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\begin{document}

\title{Elongation arrest blocking primers enhance parasite detection in 18S
rRNA metabarcoding study of Mesonauta festivus gut eukaryotic
communities.}



\author[1]{Nicolas Leroux}%
\author[1]{Sidki Bouslama}%
\author[1]{François-Étienne Sylvain}%
\author[2]{Adalberto Val}%
\author[1]{Nicolas Derome}%
\affil[1]{Laval University}%
\affil[2]{Instituto Nacional de Pesquisas da Amazonia}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{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.%
\end{abstract}%



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\section*{Introduction}

{\label{introduction}}

To date, most of the molecular microbial ecology research has focused on
the prokaryotic branch of the microbiota. Yet, previous findings have
linked the eukaryotic communities living in hosts' guts to several
pathologic conditions in fish (Scheifler et \emph{al} . 2019) and
mammals (Aivelo and Norberg 2018, Mann et \emph{al} . 2020) using a
metabarcoding approach. Furthermore, major interactions between species
from the three taxonomic domains have been reported in animal guts
(Leung et \emph{al} . 2018, Kodio et \emph{al} . 2020). However, this
field of study is slowed by the poor availability of simple and
cost-effective molecular methods to analyse gut eukaryotic communities.
While a variety of approaches are currently available, none are exempt
from the introduction of bias nor are well optimized (Green \& Minz
2005, Vestheim \& Jarman 2008). For instance, taxonomic identification
using taxonomic keys does not allow the detection of microscopic
organisms and requires \emph{a priori}knowledge of the potential
biodiversity present. Conversely, high-throughput DNA sequencing
approaches have proven their ability to identify the taxonomic richness
in samples at a higher taxonomic resolution than visual identification
in fish, while also giving additional information on diet and the
unknown diversity present (Berry et \emph{al} . 2015, Trujillo-Gonz\selectlanguage{ngerman}ález
et \emph{al} . 2022). The development of optimised molecular methods to
characterise eukaryotic gut communities is essential to better describe
the commensal eukaryotic microbiota and host-parasite interactions in
natural populations of fish.

The main challenge in analysing the eukaryotic fraction of the
microbiota comes from the homologies between the conserved ribosomal RNA
SSU sequences of the host and their associated eukaryotic organisms.
While universal primers have the advantage of amplifying taxonomically
diverse phyla, this characteristic favors the amplification of unwanted
host DNA. Indeed, DNA amplification with universal primers often leads
to a preferential amplification of the predominant host DNA in gut
samples (Su et \emph{al.} 2018). This high abundance of host DNA
contamination considerably limits the detection of DNA from other
organisms that are present in lower abundances (Vestheim and Jarman
2008, Liu et \emph{al} . 2019).

There are multiple methods to reduce the quantity of host DNA amplicons
in samples. Recently, Zhong et \emph{al} . (2021) developed CRISPR-Cas
Selective Amplicon Sequencing (CCSAS) based on taxon-specific
single-guide RNA to direct Cas9 to cut host 18S rRNA gene sequences.
Using this method, they achieved a near complete blockage of host DNA.
They also designed guide RNAs for approximately 16 000 metazoan and
plant taxa. While, CRISPR is known to recognize the wrong target gene in
certain occurrences (Fu et \emph{al} . 2013), this major advance to the
field could rapidly become the go-to approach to inhibit the
amplification of specific genes. Also, Green \& Mitz (2005) developed a
similar method using a Suicide Polymerase Endonuclease Restriction
(SuPER), in which specific restriction enzymes cut the host's amplicons
before PCR amplification. However, this method requires a unique cutting
site in the host's target gene and has only been applied once in another
study (Guedegbe et \emph{al} . 2009). Another way is to use a cocktail
of multiple group-specific amplification primers to detect a wide range
of organisms in samples (Koester \& Gergs 2017). While this method has
proven to be useful, the use of multiple primers can lead to an
amplification bias and their usage requires in-depth \emph{a
priori}knowledge of the eukaryotic communities under study. Since there
is scarce information about fish eukaryotic gut communities, the usage
of a multiple group-specific primers for highly conserved regions such
as the 18S rRNA SSU is suboptimal compared to methods that allow for
explorative and integrative research.

