Amplicon_sorter: A tool for reference‐free amplicon sorting based on sequence similarity and for building consensus sequences

Vierstraete, Andy; Braeckman, Bart P.

doi:10.1002/ece3.8603

Cited by 32 publications

(20 citation statements)

References 62 publications

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“…Lastly, we assessed Nanopore performance at producing accurate consensus full-length amplicon sequences, focusing on csp and msp1 in the Validation samples. The Amplicon_sorter tool [80] was used to produce consensus sequences with a similarity threshold of 96% (details in Methods ). Consensus sequences produced from reads in the expected ~5Kb size range of the msp1 amplicon (covering almost the entire msp1 gene) had 100% base perfect mapping back to the reference sequences for all of the laboratory clones tested.…”

Section: Resultsmentioning

confidence: 99%

Section: Antigens and Vaccine Targetsmentioning

confidence: 99%

“…Consensus sequences for csp and msp1 were produced for the laboratory clones using Amplicon_sorter [80], a tool for building reference-free consensus sequences using ONT sequenced amplicons based on read similarity and length. Reads mapping to the 3D7 csp reference sequence were extracted and used as input for Amplicon_sorter using a similarity cut-off of 96% (the threshold for merging sequences to generate consensus).…”

Section: Consensus Sequence Generation For Csp and Msp1mentioning

confidence: 99%

See 2 more Smart Citations

Nanopore sequencing for real-time genomic surveillance ofPlasmodium falciparum

Girgis

Adika

Nenyewodey

et al. 2022

Preprint

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Malaria is a global public health priority causing over 600,000 deaths annually, mostly young children living in Sub-Saharan Africa. Molecular surveillance can provide key information for malaria control, such as the prevalence and distribution of antimalarial drug resistance. However, genome sequencing capacity in endemic countries can be limited. Here, we have implemented an end-to-end workflow for Plasmodium falciparum genomic surveillance in Ghana using Oxford Nanopore Technologies, targeting antimalarial resistance markers and the leading vaccine antigen circumsporozoite protein (csp). The workflow was rapid, robust, accurate, affordable and straightforward to implement. We found that P. falciparum parasites in Ghana had become largely susceptible to chloroquine, with persistent sulfadoxine-pyrimethamine (SP) resistance, and no evidence of artemisinin resistance. Multiple Single Nucleotide Polymorphism (SNP) differences from the vaccine csp sequence were identified, though their significance is uncertain. This study demonstrates the potential utility and feasibility of malaria genomic surveillance in endemic settings using Nanopore sequencing.

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Section: Resultsmentioning

confidence: 99%

Section: Antigens and Vaccine Targetsmentioning

confidence: 99%

Section: Consensus Sequence Generation For Csp and Msp1mentioning

confidence: 99%

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Nanopore sequencing for real-time genomic surveillance ofPlasmodium falciparum

Girgis

Adika

Nenyewodey

et al. 2022

Preprint

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“…After sequencing, all reads were demultiplexed using minibar software (), based on the specified combination of sequences of barcodes and primers at the 5′ and 3′ ends for each isolate. The predominant consensus sequence was derived for each isolate using Amplicon_sorter software …”

Section: Methodsmentioning

confidence: 99%

Highly Parallelized Construction of DNA from Low-Cost Oligonucleotide Mixtures Using Data-Optimized Assembly Design and Golden Gate

Lund,

Potapov,

Johnson

et al. 2024

ACS Synth. Biol.

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Commercially synthesized genes are typically made using variations of homology-based cloning techniques, including polymerase cycling assembly from chemically synthesized microarray-derived oligonucleotides. Here, we apply Data-optimized Assembly Design (DAD) to the synthesis of hundreds of codonoptimized genes in both constitutive and inducible vectors using Golden Gate Assembly. Starting from oligonucleotide pools, we synthesize genes in three simple steps: (1) amplification of parts belonging to individual assemblies in parallel from a single pool;(2) Golden Gate Assembly of parts for each construct; and (3) transformation. We construct genes from receiving DNA to sequence confirmed isolates in as little as 4 days. By leveraging the ligation fidelity afforded by T4 DNA ligase, we expect to be able to construct a larger breadth of sequences not currently supported by homology-based methods, which require stability of extensive single-stranded DNA overhangs.

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“…Basecalling and demultiplexing was carried out on the GridION sequencing device (ONT) using Guppy (v2.24-r1122) on super accurate mode, specifying --require_barcodes_both_ends and using appropriate super accurate basecalling models for each of the different sequencing methods used. Following basecalling, amplicon_sorter 53 (v2023-06-19) was used to identify consensus amplicons between 3kb and 5kb without using a reference. Spike amplicons were identified from the output via alignment with minimap2 54 (v2.22) to the spike from the first sample we sequenced (Cypriot_G7_Gi_6739) for which a consensus was made using the same process, with the correct amplicon identified via BLAST 55 .…”

Section: Methodsmentioning

confidence: 99%

Emergence and spread of feline infectious peritonitis due to a highly pathogenic canine/feline recombinant coronavirus

Attipa,

Warr,

Epaminondas

et al. 2023

Preprint

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Cross-species transmission of coronaviruses (CoVs) poses a serious threat to both animal and human health1-3. Whilst the large RNA genome of CoVs shows relatively low mutation rates, recombination within genera is frequently observed and demonstrated4-7. Companion animals are often overlooked in the transmission cycle of viral diseases; however, the close relationship of feline (FCoV) and canine CoV (CCoV) to human hCoV-229E5,8, as well as their susceptibility to SARS-CoV-29highlight their importance in potential transmission cycles. Whilst recombination between CCoV and FCoV of a large fragment spanning orf1b to M has been previously described5,10, here we report the emergence of a novel, highly pathogenic FCoV-CCoV recombinant responsible for a rapidly spreading outbreak of feline infectious peritonitis (FIP), originating in Cyprus11. The recombination, spanning spike, shows 97% sequence identity to the pantropic canine coronavirus CB/05. Infection is spreading fast and infecting cats of all ages. Development of FIP appears rapid and likely non-reliant on biotype switch12. High sequence identity of isolates from cats in different districts of the island is strongly supportive of direct transmission. A deletion and several amino acid changes in spike, particularly the receptor binding domain, compared to other FCoV-2s, indicate changes to receptor binding and likely cell tropism.

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Amplicon_sorter: A tool for reference‐free amplicon sorting based on sequence similarity and for building consensus sequences

Cited by 32 publications

References 62 publications

Nanopore sequencing for real-time genomic surveillance ofPlasmodium falciparum

Nanopore sequencing for real-time genomic surveillance ofPlasmodium falciparum

Highly Parallelized Construction of DNA from Low-Cost Oligonucleotide Mixtures Using Data-Optimized Assembly Design and Golden Gate

Emergence and spread of feline infectious peritonitis due to a highly pathogenic canine/feline recombinant coronavirus

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