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Comparison between COI Metabarcoding and Reverse Metabarcoding with Nanopore’s MinION
University of Skövde, School of Bioscience.
2024 (English)Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
Abstract [en]

Metabarcoding have emerged as a vital tool for assessing biodiversity and early detecting invasive species. It is a technique where a certain marker gene, or barcode, is amplified from a DNA sample, sequenced and subsequently classified by comparisons to known reference genomes. This allows for identification of species by sampling water, soil, faeces etc. Compared to the traditional visual surveying, it is more cost and time efficient, in addition to scalable. One other benefit is the potential of discovering species that are elusive, scarce or too small or difficult to identify. In the recent years, the Illumina sequencing platform has been used with high accuracy, and has produced very reliable results. The Oxford Nanopore Technologies third generation sequencing platform allows for the sequencing of very long reads, although at a higher error rate. The aim of this project was to compare COI metabarcoding from Illumina sequencing to that of COI metabarcoding using the longread sequencer MinION from Oxford Nanopore Technologies. The comparisons were made on the classification performance, regarding biodiversity metrics and number of non-indigenous species detected, and total number of species detected. A secondary aim was to evaluate the performance of a metagenomic approach, a reverse metabarcoding, where sequenced longreads are classified by multiple barcodes. The DNA in this project was received from the research group SWEDNA, and had been used in their metabarcoding study using the Illumina platform. The DNA originates from three different sample types with varying integrity. Both the gDNA and amplicons of the Folmer region were sequenced with the MinION. Three datasets were generated in total by post sequencing basecalling, one of amplicon and two of gDNA but with different levels of accuracy. These were classified using Kraken2 and a custom made database of reference sequences. The results show that amplicon sequencing is well adapted to community DNA samples of high concentration and integrity. When compared, eDNA samples are ill fitted to the same method and will most likely benefit from a metagenomic approach, preferably using multiple barcodes. The new approach of reverse metabarcoding shows promise but could be further improved and evaluated.

Place, publisher, year, edition, pages
2024. , p. 30
National Category
Bioinformatics and Computational Biology
Identifiers
URN: urn:nbn:se:his:diva-24594OAI: oai:DiVA.org:his-24594DiVA, id: diva2:1903481
External cooperation
University of Gothenburg
Subject / course
Systems Biology
Educational program
Systems Biology with specialization in Bioinformatics - Master's Programme
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Examiners
Available from: 2024-10-04 Created: 2024-10-04 Last updated: 2025-09-29Bibliographically approved

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