A method of growing interest, developed by Vestheim \& Jarman (2008) is
the usage of blocking primers to inhibit the PCR amplification of
undesired DNA in metabarcoding studies. These species-specific primers
are modified with a C3 spacer at the 3' end, a short three carbon chain
that prevents the elongation of the DNA during PCR amplification. While
it is slowly getting a wide-range acceptance (Boessenkool et \emph{al} .
2012, Belda et \emph{al} . 2017, Su et \emph{al} . 2018, Liu et
\emph{al} . 2019, Mayer et \emph{al} . 2021, Rojahn et \emph{al} . 2021,
Stewart et\emph{al} . 2021), the usage of blocking primers to describe
host-parasite interactions could still benefit from additional testing
of the method for other non-referenced species. Moreover, additional
data should help compare this method to the newly developed approach
based on CRISPR-Cas (Zhong et \emph{al} . 2021). Studies have used
annealing inhibiting blocking primers, primers overlapping with the 3'
end of universal primers and competing directly for annealing, to reduce
host DNA amplification in the krill (\emph{Euphausia} \emph{superba} )
(Vestheim and Jarman 2008), mosquitoes (\emph{Anopheles gambiae} )
(Belda et \emph{al} . 2017), fish species (Su et \emph{al} . 2018) and
humans (Boessenkool et \emph{al} . 2012). On the contrary, elongation
arrest blocking primers are primers annealing anywhere in the target
sequence and stopping the advancement of the DNA polymerase. This type
of blocking primers is less studied since their effectiveness was
seemingly lower than for annealing inhibition blocking primers (Vestheim
and Jarman 2008). However, the development path for elongation arrest
blocking primer is simpler since they can be developed in ultra-variable
regions of the target gene, facilitating the discovery of unique
sequences in the host target gene.

Here, we successfully developed and tested specific elongation arrest
blocking primers based on the methodology suggested by Vestheim \&
Jarman (2008) to block \emph{Mesonauta festivus} 18S rRNA SSU during PCR
amplification. This integrative and easily implemented method allowed a
significant reduction of host's DNA amplification, while not interfering
with the amplification of other target sequences. Furthermore, the
blocking primers enhanced the detectability of other species in gut
samples and helped dress a short portrait of the commensal eukaryotic
diversity present in \emph{M. festivus} gut. Overall, this method
represents a good approach to enhance the detection of parasitic species
and ensure a cost-effective sequencing of samples enriched with host
DNA. Although developed for \emph{Mesonauta festivus} , a good model
species for ecological and evolution studies in the Amazon basin (Pires
et \emph{al} . 2015), this approach could potentially be modified for
usage with various other Teleosts.

\section*{Methods}

{\label{methods}}

\subsection*{DNA extraction}

{\label{dna-extraction}}

Eight \emph{M. festivus} were collected at Anavilhanas National Park in
the \emph{Rio Negro} (AM, Brazil) (2°41'46.1S, 60°46'33.3W) in 2015,
using small seine net-fishing. Shortly after collection, fish were
dissected using sterile instruments, under a flame, to extract a fin and
a gut sample according to the methodology described by Sylvain
et\emph{al.} (2019). Samples were frozen at -80~°C right after the
dissection and until the DNA extraction. Genomic DNA was extracted
following the manufacturer's instructions of the QIAGEN DNeasy® Blood
and Tissues kit and stored at \selectlanguage{english}-80degC until amplification. Double strand
DNA was quantified using the Qubit(r) 2.0 Fluorometer.

