PMID- 33092529 OWN - NLM STAT- MEDLINE DCOM- 20210514 LR - 20210514 IS - 1471-2164 (Electronic) IS - 1471-2164 (Linking) VI - 21 IP - 1 DP - 2020 Oct 22 TI - Daring to be differential: metabarcoding analysis of soil and plant-related microbial communities using amplicon sequence variants and operational taxonomical units. PG - 733 LID - 10.1186/s12864-020-07126-4 [doi] LID - 733 AB - BACKGROUND: Microorganisms are not only indispensable to ecosystem functioning, they are also keystones for emerging technologies. In the last 15 years, the number of studies on environmental microbial communities has increased exponentially due to advances in sequencing technologies, but the large amount of data generated remains difficult to analyze and interpret. Recently, metabarcoding analysis has shifted from clustering reads using Operational Taxonomical Units (OTUs) to Amplicon Sequence Variants (ASVs). Differences between these methods can seriously affect the biological interpretation of metabarcoding data, especially in ecosystems with high microbial diversity, as the methods are benchmarked based on low diversity datasets. RESULTS: In this work we have thoroughly examined the differences in community diversity, structure, and complexity between the OTU and ASV methods. We have examined culture-based mock and simulated datasets as well as soil- and plant-associated bacterial and fungal environmental communities. Four key findings were revealed. First, analysis of microbial datasets at family level guaranteed both consistency and adequate coverage when using either method. Second, the performance of both methods used are related to community diversity and sample sequencing depth. Third, differences in the method used affected sample diversity and number of detected differentially abundant families upon treatment; this may lead researchers to draw different biological conclusions. Fourth, the observed differences can mostly be attributed to low abundant (relative abundance < 0.1%) families, thus extra care is recommended when studying rare species using metabarcoding. The ASV method used outperformed the adopted OTU method concerning community diversity, especially for fungus-related sequences, but only when the sequencing depth was sufficient to capture the community complexity. CONCLUSIONS: Investigation of metabarcoding data should be done with care. Correct biological interpretation depends on several factors, including in-depth sequencing of the samples, choice of the most appropriate filtering strategy for the specific research goal, and use of family level for data clustering. FAU - Joos, Lisa AU - Joos L AD - Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Plant Sciences Unit, Burgemeester Van Gansberghelaan 92, 9820, Merelbeke, Belgium. AD - Department of Applied Mathematics, Computer Science and Statistics, Ghent University, Krijgslaan 281, 9000, Ghent, Belgium. FAU - Beirinckx, Stien AU - Beirinckx S AD - Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Plant Sciences Unit, Burgemeester Van Gansberghelaan 92, 9820, Merelbeke, Belgium. AD - Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 71, 9052, Ghent, Belgium. AD - Center for Plant Systems Biology, VIB, Ghent, Technologiepark 71, 9052, Ghent, Belgium. FAU - Haegeman, Annelies AU - Haegeman A AD - Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Plant Sciences Unit, Burgemeester Van Gansberghelaan 92, 9820, Merelbeke, Belgium. FAU - Debode, Jane AU - Debode J AD - Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Plant Sciences Unit, Burgemeester Van Gansberghelaan 92, 9820, Merelbeke, Belgium. FAU - Vandecasteele, Bart AU - Vandecasteele B AD - Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Plant Sciences Unit, Burgemeester Van Gansberghelaan 92, 9820, Merelbeke, Belgium. FAU - Baeyen, Steve AU - Baeyen S AD - Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Plant Sciences Unit, Burgemeester Van Gansberghelaan 92, 9820, Merelbeke, Belgium. FAU - Goormachtig, Sofie AU - Goormachtig S AD - Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 71, 9052, Ghent, Belgium. AD - Center for Plant Systems Biology, VIB, Ghent, Technologiepark 71, 9052, Ghent, Belgium. FAU - Clement, Lieven AU - Clement L AD - Department of Applied Mathematics, Computer Science and Statistics, Ghent University, Krijgslaan 281, 9000, Ghent, Belgium. FAU - De Tender, Caroline AU - De Tender C AUID- ORCID: 0000-0003-4804-1381 AD - Flanders Research Institute for Agriculture, Fisheries and Food (ILVO), Plant Sciences Unit, Burgemeester Van Gansberghelaan 92, 9820, Merelbeke, Belgium. caroline.detender@ilvo.vlaanderen.be. AD - Department of Applied Mathematics, Computer Science and Statistics, Ghent University, Krijgslaan 281, 9000, Ghent, Belgium. caroline.detender@ilvo.vlaanderen.be. LA - eng GR - 12S9418N/Research Foundation-Flanders (FWO)/ GR - 151553/Research Foundation-Flanders (FWO)/ PT - Journal Article DEP - 20201022 PL - England TA - BMC Genomics JT - BMC genomics JID - 100965258 RN - 0 (Soil) SB - IM MH - Bacteria/genetics MH - Fungi/genetics MH - Humans MH - *Microbiota/genetics MH - *Soil MH - Soil Microbiology PMC - PMC7579973 OTO - NOTNLM OT - ASV OT - Metabarcoding analysis OT - OTU OT - Rhizosphere and endosphere microbiome OT - Soil COIS- The authors declare that they have no competing interests. EDAT- 2020/10/24 06:00 MHDA- 2021/05/15 06:00 PMCR- 2020/10/22 CRDT- 2020/10/23 05:31 PHST- 2020/03/20 00:00 [received] PHST- 2020/10/08 00:00 [accepted] PHST- 2020/10/23 05:31 [entrez] PHST- 2020/10/24 06:00 [pubmed] PHST- 2021/05/15 06:00 [medline] PHST- 2020/10/22 00:00 [pmc-release] AID - 10.1186/s12864-020-07126-4 [pii] AID - 7126 [pii] AID - 10.1186/s12864-020-07126-4 [doi] PST - epublish SO - BMC Genomics. 2020 Oct 22;21(1):733. doi: 10.1186/s12864-020-07126-4.