{"doi":"10.1111/nph.16731","title":"Optimized metabarcoding with Pacific biosciences enables semi‐quantitative analysis of fungal communities","abstract":"<jats:title>Summary</jats:title><jats:p>\n<jats:list list-type=\"bullet\">\n\n<jats:list-item><jats:p>Recent studies have questioned the use of high‐throughput sequencing of the nuclear ribosomal internal transcribed spacer (ITS) region to derive a semi‐quantitative representation of fungal community composition. However, comprehensive studies that quantify biases occurring during PCR and sequencing of ITS amplicons are still lacking.</jats:p></jats:list-item>\n\n<jats:list-item><jats:p>We used artificially assembled communities consisting of 10 ITS‐like fragments of varying lengths and guanine‐cytosine (GC) contents to evaluate and quantify biases during PCR and sequencing with Illumina MiSeq, PacBio RS II and PacBio Sequel I technologies.</jats:p></jats:list-item>\n\n<jats:list-item><jats:p>Fragment length variation was the main source of bias in observed community composition relative to the template, with longer fragments generally being under‐represented for all sequencing platforms. This bias was three times higher for Illumina MiSeq than for PacBio RS II and Sequel I. All 10 fragments in the artificial community were recovered when sequenced with PacBio technologies, whereas the three longest fragments (&gt; 447 bases) were lost when sequenced with Illumina MiSeq. Fragment length bias also increased linearly with increasing number of PCR cycles but could be mitigated by optimization of the PCR setup. No significant biases related to GC content were observed.</jats:p></jats:list-item>\n\n<jats:list-item><jats:p>Despite lower sequencing output, PacBio sequencing was better able to reflect the community composition of the template than Illumina MiSeq sequencing.</jats:p></jats:list-item>\n</jats:list>\n</jats:p>","journal":"New Phytologist","year":2020,"id":676857,"datarank":0.6937459219926407,"base_score":4.624972813284271,"endowment":4.624972813284271,"self_citation_contribution":0.6937459219926407,"citation_network_contribution":0.0,"self_endowment_contribution":0.6937459219926407,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":101,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1768485,"name":"Anna Berlin","orcid":"0000-0002-9518-5719","position":1,"is_corresponding":false},{"id":54324,"name":"Mikael Brandström Durling","orcid":"0000-0001-6485-197X","position":2,"is_corresponding":false},{"id":1768489,"name":"Katharina Ihrmark","orcid":null,"position":3,"is_corresponding":false},{"id":840133,"name":"Björn D. 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Clemmensen","orcid":"0000-0002-9627-6428","position":6,"is_corresponding":false},{"id":1768500,"name":"Åke Olson","orcid":"0000-0001-8998-6096","position":7,"is_corresponding":false},{"id":1768482,"name":"Carles Castaño","orcid":"0000-0002-2403-7006","position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Optimized metabarcoding with Pacific biosciences enables semi‐quantitative analysis of fungal communities","abstract":"<jats:title>Summary</jats:title><jats:p>\n<jats:list list-type=\"bullet\">\n\n<jats:list-item><jats:p>Recent studies have questioned the use of high‐throughput sequencing of the nuclear ribosomal internal transcribed spacer (ITS) region to derive a semi‐quantitative representation of fungal community composition. However, comprehensive studies that quantify biases occurring during PCR and sequencing of ITS amplicons are still lacking.</jats:p></jats:list-item>\n\n<jats:list-item><jats:p>We used artificially assembled communities consisting of 10 ITS‐like fragments of varying lengths and guanine‐cytosine (GC) contents to evaluate and quantify biases during PCR and sequencing with Illumina MiSeq, PacBio RS II and PacBio Sequel I technologies.