\subsection*{Blocking primers
development}

{\label{blocking-primers-development}}

Using purified DNA from six \emph{M. festivus} fin samples, the V4-V5
region of the rRNA 18S gene of \emph{M. festivus} was amplified by
polymerase chain reaction (PCR) to precisely describe the target gene.
We used the following amplification primers: 566F and 1289R {[}Appendix
B{]} (Hadziavdic et \emph{al} . 2014) in order to have a sequence of the
target gene which should have 723 base pairs. Having a longer sequence
was meant to obtain a high sequence quality to help at designing the
blocking primers. The final concentrations of the PCR reaction (50 \selectlanguage{greek}µ\selectlanguage{english}L
total volume) contained: 1X of Q5 Reaction Buffer, 1X of High GC
Enhancer, 200 \selectlanguage{greek}µ\selectlanguage{english}M of dNTPs, 0.5 \selectlanguage{greek}µ\selectlanguage{english}M of each amplification primer, 0.02
U/\selectlanguage{greek}µ\selectlanguage{english}l of Q5 High-Fidelity DNA Polymerase and 3 \selectlanguage{greek}µ\selectlanguage{english}l of DNA template (lower
than 1000 ng of DNA in total). We used the following PCR program: (a)
30s 98\selectlanguage{ngerman}°C; (b) 10s 98°C; (c) 30s 60°C; (d) 20s 72°C; (e) 2min at 72°C;
using 30 amplification cycles. The amplicons were purified with 0.7X
AMPure beads (Beckman Coulter Genomics) and DNA quality was tested using
Nanodrop~2000. The six \emph{M. festivus} 18S rRNA V4-V5 gene amplicons
were sequenced by Sanger sequencing at the \emph{IBIS Plateforme
d'Analyse Génomique} to precisely describe the target gene. The six
resulting sequences' electropherograms were assembled jointly using the
CAP3 \emph{denovo} assembler implemented in the UGENE suite to obtain a
robust consensus sequence (Sequences are available on Genbank NCBI with
the following accession numbers: MZ736888:MZ736899{[}accn{]}).

The consensus sequence was aligned to known 18S rRNA gene sequences of 7
genera of parasites typically found in fish's gut,
i.e.,\emph{Cryptosporidium hominis} , \emph{Myxobolus cultus}
,\emph{Lepeophtheirus salmonis} , \emph{Myxobolus cerebralis}
,\emph{Caenorhabditis elegans} , \emph{Ichthyophthirius multifiliis}
and\emph{Aglaiogyrodactylus forficuloides} . We used the MUSCLE (Madeira
et\emph{al} . 2019) alignment program, also implemented in UGENE
(Okonechnikov et \emph{al.} 2012). The consensus sequence is available
in supporting information (Parasite\_Alignment.fa). We manually compared
the \emph{M. festivus} 18S rRNA gene V3-V4 region consensus sequence to
the seven parasite sequences to search for regions which can strongly
differentiate \emph{M. festivus} from other taxa. Three unique regions
were detected and used as templates to develop blocking primers {[}Fig.
1{]}. From these unique genomic regions, we designed four blocking
primers, two forward and two reverse primers, to specifically bind to
the chosen regions of \emph{M. festivus'} V4-V5 rRNA gene {[}Fig. 1{]}.
These primers, modified with a C3-spacer to inhibit DNA amplification,
were manufactured by \emph{Sigma-Aldrich} Corp. in Canada.

The first combination of primers {[}635F-C3 and 1062R-C3{]}, henceforth
represented by the letter \emph{F} , binds to DNA regions far from the
universal primers binding site. On the contrary, the other combination
{[}816F-C3 and 846R-C3{]}, henceforth represented by the letter\emph{M}
, is composed of primers binding at the center of the target gene and
stopping the elongation midway.

