Nanopore sequencing of PCR products enables multicopy gene family reconstruction

Namias, Alice; Sahlin, Kristoffer; Makoundou, Patrick; Bonnici, Iago; Sicard, Mathieu; Belkhir, Khalid; Weill, Mylène

doi:10.1016/j.csbj.2023.07.012

Cited by 4 publications

(16 citation statements)

References 49 publications

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“…To identify the different haplotypes present in the ace-1 PCR products of D i carriers (i.e. “triple-peak” individuals), we used two approaches: (i) Sanger sequencing, which requires a preliminary TA cloning step to provide individual haplotypes from the mixed products, and (ii) an approach initially developed to assess the diversity of Wolbachia cid genes multigenic family, which is less tedious and more sensitive than TA cloning (Namias et al 2023 ). In this approach, the PCR product is directly sequenced using Nanopore long-reads: each read then corresponds to one individual haplotype.…”

Section: Methodsmentioning

confidence: 99%

“…By cloning and sequencing a large part of the ace-1 locus for several individuals, we obtained the various haplotypes of each of their D(R) and D(S) copies. We also tested a more recently developed, and logistically easier approach, based on long-read sequences of PCR products (Namias et al 2023 ). We revealed that at least nine different ace-1 D alleles segregate in these two populations.…”

Section: Introductionmentioning

confidence: 99%

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Despite structural identity, ace-1 heterogenous duplication resistance alleles are quite diverse in Anopheles mosquitoes

Claret,

Di-Liegro,

Namias

et al. 2024

Heredity

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Anopheles gambiae s.l. has been the target of intense insecticide treatment since the mid-20th century to try and control malaria. A substitution in the ace-1 locus has been rapidly selected for, allowing resistance to organophosphate and carbamate insecticides. Since then, two types of duplication of the ace-1 locus have been found in An. gambiae s.l. populations: homogeneous duplications that are composed of several resistance copies, or heterogeneous duplications that contain both resistance and susceptible copies. The substitution induces a trade-off between resistance in the presence of insecticides and disadvantages in their absence: the heterogeneous duplications allow the fixation of the intermediate heterozygote phenotype. So far, a single heterogeneous duplication has been described in An. gambiae s.l. populations (in contrast with the multiple duplicated alleles found in Culex pipiens mosquitoes). We used a new approach, combining long and short-read sequencing with Sanger sequencing to precisely identify and describe at least nine different heterogeneous duplications, in two populations of An. gambiae s.l. We show that these alleles share the same structure as the previously identified heterogeneous and homogeneous duplications, namely 203-kb tandem amplifications with conserved breakpoints. Our study sheds new light on the origin and maintenance of these alleles in An. gambiae s.l. populations, and their role in mosquito adaptation.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Despite structural identity, ace-1 heterogenous duplication resistance alleles are quite diverse in Anopheles mosquitoes

Claret,

Di-Liegro,

Namias

et al. 2024

Heredity

Self Cite

View full text Add to dashboard Cite

show abstract

“…To identify the different haplotypes present in the ace-1 PCR products of D i carriers (i.e. "triple-peak" individuals), we used two approaches: i) classic Sanger sequencing, which requires a preliminary TA cloning step to provide individual haplotypes from the mixed products, and ii) an approach initially developed to assess the diversity of Wolbachia cid genes multigenic family, which is less tedious and more sensitive than TA cloning (Namias et al, 2023). In this approach, the PCR product is directly sequenced using Nanopore long-reads: each read then corresponds to one individual haplotype.…”

Section: Heterogeneous Duplication Detection and Frequency Estimationmentioning

confidence: 99%

“…As mentioned in Namias et al ( 2023), some heterozygous SNPs on the S haplotypes were not called: although they were supported by a high number of reads, the read distribution did not t with a diploid framework (with only two S copies). We used the script provided by Namias et al (2023) to recover those SNPs.…”

