelFinder
- IQ-TREE2
- MCMC-mPTP web server
- FastANI
- MAFFT v7.526
- PAL2NAL v14
- Modkit pileup v0.5.0
- Modkit motif search v0.5.0
Before start
- Contact community leaders for permission and access.
- Prepare mosquito collection methods according to manufacturer's instructions.
- Label tubes for specimen storage.
Mosquito Processing
Mosquitoes collected were transported live to the Centro de Investigaciones de Recursos Naturales, Universidad Nacional de la Amazonía Peruana, where they were euthanized in a −80 °C freezer.
The mosquitoes was preserved in DNA/RNA Shield, each one was labelled with a unique specimen ID before being stored overnight at 4 °C and subsequently transferred to −20 °C for up to four months prior to transport to Universidad Peruana Cayetano Heredia at room temperature for downstream molecular processing.
DNA extraction
The specimens were washed three times with PBS (1×) and incubated in lysis buffer containing 20 µL of Proteinase K (20 mg/mL) and 200 µL of PBS at 55 °C for 1 h.
Then, the mosquito was squished by mechanical disruption using Zymo Research's individual squisher molecules.
An equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added and mixed by inversion 10 times.
It was centrifuged at 12000 g for 10 min at 4 °C and the aqueous upper phase was recovered in a new tube without touching the interface.
An equal volume of chloroform was added and mixed 10 times by inversion.
It was centrifuged at 12000 g for 10 min at 4 °C and the aqueous upper phase was recovered in a new tube without touching the interface.
An equal volume of 100% ethanol was added, mixed 10 times by inversion, and allowed to precipitate for 30 minutes at -70 °C.
It was centrifuged at 16000 g for 10 min at 4 °C and the ethanol was discarded.
Pellets were washed twice with 75% ethanol, air-dried, and resuspended with 24 µL of nuclease-free water.
To further improve purity, DNA was cleaned using the DNA Clean and Concentrator™-5 kit (Zymo Research) and eluted in 14 µL of nuclease-free water.
DNA concentration, purity, and integrity were assessed using Qubit, NanoDrop, and Agilent TapeStation. Samples were stored at −20 °C until further use.
Library preparation and Sequencing
The DNA inputs ranged from 40 to 1180 nanograms with fragment sizes between 5592 bp and 24138 basepairs were used to prepare the library using the Oxford Nanopore Technologies (ONT) Ligation Sequencing Kit (SQK-LSK114) following the Native DNA sequencing protocol, without fragmentation or PCR amplification.
Final libraries were loaded onto PromethION flow cells (R10.4.1) and the sequence data was analyzed using an external computer running Ubuntu 22.04.4 LTS, equipped with an Intel Core i9-14900K CPU (24 cores/32 threads), 128 GB RAM, and two NVIDIA GeForce RTX 4090 GPUs with 24 GB VRAM each.
Protocol references
[14] Manufacturer's instructions for BG-Sentinel traps
[15] Manufacturer's instructions for Prokopack aspirators
[16] Standard taxonomic keys