\subsection*{Blocking primer
evaluation}

{\label{blocking-primer-evaluation}}

\subsubsection*{PCR amplification and DNA
sequencing}

{\label{pcr-amplification-and-dna-sequencing}}

DNA from eight \emph{M. festivus} gut samples collected at Anavilhanas
National Park in the \emph{Rio Negro} was used as PCR material to test
the efficiency of the blocking primer combinations. Four gut samples
were filled with food (Sample ID 1 to 4), while the other four samples
had a moderate to null food bolus, leading to a higher host DNA to gut
content ratio (Sample ID 5 to 8). The V4-V5 region (\selectlanguage{english}[?] 550 base pairs) of
the 18S rRNA gene was amplified by PCR using the amplification primers
616*F, 574*F and 1132R {[}Appendix B{]} (Hugerth et \emph{al} . 2014).
We used two forward primers to amplify a higher diversity of organisms
in samples since 616*F and 574*F does not amplify the same range of
organisms. This set of primers was designed to optimise parasite species
detection (Kounosu et \emph{al} . 2019). We produced seven different PCR
reaction mixes for each sample. For each PCR reaction mix, the final
concentrations were (50 \selectlanguage{greek}µ\selectlanguage{english}l total volume): 1X of Q5 Reaction Buffer, 1X
of High GC Enhancer, 200 \selectlanguage{greek}µ\selectlanguage{english}M of dNTPs, 0.5 \selectlanguage{greek}µ\selectlanguage{english}M of each amplification
primer, 0.02 U/\selectlanguage{greek}µ\selectlanguage{english}l of Q5 High-Fidelity DNA Polymerase and 3 \selectlanguage{greek}µ\selectlanguage{english}l of DNA
template (lower than 1000 ng of DNA in total). To this reaction mix we
added either 2X, 5X or 10X, in comparison to amplification primers, of
each of the two combination of blocking primers (i.e.,
\emph{F-primers}and \emph{M-primers} ) and included a control group
without blocking primers. The following PCR program was used: (a) 30s
98\selectlanguage{ngerman}°C; (b) 10s 98°C; (c) 30s 57°C; (d) 20s 72°C; (e) 2min at 72°C; 35
amplification cycles. Amplified DNA was purified with 0.5X AMPure beads
(Beckman Coulter Genomics) since using a lower PCR product to beads
volume ratio helps to eliminate shorter sequences, primers dimers,
proteins, and phenols. Post\selectlanguage{english}-PCR DNA concentration and quality were
assessed on Nanodrop and by electrophoresis on 2\% agarose gels. Dual
indexing PCR was completed using the following concentrations in the
reaction mix (25 \selectlanguage{greek}µ\selectlanguage{english}l total volume): 1X of Q5 Reaction Buffer, 1X of High
GC Enhancer, 200 \selectlanguage{greek}µ\selectlanguage{english}M of dNTPs, 0.5 \selectlanguage{greek}µ\selectlanguage{english}M of each unique indexing primer,
0.02 U/\selectlanguage{greek}µ\selectlanguage{english}l of Q5 High-Fidelity DNA Polymerase and 1 \selectlanguage{greek}µ\selectlanguage{english}l of DNA template
from the amplifying PCR. We used the following PCR program: 30~s 98\selectlanguage{ngerman}°C;
(b) 10~s 98°C; (c) 30~s 60°C; (d) 20~s 72°C; (e) 2~min at 72°C;
12~amplification cycles. We ran the PCR product on a 2\% electrophoresis
gel to assess the efficiency of the PCR reaction, purified the amplified
DNA with 0.7X AMPure beads (Beckman Coulter Genomics) and verified the
DNA concentration and quality on Nanodrop\textsuperscript{TM} 2000.
Positive and negative controls were included at each step of the PCR
amplifications. After purification, multiplex sequencing was performed
using the MiSeq platform from Illumina\textsuperscript{\textbf{®}}
~(Illumina), by the~\emph{Plateforme d'analyses génomiques} ~at
the~\emph{Institut de Biologie Intégrative et des Systèmes} ~(IBIS)
of~\emph{Université Laval} using the MiSeq Reagent V3 (2 x 300 bp) Kit
according to the manufacturer's protocols.~Positive and negative
controls were also sequenced but did not result in any sequence
including amplification primers, supporting the absence of contamination
during PCRs.

