DOI: 10.5061/dryad.c59zw3rp8
title: A multiplex assay to detect mosquito species, bloodmeal host source and <em>Plasmodium</em> in malaria vectors using nanopore amplicon sequencing

description: Multiple species of Anopheles mosquitoes transmit malaria around the
 world. Molecular methods are often employed to confirm vector species,
 detect parasites, and determine bloodmeal host sources; these assays are
 often performed separately and can be time-consuming and expensive.
 However, in this study, we show that the Oxford Nanopore Technologies
 (ONT) MinION Sequencer offers a cost-effective and efficient alternative
 to accurately identify mosquito species, host bloodmeal sources, and
 detect parasites simultaneously in malaria vectors. We sequenced 150
 insectary-reared mosquitoes representing nine species and 150 blood-fed
 mosquitoes with one of five vertebrate blood sources. We also analyzed the
 presence of Plasmodium falciparum in 40 mosquito samples. A final combined
 assay integrated all three previously optimized assays into a single
 sequencing run, demonstrating the high throughput capability of the
 Nanopore sequencing platform. This run included 32 samples for each
 targeted amplicon, totaling 96 samples. For comparison, we sequenced all
 samples using a standard Sanger sequencing protocol. Our results showed
 that the MinION sequencing platform accurately identified all 9 mosquito
 species, five different bloodmeal hosts from the blood-fed mosquitoes up
 to 48 hours post blood feed, and detected P. falciparum in the 40 positive
 controls. The sequencing results obtained using the MinION platform
 exhibited high concordance with those from standard Sanger sequencing, as
 demonstrated by comparable similarity scores and correct mosquito species
 identification. This demonstrates that our MinION sequencing and analysis
 protocol offers a novel, highly precise, cost-effective solution for
 mosquito species identification, bloodmeal analysis, and parasite
 detection.
description: # A multiplex assay to detect mosquito species, bloodmeal host source and
 Plasmodium in malaria vectors using nanopore amplicon sequencing [Access
 this dataset on Dryad](https://doi.org/10.5061/dryad.c59zw3rp8) ##
 Description of the data and file structure The data were generated to
 support a multiplex amplicon sequencing assay designed to detect mosquito
 species, bloodmeal host source, and Plasmodium infection using Oxford
 Nanopore sequencing technology. Experimental efforts included the
 collection of 150 mosquito specimens representing nine species, DNA
 extraction, targeted amplification of species-specific, host-specific, and
 Plasmodium-specific loci, and sequencing on the Oxford Nanopore MinION
 platform. The dataset includes processed consensus sequences for mosquito
 species identification, bloodmeal source determination, and pathogen
 detection, along with associated PCR results. ## Description of the data
 and file structure 1. Consensus Sequence Files (`.txt`) Files: *
 `minION_Sequences_TB_Uploaded_gDNA_20252901.txt` *
 `minION_Sequences_TB_Uploaded_rRNA_Bloodmeal_20252101.txt` *
 `minION_Sequences_TB_Uploaded_rRNA_PLasmodium_20252101.txt` These files
 contain consensus sequences generated from Nanopore reads using ONTrack
 and Geneious Prime. Users can perform BLAST searches on these sequences to
 identify mosquito species, bloodmeal hosts, and Plasmodium infection. 2.
 PCR Results (`PCR_Data_Table_Final.csv`) This CSV file contains processed
 PCR results, including sample identifiers, target loci, and amplification
 outcomes. ## Workflow Overview: 1. Raw sequencing data were basecalled
 using Guppy to generate FASTQ files. 2. Read quality and length
 distributions were visualized using NanoPlot. 3. Consensus sequences were
 generated using ONTrack or Geneious Prime. 4. Consensus sequences were
 compared to reference databases using BLASTn for species, host, and
 Plasmodium identification. ## Access information Other publicly accessible
 locations of the data: * Consensus sequences generated in this study have
 been deposited in NCBI GenBank and are publicly accessible under their
 respective accession numbers Data was derived from the following sources:
 * Reference sequences used for mosquito species, bloodmeal host, and
 Plasmodium identification were obtained from publicly available NCBI
 GenBank records. ## Code/software All executable code associated with this
 dataset has been removed from the README and is provided as a separate
 script file to improve readability and reusability. Scripts included in
 this repository: ONTrack_pipeline.sh NanoPlot_QC.sh Script descriptions:
 NanoPlot_QC.sh This shell script generates read quality metrics and read
 length distribution plots from Oxford Nanopore FASTQ files using NanoPlot.
 The script was used to assess sequencing quality prior to downstream
 consensus sequence generation. Outputs include summary statistics and
 graphical visualizations of read length and quality. ONTrack_pipeline.sh
 This shell script implements the ONTrack pipeline for processing Oxford
 Nanopore MinION amplicon sequencing data. The script concatenates FASTQ
 files when multiple files per barcode are present, converts FASTQ files to
 FASTA format, prepares an input directory for ONTrack, activates a conda
 environment, and executes the ONTrack R-based workflow to generate
 consensus sequences and BLAST-based species identifications. Workflow
 relationship: NanoPlot quality control was performed prior to consensus
 sequence generation. FASTQ files passing quality assessment were
 subsequently processed using the ONTrack pipeline. Key software and tools
 include: * Oxford Nanopore MinKNOW (v22.12.5) – Used for raw sequencing
 data acquisition and basecalling. * Guppy (v6.4.6) – Performed basecalling
 of raw FAST5 files to generate FASTQ reads. * ONTrack (v1.2) – Used to
 generate consensus sequences from Nanopore reads. * Geneious Prime
 (v2024.1.1) – Assisted with sequence assembly, alignment, and
 visualization of consensus sequences. * NanoPlot (v1.41.0) – Visualized
 read length distributions and quality metrics. * Python (v3.11) with
 packages: * `pandas` – Data handling and organization * `matplotlib` and
 `seaborn` – Plotting and visualization * `Biopython` – Sequence
 manipulation and BLAST integration The script assumes that
 barcode-specific FASTQ files produced by Oxford Nanopore MinION sequencing
 are present in the working directory. Input files are copied into an
 ONtrack_input/ directory prior to execution. File paths referencing local
 installations of ONTrack may require modification by the user. Relative
 file paths are recommended to ensure reproducibility across computing
 environments.