Nanopore sequencing in non-human forensic genetics

Ogden, Rob; Vasiljevic, Nina; Prost, Stefan

doi:10.1042/etls20200287

Cited by 9 publications

(9 citation statements)

References 42 publications

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“…While several studies have compared nanopore sequencing with NGS such as Illumina sequencing, [37][38][39] relatively few works are directed toward the comparison of MinION sequencing and Sanger sequencing. 40 The interest in using nanopore as a potential alternative sequencing tool to Sanger sequencing are primarily for amplicon-based assays, commonly used in forensic genetics or tracking species in the field. 36,40 Since Sanger and nanopore sequencing are based on completely different technologies and generate a different form of output, the comparison is not straightforward and done through the generation of a consensus sequence from nanopore read sequences.…”

Section: Discussionmentioning

confidence: 99%

“…40 The interest in using nanopore as a potential alternative sequencing tool to Sanger sequencing are primarily for amplicon-based assays, commonly used in forensic genetics or tracking species in the field. 36,40 Since Sanger and nanopore sequencing are based on completely different technologies and generate a different form of output, the comparison is not straightforward and done through the generation of a consensus sequence from nanopore read sequences. 20 Among the few comparisons being made for targeted amplicon analysis, Vasiljevic et al reported using nanopore sequencing to identify animal species via the species-diagnostic region of mitochondrial cytochrome b (mtDNA cyt b) gene with an amplicon length of approximately 421 bp.…”

Section: Discussionmentioning

confidence: 99%

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Application of nanopore sequencing for accurate identification of bacterial colonies

Marshall

Fuller

Dougall

et al. 2023

Preprint

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Culture based detection remains to be one of the most reliable and acceptable techniques to detect extremely low quantity pathogens present in a sample. The process typically involves inoculating the sample on an agar plate to allow growth of the microorganisms to form colonies, followed by the identification of the individual colonies, commonly by DNA sequencing of a PCR-amplified targeted gene. Sanger method is often the default choice of sequencing as it offers affordable and accurate results for a single species. However, the technique could pose limitations in certain situations such as identification of multi-species microbial colonies. In this work, we compared the performance of Sanger sequencing with MinION nanopore sequencing in identifying bacterial colonies derived from bioaerosol samples. We conducted Sanger and nanopore sequencing of full-length 16S rRNA genes from seven bacterial colonies derived from bioaerosol samples and compared the outcome by alignment against NCBI 16S reference database. We found that for five out of seven colonies both techniques indicated the presence of the same bacterial genus. For one of the remaining colonies, a noisy Sanger electropherogram failed to generate a meaningful sequence, but nanopore sequencing identified it to be a mix of two bacterial genera Alkalihalobacillus and Kocuria. For the other remaining colony, the Sanger sequencing suggested Micrococcus with a clean electropherogram, however, the nanopore sequencing suggested the presence of an additional genus Paraburkholderia. Further corroborating these findings with mock multispecies colonies from pure bacterial DNA samples, we confirm that nanopore sequencing is comparable to the Sanger method in identifying colonies with single bacterial species but is the superior method in classifying individual bacterial components with their relative abundances in multispecies colonies. Our results suggest that nanopore sequencing could be advantageous over Sanger sequencing for colony identification in culture-based analysis of environmental samples such as bioaerosol where direct inoculation of the sample to culture plate might lead to formation of multispecies colonies.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Application of nanopore sequencing for accurate identification of bacterial colonies

Marshall

Fuller

Dougall

et al. 2023

Preprint

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“…MinION's main disadvantage is a relatively low read accuracy compared with the highly accurate sequencing of Sanger and Illumina methods, the estimated error rate being between 5 and 15% [30][31][32] despite its constant improvement. Therefore, the analysis of ONT sequence reads is a complex task, particularly when variant detection is required.…”

