Haplotype-level metabarcoding of freshwater macroinvertebrate species: a
prospective tool for population genetic analysis
Abstract
The development and evaluation of DNA metabarcoding protocols for
haplotype-level resolution require attention, specifically for
population genetic analysis, i.e., parallel estimation of genetic
diversity and dispersal patterns among multiple species present in a
bulk sample. Further exploration and assessment of the laboratory and
bioinformatics strategies are warranted to unlock the potential of
metabarcoding-inferred population genetic analysis. Here, we assessed
the inference of freshwater macroinvertebrate haplotypes from DNA
metabarcoding data using mock samples with known Sanger-sequenced
haplotypes. We also examined the influence of different DNA template
concentrations and PCR cycles on detecting true haplotypes and the
reduction of spurious haplotypes obtained from DNA metabarcoding. We
tested our haplotyping strategy on a mock sample containing 20 specimens
from four species with known haplotypes based on the 658-bp Folmer
region of the mitochondrial cytochrome c oxidase gene. The read
processing and denoising step resulted in 14 zero-radius operational
taxonomic units (ZOTUs) of 421-bp length, with 12 ZOTUs having 100%
match with 12 of the Sanger haplotype sequences. Quality passing reads
relatively increased with increasing PCR cycles, and the relative
abundance of each ZOTUs was consistent for each cycle number. This
suggests that increasing the cycle number from 24 to 64 did not affect
the relative abundance of quality passing filter reads of each ZOTUs.
Our study demonstrated the ability of DNA metabarcoding to infer
intraspecific variability while highlighting the challenges that need to
be addressed before its possible applications to population genetic
studies.