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 <i>Fagus </i>(Fagaceae). We compared Operational Taxonomic Units (OTUs) and Amplicon Sequence Variants (ASVs) of seven species-samples (<i>F. caspica </i>Denk &amp; G.W.Grimm<i>, </i><i>F. crenata </i>Blume<i>, F. hayatae </i>Palib.<i>, </i><i>F. hohenackeriana </i>Palib.<i>, F. japonica </i>Maxim.<i>, F. orientalis </i>Lipsky<i>, F. sylvatica </i>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 &gt;4) and ASVs. Despite a strong reduction (&gt;80%) of the representative sequences, DADA2-ASVs identified all main 5S-IGS variants known for <i>Fagus</i>, 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 &gt;25.This fileset includes basic information on the produced data, the used alignments and resultant inference files.<b>Supplementary Data Archive (SDA)</b>—zipped analysis files, see _content.txt for archive's content and naming conventions<b>File S1</b>—multiple spread-sheet (XLSX) file listing the abundance of detected OTU and ASV representative sequence sets<b>File S2</b>—multiple spread-sheet (XLSX) file summarising the inference difficulty scores and non-parametric bootstrap supports for (alternative) topological aspects seen in all inferred ML trees<b>File S3</b>–PDF with graphically enhanced versions of the inferred per-sample ML trees (subtrees representing known (Cardoni et al. 2022, <i>The Plant Journal</i> <b>109</b>:909–926; Denk et al. 2024, <i>Willdenowia </i><b>54</b>:151–181), and new 5S-IGS main types (<i>F. hayatae </i>sample) collapsed<br>