Comparison of short and long-read metabarcoding sequencing: challenges and solutions for plastid read removal and microbial community exploration of seaweed samples

Comparison of short and long-read metabarcoding sequencing: challenges and solutions for plastid read removal and microbial community exploration of seaweed samples | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Molecular Ecology Resources This is a preprint and has not been peer reviewed. Data may be preliminary. 3 September 2025 V2 Latest version Share on Comparison of short and long-read metabarcoding sequencing: challenges and solutions for plastid read removal and microbial community exploration of seaweed samples Authors : Coralie Rousseau 0009-0005-8568-3449 [email protected] , Nicolas Henry 0000-0002-7702-1382 , Sylvie Rousvoal , Gwenn Tanguy 0009-0003-0444-3810 , Erwan Legeay , Catherine Leblanc 0000-0001-7149-0332 , and Simon Dittami 0000-0001-7987-7523 Authors Info & Affiliations https://doi.org/10.22541/au.172675397.78191661/v2 Published Molecular Ecology Resources Version of record Peer review timeline 904 views 459 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Short-read metabarcoding analysis is the gold standard to access to partial 16S and ITS genes with high read quality. With the advent of long-read sequencing, the amplification of full-length target genes is possible but with low read accuracy. Moreover, the amplification of 16S rDNA genes in seaweed or plant samples results in a large proportion of plastid reads, which are directly or indirectly derived from cyanobacteria. Primers designed not to amplify plastid sequences are available for short-read sequencing, while Oxford Nanopore Technology offers adaptive sampling, a unique way to remove reads in real-time. In this study, we compare three options to address the plastid read issue: deleting plastid reads with adaptative sampling, using optimized primers with Illumina MiSeq technology, and sequencing large numbers of reads with Illumina NovaSeq technology with universal primers. We showed that adaptive sampling using default settings of the MinKNOW software was ineffective for plastid depletion. We also demonstrated with a mock community that the SAMBA workflow provided the most accurate taxonomic assignment at the bacterial genus level compared to the IDTAXA and KRAKEN2 pipelines, but many false positives were generated at species level. Although NovaSeq sequencing with universal primer stood out for studying the algal bacterial community due to its deep coverage, the inclusion of eukaryotes and bacteria in the same sequencing run, and the low error rate. The combination of Illumina and ONT sequencing helped us explore the fungal diversity and allowed for the retrieval taxonomic information for genera poorly represented in the sequence databases. Supplementary Material File (c_rousseau_2024_comparison_sequencing_seaweeds.pdf) Download 3.79 MB Information & Authors Information Version history V1 Version 1 19 September 2024 V2 Version 2 03 September 2025 Peer review timeline Published Molecular Ecology Resources Version of Record 4 Jun 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Collection Molecular Ecology Resources Keywords adaptive sampling bacteria fungi illumina macroalgae oxford nanopore technology Authors Affiliations Coralie Rousseau 0009-0005-8568-3449 [email protected] Integrative Biology of Marine Models (LBI2M, UMR 8227), Station Biologique de Roscoff, Sorbonne Université, CNRS, 29680 Roscoff, France View all articles by this author Nicolas Henry 0000-0002-7702-1382 ABiMS, FR2424, Station Biologique de Roscoff, Sorbonne Université, CNRS, 29680 Roscoff, France View all articles by this author Sylvie Rousvoal Integrative Biology of Marine Models (LBI2M, UMR 8227), Station Biologique de Roscoff, Sorbonne Université, CNRS, 29680 Roscoff, France View all articles by this author Gwenn Tanguy 0009-0003-0444-3810 Genomer Platform, FR2424, Station Biologique de Roscoff, Sorbonne Université, CNRS, 29680 Roscoff, France View all articles by this author Erwan Legeay Genomer Platform, FR2424, Station Biologique de Roscoff, Sorbonne Université, CNRS, 29680 Roscoff, France & Adaptation and Diversity in the Marine Environment (UMR 7144), Station Biologique de Roscoff, Sorbonne Université, CNRS, 29680 Roscoff, France View all articles by this author Catherine Leblanc 0000-0001-7149-0332 Integrative Biology of Marine Models (LBI2M, UMR 8227), Station Biologique de Roscoff, Sorbonne Université, CNRS, 29680 Roscoff, France View all articles by this author Simon Dittami 0000-0001-7987-7523 Integrative Biology of Marine Models (LBI2M, UMR 8227), Station Biologique de Roscoff, Sorbonne Université, CNRS, 29680 Roscoff, France View all articles by this author Metrics & Citations Metrics Article Usage 904 views 459 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Coralie Rousseau, Nicolas Henry, Sylvie Rousvoal, et al. Comparison of short and long-read metabarcoding sequencing: challenges and solutions for plastid read removal and microbial community exploration of seaweed samples. Authorea . 03 September 2025. DOI: https://doi.org/10.22541/au.172675397.78191661/v2 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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