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A new long-read mitochondrial-genome protocol (PacBio HiFi) for haemosporidian parasites: a tool for population and biodiversity studies
Pacheco, M. Andreína Cepeda, Axl S. Miller, Erica A. Beckerman, Scott Oswald, Mitchell London, Evan Mateus-Pinilla, Nohra E. Escalante, Ananias A.
Pacheco, M. Andreína
Cepeda, Axl S.
Miller, Erica A.
Beckerman, Scott
Oswald, Mitchell
London, Evan
Mateus-Pinilla, Nohra E.
Escalante, Ananias A.
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Journal article
Date
2024-05-04
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Department
Biology
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DOI
https://doi.org/10.1186/s12936-024-04961-8
Abstract
Background
Studies on haemosporidian diversity, including origin of human malaria parasites, malaria's zoonotic dynamic, and regional biodiversity patterns, have used target gene approaches. However, current methods have a trade-off between scalability and data quality. Here, a long-read Next-Generation Sequencing protocol using PacBio HiFi is presented. The data processing is supported by a pipeline that uses machine-learning for analysing the reads.
Methods
A set of primers was designed to target approximately 6 kb, almost the entire length of the haemosporidian mitochondrial genome. Amplicons from different samples were multiplexed in an SMRTbell® library preparation. A pipeline (HmtG-PacBio Pipeline) to process the reads is also provided; it integrates multiple sequence alignments, a machine-learning algorithm that uses modified variational autoencoders, and a clustering method to identify the mitochondrial haplotypes/species in a sample. Although 192 specimens could be studied simultaneously, a pilot experiment with 15 specimens is presented, including in silico experiments where multiple data combinations were tested.
Results
The primers amplified various haemosporidian parasite genomes and yielded high-quality mt genome sequences. This new protocol allowed the detection and characterization of mixed infections and co-infections in the samples. The machine-learning approach converged into reproducible haplotypes with a low error rate, averaging 0.2% per read (minimum of 0.03% and maximum of 0.46%). The minimum recommended coverage per haplotype is 30X based on the detected error rates. The pipeline facilitates inspecting the data, including a local blast against a file of provided mitochondrial sequences that the researcher can customize.
Conclusions
This is not a diagnostic approach but a high-throughput method to study haemosporidian sequence assemblages and perform genotyping by targeting the mitochondrial genome. Accordingly, the methodology allowed for examining specimens with multiple infections and co-infections of different haemosporidian parasites. The pipeline enables data quality assessment and comparison of the haplotypes obtained to those from previous studies. Although a single locus approach, whole mitochondrial data provide high-quality information to characterize species pools of haemosporidian parasites.
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Citation
Pacheco, M.A., Cepeda, A.S., Miller, E.A. et al. A new long-read mitochondrial-genome protocol (PacBio HiFi) for haemosporidian parasites: a tool for population and biodiversity studies. Malar J 23, 134 (2024). https://doi.org/10.1186/s12936-024-04961-8
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Springer Science and Business Media
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Malaria Journal, Vol. 23, Iss. 1
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Attribution CC BY