3.3. Comparing general diversity
As expected, the taxonomic assignment led to different outcomes depending on the analyses, as metabarcoding results were presented as OTUs, CBH-long results as reconstructed sequences, and CHB-short results as grouped (not clustered) sequences for the same taxa. Interestingly, the detection trends of taxa and phyla among the methods varied across the three analyzed kingdoms, Archaea, Bacteria and Eukaryota (synoptic visualization in Fig 3, sample-wise data listed in Appendix 5).
In the case of Archaea CHB-long, a slightly higher number of taxa was revealed than with CBH-short or MTB, but with one missing phylum (DPANN group). Most of this difference came from the Tack group, with 419 taxa identified by CBH-short, 869 taxa identified using long sequences and only 166 taxa identified with MTB. For bacteria, CBH shows generally higher diversity detection; CBH-short detected most phyla, with 18 and 11 using CBH-long and 12 with MTB. Some rare phyla were only detected by CBH-long or MTB, but all were detected by CBH-short. Additionally, over 1200 more taxa were revealed via full-length barcodes than with OTUs by MTB. More importantly was the difference in the displayed diversity itself; over 50 percent of bacteria identified with CBH belonged to Proteobacteria (3588 taxa with CBH-long; 10520 taxa with CBH short), and only 25% (1228 taxa) belonged to Proteobacteria with MTB.
In contrast, using CBH for eukaryotes showed opposite trends. Here, most detections of phyla and other taxa were obtained by MTB. Overall, less than 2% of the reconstructed fragments (n=267) were identified compared to those with metabarcoding (OTU n=13794), and only six out of 18 phyla were identified, whereas CBH-short still detected 14 phyla (n=4571). However, these newly designed 18S probes mostly focused on metazoans, as part of Opisthokonta; here, CBH showed a relatively higher percentage of detections. With a total of 2527 taxa identified using metabarcoding, 2089 were CBH short, and 181 were full-length sequences. Finally, the overall identification and displayed biodiversity varied in the amount and abundance of phyla, depending on the kingdom/gene region as well as the analytical method.