Pruning the tree: comparing OTUs and ASVs for processing High-Throughput Sequencing 5S-IGS nuclear ribosomal DNA in phylogenetic studies – online supplementary data repository

We perfomed HTS Illumina Sequencing of the nuclear 5S rDNA intergenic spacers to address taxonomy, diversity and evolution in Fagus (Fagaceae). We compared Operational Taxonomic Units (OTUs) and Amplicon Sequence Variants (ASVs) of seven species-samples (F. caspica Denk & G.W.Grimm, F. crenata Blume, F. hayatae Palib., F. hohenackeriana Palib., F. japonica Maxim., F. orientalis Lipsky, F. sylvatica L.) obtained with MOTHUR (two different workflows) and DADA2 (three different pooling options). We assessed the capacity of the derived phylogenetic inferences to enclose pivotal 5S-IGS variant types across methods. Over 70% of processed reads were shared between OTUs (100% identity; cutoff abundance >4) and ASVs. Despite a strong reduction (>80%) of the representative sequences, DADA2-ASVs identified all main 5S-IGS variants known for Fagus, fully reflecting the overall genetic diversity patterns within each sample. In contrast, large proportions of low-abundant representative amplicons appeared in MOTHUR-OTUs and -ASVs profiles and were inference-wise redundant. The higher convenience and reliability of DADA2 datasets were confirmed by further comparison with MOTHUR-datasets generated with cutoff abundance >25.This fileset includes basic information on the produced data, the used alignments and resultant inference files.Supplementary Data Archive (SDA)—zipped analysis files, see _content.txt for archive's content and naming conventionsFile S1—multiple spread-sheet (XLSX) file listing the abundance of detected OTU and ASV representative sequence setsFile S2—multiple spread-sheet (XLSX) file summarising the inference difficulty scores and non-parametric bootstrap supports for (alternative) topological aspects seen in all inferred ML treesFile S3–PDF with graphically enhanced versions of the inferred per-sample ML trees (subtrees representing known (Cardoni et al. 2022, The Plant Journal 109:909–926; Denk et al. 2024, Willdenowia 54:151–181), and new 5S-IGS main types (F. hayatae sample) collapsed