</jats:p></jats:list-item>\n\n<jats:list-item><jats:p>Fragment length variation was the main source of bias in observed community composition relative to the template, with longer fragments generally being under‐represented for all sequencing platforms. This bias was three times higher for Illumina MiSeq than for PacBio RS II and Sequel I. All 10 fragments in the artificial community were recovered when sequenced with PacBio technologies, whereas the three longest fragments (&gt; 447 bases) were lost when sequenced with Illumina MiSeq. Fragment length bias also increased linearly with increasing number of PCR cycles but could be mitigated by optimization of the PCR setup. No significant biases related to GC content were observed.</jats:p></jats:list-item>\n\n<jats:list-item><jats:p>Despite lower sequencing output, PacBio sequencing was better able to reflect the community composition of the template than Illumina MiSeq sequencing.</jats:p></jats:list-item>\n</jats:list>\n</jats:p>","is_dataset_classified":null,"base_score":4.624972813284271,"endowment":4.624972813284271,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"32531109","pmcid":null,"openalex_id":"https://openalex.org/W3006365642","authors":[],"funders":[{"funder_name":"Svenska Forskningsrådet Formas","grant_id":"grant 2011‐1747","title":null},{"funder_name":"Vetenskapsrådet","grant_id":"2015‐04411","title":null},{"funder_name":"Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning","grant_id":"unidentified","title":"unidentified"}],"total_grants":3,"fwci":6.4639,"citation_percentile":0.97971503,"influential_citations":0,"citation_trend":[{"year":2020,"count":5},{"year":2021,"count":23},{"year":2022,"count":22},{"year":2023,"count":14},{"year":2024,"count":9},{"year":2025,"count":16},{"year":2026,"count":12}],"oa_status":"hybrid","license":"cc-by-nc","oa_locations":[{"url":"https://nph.onlinelibrary.wiley.com/doi/pdfdirect/10.1111/nph.16731","host_type":"journal"},{"url":"https://nph.onlinelibrary.wiley.com/doi/pdfdirect/10.1111/nph.16731","host_type":"publisher"},{"url":"https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fnph.16731","host_type":"publisher"},{"url":"https://onlinelibrary.wiley.com/doi/pdf/10.1111/nph.16731","host_type":"publisher"},{"url":"https://onlinelibrary.wiley.com/doi/full-xml/10.1111/nph.16731","host_type":"publisher"},{"url":"https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/nph.16731","host_type":"publisher"},{"url":"https://doi.org/10.1111/nph.16731","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/32531109","host_type":"repository"},{"url":"https://pub.epsilon.slu.se/22681/1/Castano_c_et_al_210301.pdf","host_type":"repository"},{"url":"https://dx.doi.org/10.1111/nph.16731","host_type":""},{"url":"https://publications.scilifelab.se/publication/1fcb59bfe89a4aa2bc5101cd706fe80f","host_type":""}],"fields_of_study":["Plant Pathogens and Fungal Diseases","Mycorrhizal Fungi and Plant Interactions","Plant Pathogens and Resistance","0301 basic medicine","0303 health sciences","03 medical and health sciences","Base Composition","Base Sequence","High-Throughput Nucleotide Sequencing","Mycobiome","Sequence Analysis, DNA"],"mesh_terms":["Mycobiome","Base Composition","Base Sequence","Sequence Analysis, DNA","High-Throughput Nucleotide Sequencing"],"keywords":["Illumina dye sequencing","Amplicon sequencing","Biology","Amplicon","DNA sequencing","Deep sequencing","GC-content","Computational biology","Genetics","Internal transcribed spacer","Ribosomal RNA","DNA","Polymerase chain reaction","Gene","Genome","16S ribosomal RNA","ITS","High-throughput Sequencing","Pacbio","Metabarcoding","Illumina Miseq","Mock Community","Metabarcoding Biases","Base Composition","Microbiology (Microbiology in the medical area to be 30109)","Genetics (medical genetics to be 30107 and agricultural genetics to be 40402)","Base Sequence","High-Throughput Nucleotide Sequencing","Sequence Analysis, DNA","630","Mycobiome"],"sdg_mappings":[{"sdg_number":0,"sdg_label":"Life below water"}],"linked_datasets":[],"clinical_trials":[],"software_tools":[],"database_accessions":[],"source":"live","citation_network_status":"fetched"},"created_at":"2026-08-17T03:14:44.718482Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}