NGSpeciesID: DNA barcode and amplicon consensus generation from long‐read sequencing data

Sahlin, Kristoffer; Lim, Marisa; Prost, Stefan

doi:10.1002/ece3.7146

Cited by 62 publications

(65 citation statements)

References 23 publications

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“…We further compared the barcodes obtained with ONTbarcoder with those obtained via the recent software NGSpeciesID [56]. NGSpeciesID often provides multiple consensus barcodes for the same set of reads for a specimen.…”

Section: Resultsmentioning

confidence: 99%

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MinION barcodes: biodiversity discovery and identification by everyone, for everyone

Srivathsan

Lee

Katoh

et al. 2021

Preprint

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DNA barcodes are a useful tool for discovering, understanding, and monitoring biodiversity. This is critical at a time when biodiversity loss is a major problem for many countries. However, widespread adoption of barcoding programs requires the process to be cost-effective and simple to apply. We here present a workflow that satisfies these conditions. It was developed via 'innovation through subtraction' and thus requires minimal lab equipment, can be learned within days, reduces the barcode sequencing cost to <10 cents, and allows fast turnaround from specimen to sequence by using the real-time sequencer MinION. We first describe cost-effective and rapid procedures in a comprehensive workflow for obtaining tagged amplicons. We then demonstrate how a portable MinION device can be used for real-time sequencing of tagged amplicons in many settings (field stations, biodiversity labs, citizen science labs, schools). Small projects can use the flow cell dongle ('Flongle') while large projects can rely on MinION flow cells that can be stopped and re-used after collecting sufficient data for a given project. We also provide amplicon coverage recommendations that are based on several runs of MinION flow cells (R10.3) involving >24,000 specimen barcodes, which suggest that each run can generate >10,000 barcodes. Additionally, we present a novel software, ONTbarcoder, that overcomes the bioinformatics challenges posed by the sequencing errors of MinION reads. This software is compatible with Windows10, Macintosh, and Linux, has a graphical user interface (GUI), and can generate thousands of barcodes on a standard laptop within hours based on two input files (FASTQ, demultiplexing file). Next, we document that MinION barcodes are virtually identical to Sanger and Illumina barcodes for the same specimens (>99.99%). Lastly, we demonstrate how rapidly MinION data have improved by comparing the performance of sequential flow cell generations. We overall assert that barcoding with MinION is the way forward for government agencies, universities, museums, and schools because it combines low consumable and capital cost with scalability. Biodiversity loss is threatening the planet and the use of MinION barcodes will help with enabling an army of researchers and citizen scientists, which is necessary for effective biodiversity discovery and monitoring.

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Section: Resultsmentioning

confidence: 99%

“…MinION has been used for barcoding fungi, animals, and plants and alternative pipelines have been developed [36, 54, 56, 60–66], but there is one fundamental difference between these studies/pipelines and the vision presented here. These studies tended to show that MinION sequencing can be done in the field.…”

Section: Discussionmentioning

confidence: 99%

MinION barcodes: biodiversity discovery and identification by everyone, for everyone

Srivathsan

Lee

Katoh

et al. 2021

Preprint

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“…There are a variety of tools and pipelines published for the analysis of DNA barcoding data generated with the MinION sequencer (e.g. Consesion [17], ONTrack [18], SAIGA [12], NGSpeciesID [19]). In the first step of most applications using MinION data, the raw signal from the sequencer has to be transformed into nucleotide sequences.…”

Section: Applications To Species Identificationmentioning

confidence: 99%

“…Alternatively, to demultiplex libraries based on ONT or custom design barcodes, tools such as minibar [26] are available. After demultiplexing, reads below a certain quality score can be removed with programmes such as NanoFilt [29] or within consensus generation software tools such as NGSpeciesID [19].…”

Section: Data Processing and Bioinformatic Considerationsmentioning

confidence: 99%

“…In the first validation of MinION sequencing data for molecular genetic non-human forensic species identification, Vasiljevic et al [35] examined the NGSpeciesID software [19] for the generation of consensus sequences for single-donor samples. In its newest version, this software includes all necessary steps (such as quality and length filtering, read subsampling and clustering, consensus generation, primer removal and polishing) that are needed to generate reliable consensus sequences and is thus easier to use than other available software solutions (https://github.com/ksahlin/NGSpeciesID).…”

Section: Opportunities and Current Research In Non-human Forensicsmentioning

confidence: 99%

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Nanopore sequencing in non-human forensic genetics

Ogden

Vasiljevic

Prost

2021

Emerging Topics in Life Sciences

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The past decade has seen a rapid expansion of non-human forensic genetics coinciding with the development of 2nd and 3rd generation DNA sequencing technologies. Nanopore sequencing is one such technology that offers massively parallel sequencing at a fraction of the capital cost of other sequencing platforms. The application of nanopore sequencing to species identification has already been widely demonstrated in biomonitoring studies and has significant potential for non-human forensic casework, particularly in the area of wildlife forensics. This review examines nanopore sequencing technology and assesses its potential applications, advantages and drawbacks for use in non-human forensics, alongside other next-generation sequencing platforms and as a possible replacement to Sanger sequencing. We assess the specific challenges of sequence error rate and the standardisation of consensus sequence production, before discussing recent progress in the validation of nanopore sequencing for use in forensic casework. We conclude that nanopore sequencing may be able to play a considerable role in the future of non-human forensic genetics, especially for applications to wildlife law enforcement within emerging forensic laboratories.

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Complete pipeline for Oxford Nanopore Technology amplicon sequencing (ONT‐AmpSeq): from pre‐processing to creating an operational taxonomic unit table

Schacksen,

Østergaard,

Eskildsen

et al. 2024

FEBS Open Bio

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Amplicon sequencing has long served as a robust method for characterising microbial communities, and despite inherent resolution limitations, it remains a preferred technique, offering cost‐ and time‐effective insights into bacterial compositions. Here, we introduce ONT‐AmpSeq, a user‐friendly pipeline designed for processing amplicon sequencing data generated from Oxford Nanopore Technology (ONT) devices. Our pipeline enables efficient creation of taxonomically annotated operational taxonomic unit (OTU) tables from ONT sequencing data, with the flexibility to multiplex amplicons on the same barcode. The pipeline encompasses six main steps—statistics, quality filtering, alignment, clustering, polishing, and taxonomic classification—integrating various state‐of‐the‐art software tools. We provide a detailed description of each step, along with performance tests and robustness evaluations using both test data and a ZymoBIOMICS® Microbial Community Standard mock community dataset. Our results demonstrate the ability of ONT‐AmpSeq to effectively process ONT amplicon data, offering valuable insights into microbial community composition. Additionally, we discuss the influence of polishing tools on taxonomic insight and the impact of taxonomic annotation methods on the derived microbial composition. Overall, ONT‐AmpSeq represents a comprehensive solution for analysing ONT amplicon sequencing data, facilitating streamlined and reliable microbial community analysis. The pipeline, along with test data, is freely available for public use.

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NGSpeciesID: DNA barcode and amplicon consensus generation from long‐read sequencing data

Cited by 62 publications

References 23 publications

MinION barcodes: biodiversity discovery and identification by everyone, for everyone

MinION barcodes: biodiversity discovery and identification by everyone, for everyone

Nanopore sequencing in non-human forensic genetics

Complete pipeline for Oxford Nanopore Technology amplicon sequencing (ONT‐AmpSeq): from pre‐processing to creating an operational taxonomic unit table

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