Elongation arrest blocking primers enhance parasite detection in 18S
rRNA metabarcoding study of Mesonauta festivus gut eukaryotic
communities.
Abstract
The study of the eukaryotic fraction of the microbiota using a
metabarcoding approach is usually hindered by the high host to
eukaryotic microbiota DNA ratio in samples. Indeed, the 18S rRNA gene is
very similar for both the host and its eukaryotic communities, leading
to a preferential amplification of the predominant host DNA when using
universal primers. Multiple approaches have been developed to reduce
host DNA amplification. One method is based on elongation arrest
blocking primers, oligonucleotides modified with a C3 Spacer that stops
the advancement of the DNA polymerase at non-conserved regions of a
target gene. In this paper, we successfully developed and tested
species-specific elongation arrest blocking primers to block the Flag
cichlid, Mesonauta festivus, 18S rRNA SSU. Our elongation arrest
blocking primers significantly reduced the amount of host DNA amplicons
by 66 %. In addition to reducing the amount of sequencing wasted, the
blocking primers increased the detectability of potentially dangerous
parasitic taxa in fish gut, highlighting the potential of the method for
parasitic screening. For instance, we discovered a case of infection by
the parasitic ciliate Nyctotherus sp. and detected the presence of a
parasitic Trematode and an Amoebae. While our data support the
possibility of achieving a complete inhibition of host DNA amplification
using elongation arrest blocking primers, more research is still
required. Still, there is a need for the development and additional
testing of protocols to study the eukaryotic diversity present in fish
gut, a slow-growing field of study in comparison to its prokaryotic
counterpart.