A set of nematode rRNA cistron databases and a primer assessment tool to
enable more flexible and comprehensive metabarcoding
Abstract
The ITS-2-rRNA has been particularly useful for nematode metabarcoding.
but does not resolve all phylogenetic relationships and reference
sequences are not available for many nematode species. This is a
particular issue when metabarcoding complex communities such as wildlife
parasites or terrestrial and aquatic free-living nematode communities.
We have used markerDB to produce four databases of distinct regions of
the rRNA cistron; the 18S rRNA gene, the 28S rRNA gene, the ITS-1
intergenic spacer and the region spanning ITS-1_5.8S_ITS-2. These
databases comprise 2645, 254, 13461, and 10107 unique full-length
sequences representing 1391, 204, 1837, and 1322 nematode species,
respectively. The comparative analysis illustrates the complementary
value but also reveals a better representation of Clade III, IV, and V
than Clade I and Clade II nematodes in each case. Although the ITS-1
database includes the largest number of unique full-length sequences,
the 18S rRNA database provides the widest taxonomic coverage. We also
developed, PrimerTC; a tool to assess primer sequence conservation
across any reference sequence database and have applied it to evaluate a
large number of previously published rRNA cistron primers. We identified
sets of primers that currently provide the broadest taxonomic coverage
for each rRNA marker across the nematode phylum. These new resources
will facilitate more comprehensive metabarcoding of nematode communities
using either short-read or long-read sequencing platforms. Further,
PrimerTC is available as a simple WebApp to guide or assess PCR primer
design for any genetic marker and/or taxonomic group beyond the nematode
phylum.