Discussion
Blocking primer
development
From the three unique genomic regions discovered in M. festivusV4-V5 18 rRNA gene, we developed two combinations of blocking primers
that were specific enough to accurately ligate to the host’ target
region. Elongation arrest blocking primers were developed in duos, one
forward and one reverse, to block the amplification on both the 3’ and
5’ strands. They have been modified with a C3 spacer at the 3’ end, a
chain of 3 hydrocarbons, to stop the advancement of the DNA polymerase.
The blockage should result in the formation of shorter and incomplete
amplicons that will not be sequenced because they do not possess the two
adapter sequences required for Illumina Mi-seq sequencing.
Vestheim & Jarman (2008) previously tried to develop elongation arrest
blocking primers. However, their attempt was unsuccessful since these
completely inhibited the PCR reaction in their study. Likewise, the
authors suggested a lower efficiency of elongation arrest blocking
primers because they are not interacting directly with the DNA
polymerase (Vestheim and Jarman 2008). Yet, there is no scientific data
to support their lower efficiency. Also, Belda et al . (2017)
previously achieved a blockage of more than 80 % using elongation
arrest by Peptide-Nucleic Acid while they failed at reducing host DNA
using annealing blocking primers in the same study. Furthermore, the
genetic regions adjacent to universal primers are usually very conserved
(Rojahn et al . 2021). For this reason, elongation arrest primers
are more versatile and easier to develop, making them suitable for any
species and a wide range of studies. The development of a replicable
method to develop such primers should facilitate further Eukaryote
metagenomic studies.
Our first duo of primers (F-primers ) [635F-C3 and 1062R-C3]
was binding to DNA regions afar from the amplification primers annealing
site [Fig. 1]. The F-primers set was meant to block the DNA
polymerase towards the end of the amplification, forming an incomplete
amplicon that should not include the 3’ complementary sequence required
for the barcode indexing PCR. Since the indexing sequence is required
for Miseq sequencing, the incomplete amplicons should not be sequenced.
The second duo (M-primers ) [816F-C3 and 846R-C3] was composed
of primers binding at the center of the target gene and stopping the
elongation midway [Fig. 1]. This results in the formation of
incomplete amplicons of about half the length of a normal amplicon. The
formation of short amplicons has numerous advantages. Firstly, the
incomplete amplicons are visible on electrophoresis or polyacrylamide
gels, on which the efficacy of the blocking primers can be assessed
quickly after the PCR amplification. Secondly, these shorter amplicons
are easy to filter-out with AMPure beads from Beckman Coulter Genomics.
The size recovery based on bead to DNA ratio step tends to select longer
amplicons and remove primer dimers and other unwanted contaminants. A
bead to DNA ratio of 0.5X is removing most of the amplicons of less than
300 bp facilitating the removal of incomplete amplicons which could
otherwise negatively affect the indexing PCR.
Blocking primer
evaluation
There was a marked difference between the relative abundances of host
related ASVs detected in the eight guts sampled. This important
variation was caused by the state of the gut sampled. While four guts
were filled with food and contained a low ratio of host tissues to gut
content (Gut IDs 1 to 4), the four other guts contained a higher
relative mass of host tissues (Gut IDs 5 to 8). Blocking primers are
especially needed in this second case, where the detection of the
Eukaryotic communities of the gut is masked by the high abundance of
host DNA in samples.
The F-primers did not significantly reduce the amount of host DNA
in samples [Fig. 2]. This could be due to the annealing position of
the F-primers , located afar from the annealing site of the
polymerase. Here, the long distance between the initial position of the
polymerase and the blocking primers could lead to an inefficacy at
blocking the polymerase. While we have no way of verifying this
hypothesis, host DNA sequences were full length in the samples amplified
using the F-primers , confirming that there was no blockage of the
polymerase during the PCR amplification. Considering the inefficacy of
the F-primers , it would have been interesting to test for
blocking primers complementary to the one used in this study. The
complementary sequence of the F-primers would have blocked the
amplification of host DNA nearer to the polymerase annealing site. The
resulting amplicons would have been very short, approximately 50 base
pairs, and could potentially lead to a better blockage of host DNA
amplification by directly removing the cleaved sequences during the size
selection step using AMPure beads from Beckman Coulter Genomics.
Conversely, M-primers significantly reduced the relative
abundance of host DNA by 66 % in samples [Fig. 2]. Also, these
primers systematically led to a reduction in host DNA abundance, proving
their efficacy in the presence of both low and high relative abundances
of host DNA [Table 1]. While we achieved a significant reduction of
host DNA amplification in samples using elongation arrest blocking
primers, we were not able to reach a complete inhibition. Previous
studies reached near complete inhibition of host target gene
amplification using both annealing blocking primers and CCSAS (Vestheim
and Jarman 2008, Liu et al . 2019, Zhong et al . 2021).
Furthermore, Zhong et al. (2021) already developed guide RNAs for
16 000 referenced Eukaryotes. Their method could also be implemented for
unreferenced organisms using sanger sequencing, as we did in this study.
Consequently, future research should consider testing and optimizing
this novel approach, which could represent a major advancement to the
field.
Similarly to the study of Vestheim and Jarman (2008), using a
concentration of more than 2X of elongation arrest blocking primers as
the M-primers completely inhibited the PCR amplification. This
could be caused by the short complementary region between these two
blocking primers, which may favour the formation of primer dimers.