Section: Heterogeneous Duplication Detection and Frequency Estimationmentioning

confidence: 99%

“…By cloning and sequencing a large part of the ace-1 locus for several individuals, we obtained the various haplotypes of each of their D(R) and D(S) copies. We also tested a more recently developed, and logistically easier approach, based on long-read sequences of PCR products (Namias et al, 2023). We revealed that at least nine different ace-1 D alleles segregate in these two populations.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Despite structural identity, ace-1 heterogenous duplication resistance alleles are quite diverse in Anopheles mosquitoes

Labbé,

Claret,

Di-Liegro

et al. 2023

Preprint

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Anopheles gambiae s.l. has been the target of intense insecticide treatment since the mid-XXth century to try and control malaria. A substitution in the ace-1 locus has been rapidly selected for, allowing resistance to organophosphate and carbamate insecticides. Since then, two types of duplication of the ace-1 locus have been found in An. gambiae s.l. populations: homogeneous duplications that are composed of several resistance copies, or heterogeneous duplications that contain both resistance and susceptible copies. Heterogeneous duplications confer a permanent intermediate trade-off between the resistance induced by the mutation in the presence of insecticides and the disadvantages it incurs in their absence. So far, a single heterogeneous duplication has been described in An. gambiae s.l. populations (in contrast with the multiple duplicated alleles found in Culex pipiens mosquitoes). We used an innovative approach, combining long and short read sequencing with Sanger sequencing to precisely identify and describe at least nine different heterogeneous duplications, in two populations of An. gambiae s.l.. We show that these alleles share the same structure as the previously identified heterogeneous and homogeneous duplications, namely 203-kb tandem amplifications with conserved breakpoints. Our study sheds new light on the origin and maintenance of these alleles in An. gambiae s.l. populations, and their role in mosquito adaptation.

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The Genetic Puzzle of Multicopy Genes: Challenges and Troubleshooting

Partida,

Dias,

Portes

et al. 2024

Preprint

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Background: Studies with multicopy genes impose challenges related to gene redundancy and sequence similarity among copies. Recent advances in molecular biology and genomics tools associated with dedicated databases facilitate their study. Thus, the present work emphasizes the need for rigorous methodologies and standardized approaches to interpret RT-qPCR results accurately. Results: The present work in Physcomitrium patens provides a comprehensive five-step protocol, using thiamine thiazole synthase (THI1) and sucrose 6-phosphate phosphohydrolase (S6PP) genes as proof of concept, to showcase a systematic workflow for studying multicopy genes. Beyond examining genes of interest, we highlight the critical role of choosing appropriate internal controls in the analytical process for accurately interpreting gene expression patterns. We emphasized the importance of identifying the most relevant orthologous gene, recognizing the inherent challenges in determining the most functional copy for subsequent studies. Our objective is to enhance comprehension of gene redundancy by dissecting multicopy genes' genomic landscape and its characteristics. Furthermore, we address the decision-making process surrounding the quantification of expression levels of multicopy genes. Conclusions: The study of multicopy genes discloses early events of functional adaptation. We emphasize the significance of multicopy genes in plant biology and provide a practical protocol for their study. Plant systems are strongly influenced by light/dark cycles, and the challenges inherent to this process are acknowledged. In conclusion, our comprehensive approach aims to advance the understanding of multicopy gene dynamics, offering practical methodologies and contributing valuable insights to the scientific community.

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Nanopore sequencing of PCR products enables multicopy gene family reconstruction

Cited by 4 publications

References 49 publications

Despite structural identity, ace-1 heterogenous duplication resistance alleles are quite diverse in Anopheles mosquitoes

Despite structural identity, ace-1 heterogenous duplication resistance alleles are quite diverse in Anopheles mosquitoes

Despite structural identity, ace-1 heterogenous duplication resistance alleles are quite diverse in Anopheles mosquitoes

The Genetic Puzzle of Multicopy Genes: Challenges and Troubleshooting

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