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.