\subsubsection*{Data processing and taxonomic
assignation}

{\label{data-processing-and-taxonomic-assignation}}

The analysis of amplicon sequences was done at the~\emph{Institut de
Biologie Intégrative et des Systèmes} ~(IBIS) at~\emph{Université Laval}
. After trimming with CutAdapt (Martin 2011), 2,835,254 sequences were
obtained (mean of 33,753 paired end sequences per sample). The
demultiplexed fastq paired sequence files were processed through dada2
(Callahan et \emph{al} . 2016) using the function\emph{ytfilterAndTrim}
() with the following parameters: 280 for the read truncation length,
total removal of reads including a nucleotide with a Phred score of two
of lower and a maximum expected error of two for forward reads and four
for reverse reads. The filtered reads were then fed to the error rate
learning, dereplication and ASV inference steps using the functions
\emph{learnErrors} (), \emph{derepFastq} () and\emph{dada} (). Then,
forward and reverse reads were merged using the function
\emph{mergePairs()} using the default parameters. Chimeric sequences
were removed using the \emph{removeBimeraDenovo} () function with the
``consensus'' method parameter. Taxonomic assignation was done using the
function \emph{assignTaxonomy} from DADA2 in R (Wang et\emph{al} . 2007)
based on the SILVA Release 138.1 Ref NR 99 for SSU, which is based on a
99\% criterion to remove redundant sequences in the database. We
verified that the sequencing depth of every sample was sufficient using
a rarefaction analysis {[}Fig. S1{]}. We calculated a taxonomic tree
based on the DNA sequences using the \emph{R} package\emph{ape} (Paradis
et \emph{al} . 2004, Paradis \& Schliep 2019) and a\emph{Phyloseq}
(McMurdie \& Holmes 2013) object was produced in\emph{R} to do
subsequent analyses.

\subsubsection*{Statistical analyses}

{\label{statistical-analyses}}

We estimated blocking primers efficiency by calculating the relative
abundance of the class Actinopterygii, the class of \emph{M. festivus} ,
in samples using the R package \emph{Phyloseq} . Then, we grouped
samples according to the blocking primer used during their amplification
and produced barplots representing the mean relative abundances of
Actinopterygii in each group. Since the relative abundances were not
normally distributed, we used a non-parametric paired Wilcoxon test to
verify for significant differences in host amplicons relative abundance
between blocking primer groups.

Using the \emph{R} functions \emph{plot\_richness} from
\emph{Phyloseq}and \emph{estimate\_pd} from \emph{btools} , we estimated
the observed (Total number of ASVs), Faith (Phylogenetic diversity),
Shannon, and Simpson alpha diversity indexes of the 5 blocking primer
groups. We calculated a two-way ANOVA using both the blocking primer
groups and the Gut-ID as explicative variables for each diversity index
estimated. We verified the assumptions of normality and homoscedasticity
of variances using Shapiro-Wilk tests on residuals and Levene tests for
each group. In cases where the assumptions were not respected, we used a
square root transformation which lowered the impact of anomaly high
alpha diversity values. When the ANOVA was significant for a given Alpha
diversity metric, we used a Tukey's HSD to verify for pairwise
differences between groups considering a 95~\% confidence interval.
Afterward, we computed the same diversity analyses based on a dataset
where we removed the class Actinopterygii, using the function
``subset\_taxa()'' from\emph{Phyloseq} , to verify if host related ASVs
diversity were driving the differences between groups. For both the
datasets with and without host related ASVs, we produced boxplots
representing the four alpha diversity metrics in function of the
experimental groups considered.

A two-way PERMANOVA based on Bray-Curtis dissimilarity indexes was
calculated using the function \emph{adonis} in the \emph{R}
package\emph{vegan} considering the blocking primer groups and the gut
IDs of samples as the independent variables. To verify for significant
differences between blocking primer groups, we used a post-hoc pairwise
Adonis test with the function \emph{pairwise.adonis2} . Afterwards,
PCoAs based on Bray-Curtis dissimilarity indexes and Weighted Unifrac
phylogenetic distances were produced using the
function\emph{plot\_ordination} from the \emph{R} package
\emph{PHYLOSEQ} to illustrate the results of the PERMANOVA. Again, we
produced another PCoA based on Bray-Curtis dissimilarity index using the
dataset without host related ASVs. More specifically, we removed the
class Actinopterygii from the Phyloseq object and produced a PCoA to
assess the impact of blocking primers while removing the influence of
host related ASVs.

