Experimental evaluation of species and genetic variability based on DNA metabarcoding from the aquatic environment: extra OTUs formed by NUMTS may reduce the diversity of ASVs
Authors: Turanov Sergei , Rutenko Olesia , Koltsova Marina 
Published: November 2022

The data on the intraspecific genetic variation for monitoring and
conservation of wild populations is an important link for the assessment
of the organisms resistance to changing environmental conditions and
anthropogenic pressures. The metabarcoding of DNA from the aquatic
environment provides a gradual transition to non-invasive methods of
biodiversity research, including within-species level. However, the
degradation of DNA under UV light in the aquatic environment limits the
choice of markers in favor of short standardized regions. Hence, the
consequences of information loss when shifting from barcode to
metabarcode are not entirely clear. The efforts on approbation and
calibration at the intraspecies level under experimental conditions are
limited to molecular genetic markers designed for target species. In
this study, we aimed to address these challenges: to assess the
intraspecific variation in different taxa based on the COI
barcode reduced to Leray region (~313bp), accessible
from the GenBank, as well as experimentally evaluate the possibility to
identify Operational Taxonomic Units (OTUs) and Amplicon Sequence
Variant (ASVs) in marine eDNA among abundant species of the
Zostera sp. community in the northern Sea of Japan:
Hexagrammos octogrammus, Pholidapus dybowskii (Teleostei:
Perciformes), and Pandalus latirostris (Arthropoda: Decapoda).
The three abovementioned species were collected at two distant locations
in the Great Bay of the Japan Sea and placed into a separate 150-liter
aquaria to produce both – individual and mock communities eDNA samples.
Then all individuals were euthanized and genotyped individually for 650
bp and 313 bp COI gene regions. The COI Leray region was
amplified based on the eDNA of mock communities and individual
specimens. The resulting amplicons were sequenced on the Illumina 250 bp
pair-end platform and processed based on the Begum pipeline. Along with
the OTUs based on both global and local references we tried to retrieve
individual haplotypes from the obtained reads. We found that eDNA
samples from the experiment when blasting on local reference produce
additional OTUS which we consider to be NUMTS. Surprisingly, the
presence of NUMTS in the eDNA samples reduces the detection of ASVs,
which may be related both to the low sequencing coverage in the
experiment and probably to the natural competition of pseudogenes for
primer binding sites during amplification. Perhaps a PCR-free,
metagenomic approach, despite poor accessibility, might solve these
difficulties. In addition, we have gathered and analyzed natural water
samples from one of the sample locations of Zostera sp. community
with a little sequence coverage and failed to retrieve any reliable
information about OTUs and ASVs of taxa in mock communities, which may
indicate much higher biomass of non-target organisms in the studied
community.

A total of 90 sequence data sets were collected for some common groups
of multicellular organisms (Mollusca, Echinodermata, Crustacea,
Polychaeta and Actinopterygii) through the search on the mitochondrial
COI gene in the popset database of the NCBI. The separate sets of
sequences of Leray region were generated. Then, the values of haplotypic
variability, as well as the number of population clusters of the same
dataset were calculated for the region of original length and Leray
region. The produced results reflect the decrease of population
diversity by 1 cluster in average while switching from barcode to
metabarcode. In addition we found that the length of the Leray fragment
can vary in the Echinoderms.