Section: Discussionmentioning

confidence: 99%

Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology

Rosa

Brandtner²,

Mancini

et al. 2021

Water

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The emergence of SARS-CoV-2 variants of concern (VOCs) and variants of interest (VOIs) poses an increased risk to global public health and underlines the need to prioritise monitoring and research to better respond to the COVID-19 pandemic. Wastewater monitoring can be used to monitor SARS-CoV-2 spread and to track SARS-CoV-2 variants. A long read amplicon sequencing approach based on the Oxford Nanopore technology, targeting the spike protein, was applied to detect SARS-CoV-2 variants in sewage samples collected in central Italy on April 2021. Next-generation sequencing was performed on three pooled samples. For variant identification, two approaches–clustering (unsupervised) and classification (supervised)–were implemented, resulting in the detection of two VOCs and one VOI. Key mutations of the Alpha variant (B.1.1.7) were detected in all of the pools, accounting for the vast majority of NGS reads. In two different pools, mutations of the Gamma (P.1) and Eta (B.1.525) variants were also detected, accounting for 22.4%, and 1.3% of total NGS reads of the sample, respectively. Results were in agreement with data on variant circulation in Italy at the time of wastewater sample collection. For each variant, in addition to the signature key spike mutations, other less common mutations were detected, including the amino acid substitutions S98F and E484K in the Alpha cluster (alone and combined), and S151I in the Eta cluster. Results of the present study show that the long-read sequencing nanopore technology can be successfully used to explore SARS-CoV-2 diversity in sewage samples, where multiple variants can be present, and that the approach is sensitive enough to detect variants present at low abundance in wastewater samples. In conclusion, wastewater monitoring can help one discover the spread of variants in a community and early detect the emerging of clinically relevant mutations or variants.

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“…The other major cost reduction comes from extremely affordable nanopore sequencing, which has been proposed for use in wildlife forensics (Hall et al, 2020;Ogden et al, 2021). Although we used more flowcells to recover the 2346 barcodes than Srivathsan et al (2021), owing to the overall poor quality of our samples, each barcode still costs <USD1 to sequence.…”

Section: Rapid Genotyping With Ont Barcodes For Large Seizuresmentioning

confidence: 99%

Uncovering the magnitude of African pangolin poaching with extensive nanopore DNA genotyping of seized scales

Yeo,

Chan,

Hiong

et al. 2023

Conservation Biology

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Pangolins are among the most highly trafficked animals, with shipments of pangolin scales and products weighing several metric tonnes being seized at various ports. These large seizures are challenging to process and comprehensively genotype for both upstream provenance tracing and species identification for prosecution. In this study, we implemented a scalable DNA barcoding pipeline that leverages rapid DNA extraction and MinION sequencing to genotype a substantial proportion of scales subsampled from two record consignments seized in Singapore in 2019 (37.5 tonnes). This yielded a total of 2,346 cytochrome b (cytb) barcodes from white‐bellied (Phataginus tricuspis; 1,091 scales), black‐bellied (Phataginus tetradactyla; 227 scales) and giant (Smutsia gigantea; 1,028 scales) pangolin scales. Clustering analyses reveal rich haplotype diversity in the white‐bellied pangolin scales (121 haplotypes, 66 novel), while the genetic diversity of black‐bellied and giant pangolin scales appear more conserved (black‐bellied: 22 haplotypes, 15 novel; giant: 25 haplotypes, 21 novel). Using reference sequences, these scales could be matched to phylogeographical regions of origin. We find a large proportion of novel haplotypes likely from Western and Western Central Africa, suggesting potential resurgence of poaching and newly‐exploited populations in these regions. Our study illustrates the utility of extensively subsampling large seizures and outlines an efficient molecular approach for rapid genetic screening that should be accessible to most forensic laboratories and enforcement agencies.This article is protected by copyright. All rights reserved

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Nanopore sequencing in non-human forensic genetics

Cited by 9 publications

References 42 publications

Application of nanopore sequencing for accurate identification of bacterial colonies

Application of nanopore sequencing for accurate identification of bacterial colonies

Key SARS-CoV-2 Mutations of Alpha, Gamma, and Eta Variants Detected in Urban Wastewaters in Italy by Long-Read Amplicon Sequencing Based on Nanopore Technology

Uncovering the magnitude of African pangolin poaching with extensive nanopore DNA genotyping of seized scales

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