Consequently, the PCR may be inhibited by the formation of secondary
structures at high concentrations, ultimately leading to unwanted
interactions with the polymerase. Also, there could be non-specific
binding in presence of a high concentration of the M-primers . Not
achieving to include higher concentrations of blocking primers is a
major shortcoming since previous studies systematically obtained a
higher amplification inhibition when using 10X of blocking primers
(Vestheim and Jarman 2008, Clerissi et al . 2018, Su et al .
2018). In this sense, it is possible that achieving to use a higher
concentration of elongation arrest blocking primers could lead to a near
complete inhibition of host target gene amplification.
Alpha diversity
There was a reduction in Faith’s phylogenetic alpha diversity in samples
from the M-primers group. However, the reduction in this alpha
diversity metric was mainly caused by the lower number of host-related
ASVs present in samples amplified with the M-Primers . Indeed, the
Shannon and Simpson alpha diversity indexes, two metrics that correct
for the evenness, were not significantly different. Here, the evenness
gained from the reduced abundance of overrepresented host ASVs is
counterbalancing for the loss of some ASVs from the class Actinopterygii
in the M-primers group. To support this, there is no significant
difference in alpha diversity between groups when omitting
Actinopterygii in the dataset. This confirms that the differences in
alpha diversity between M-primers and control groups were mainly
caused by the presence of many ASVs from this class. According to these
alpha diversity results, M-primers specifically inhibited the
amplification of host DNA sequences since their usage did not hinder the
detection of other ASVs in samples.
However, the usage of blocking primers did not lead to the detection of
an increased alpha diversity in samples. Moreover, we only detected a
mean of 30 ASVs per sample, which is a low total diversity. For this
reason, the removal of a small number of host-related ASVs in theM-primers group could represent an important shift in the
communities observed in samples. Strikingly, 20 % of all ASVs in the
dataset are related to vertebrates, while M. festivus diet should
not include vertebrates. Indeed, the species has a generalist diet
mostly composed of detritus and periphyton (Pires et al. 2015).
For this reason, most of these Vertebrata ASVs probably originate from
host DNA present in samples. They could also come from M.
festivus picking on other fish, as M. festivus was previously
observed cleaning other fish from endoparasites (Severo-Neto and
Froehlich 2015). Overall, alpha diversity data support that using
blocking primers facilitated the unravelling of all the targeted
Eukaryotic diversity while requiring a lower sequencing depth.
Beta diversity
M-primers led to a significant shift in beta diversity in the
four samples which contained a high ratio of host tissues to gut content
(Gut IDs 5 to 8) but had a limited influence on the beta diversity of
the four other samples (Gut IDs 1 to 4) [Fig. 3]. Consequently, theM-primers reduced the impact of a starting high relative mass of
host tissues in samples by homogenizing both types of samples. This is a
major advantage of using blocking primers as it mitigates the sampling
bias that occur when we collect fish on the field. This is even more
important when working with samples that have a low diversity as the
background noise caused by the important abundance of host sequences has
a major influence on the community analysed.
In parallel to what we observed in the alpha diversity data, omitting
the class Actinopterygii negated the effect of the M-Primers on
the beta diversity [Fig. S5]. While these results support a high
specificity of the blocking primers to the host target gene, this does
not rule out the possibility that our blocking primers would also
inhibit the amplification of other teleosteans. This limits the method
for the description carnivorous Teleostean diet. Still, diet studies
should rather focus on the study of the food bolus, which contains a
reduced amount of host sequences and a higher prey DNA quality as it was
not completely digested yet.
Blocking primers for parasite
detection
The use of M. primers enhanced the detection of low abundance
parasitic taxonomic classes in gut IDs 5 to 8 by reducing the background
noise caused by the high relative abundance of host DNA [Fig. 4].
Indeed, the M-primers enhanced the detection of parasitic classes
of high interest as Trematodes in gut ID 6 [Fig. 4]. This class of
parasitic helminth is known to infect fish species as an intermediate
host and Mammals as definitive host, leading to potential risk for
Amazonian communities. We also detected Arcellinida in gut ID 7, a class
of Amoebae that could be a parasitic taxon infecting M. festivus .
This supports the usefulness of blocking primers for parasitic
screening, as parasitic taxa are usually present in lower abundances
than host tissues and food in gut samples.
More broadly, the implementation of a metataxonomic approach combined
with blocking primers optimized for parasite screening allowed the
detection of multiple other potential parasitic infections of M.
festivus . For instance, we detected the presence of a Ciliophora from
the genus Nyctotherus sp. , a parasite known to cause diarrhea and
digestive problems in pet turtles (Satbige et al . 2017, Suzuki etal . 2020). To our knowledge, this genus was only documented once
infecting a fish (Earl & Jiménez 1969). Also, we detected Microsporidia
in very low abundance in gut ID 4 but cannot conclude about its
parasitic role in M. festivus considering the low amount of data
that we collected. These results highlight the potential of developing
metataxonomic approaches to describe the host-parasite relationships in
a region as diversified as the Amazonian rainforest. The usage of a
metataxonomic pipeline favors the detection of unknown parasitic
infections. These species would potentially not have been detected using
conventional taxonomic identification methods, which rely on light
microscopy and are still the main method used in tropical parasite
ecology studies. However, using DNA based methods is not conclusive
about the infectiousness of the taxa that are detected in the gut as
dead specimens could also be amplified. Here, the gold standard would be
a combination of DNA based and phenotypic data confirming the presence
of a parasite at the infectious stage.