We produced a taxonomic barplot considering the top 10~\% ASVs in
abundance (43 most abundant ASVs) present in both the
\emph{M-primers}and control groups for the four samples with a high
relative abundance of host DNA using the function \emph{plot\_bar} from
\emph{Phyloseq} in\emph{R} . We produced the barplot considering the
Class as the taxonomic resolution. We omitted the Phylum
\emph{Streptophyta} since its overabundance in most samples masked the
detectability of other more-interesting taxonomic units and is food
related.

\section*{Results}

{\label{results}}

\subsection*{Blocking primers
development}

{\label{blocking-primers-development-1}}

We discovered three unique regions in \emph{M. festivus} rRNA 18S V4-V5
gene consensus sequence following its comparison with seven potential
fish parasites {[}Fig. 1{]}. One region was located 5' of the consensus
sequence and had a length of 24 pb, a second one was located at the
middle of the sequence and had a length of 40 pb, and the third was
located 3'and had a length of 28 pb. From these regions, two
combinations of blocking primers were developed, the
\emph{F-primers}{[}635F-C3 and 1062R-C3{]} and the \emph{M-primers}
{[}816F-C3 and 846R-C3{]}, respectively located at the extremities and
at the middle of the target gene.

\subsection*{Blocking primer
evaluation}

{\label{blocking-primer-evaluation-1}}

The relative abundance of host DNA in samples amplified using
the\emph{F-Primers} was not significantly different from the control
group {[}Fig. 2{]}. Indeed, none of the three concentrations
of\emph{F-primers} significantly reduced the number of host amplicons in
samples according to the paired samples Wilcoxon test (p-values
\textgreater{} 0.05). Also, using an increasing concentration
of\emph{F-primers} , from 2X to 10X, did not improve its effectiveness
and led to inconsistent results in samples with a low host DNA to gut
content ratio {[}Table 1{]}.

According to the paired sample Wilcoxon test, the \emph{M-primers} at a
concentration of 2X allowed a significant reduction of the mean relative
abundance of host sequences present in samples when compared to the
control group (p-value = 0.004). The mean relative abundance of the
class Actinopterygii, the taxonomic class of \emph{M. festivus} , in
the\emph{M-primers} group represented 34.01 \% of the abundance of the
control group {[}Fig. 2{]}. Also, the M-Primers efficacy at inhibiting
host's DNA amplification was significative in samples starting with a
low and a high host DNA to gut content ratio {[}Table 1{]}. For example,
the mean relative abundance of the class Actinopterygii in gut IDs 1 to
4 of the \emph{M-primers} group represents 14.58 \% of the relative
abundance of the control group. Furthermore, host DNA relative abundance
in gut IDs 7 and 8 respectively reduced from 72 and 77.9~\% to 24 and
21.5~\%. However, using a concentration of more than 2X of
the\emph{M-primers} completely inhibited the PCR amplification {[}Table
1{]}.

\subsection*{Alpha diversity}

{\label{alpha-diversity}}

The Faith phylogenetic alpha diversity index was significantly lower in
the \emph{M-Primers} group than in the control group in the Tukey HSD
test (p-value = 0.02) {[}Fig. S2{]}. However, the total number of ASVs
(observed diversity), the Shannon and the Simpson alpha diversity
indexes were not significantly lower when comparing the same groups
(p-value \textgreater{} 0.05). Furthermore, when omitting the class
Actinopterygii, there was no significant difference in alpha diversity
between the \emph{M-primers} group and the control group in the Tukey
HSD test (p-values \textgreater{} 0.05) {[}Fig.~S3{]}. For
the\emph{F-Primers} , their effect was inconsistent at different
concentrations and did not significantly influence the alpha diversity
in samples. Overall, we observed a mean of 30 ASVs per sample, with
20~\% of these from the Phylum Vertebrata. Also, two genera of
plants,\emph{Cyperus sp.} and \emph{Oryza sp.} , were the most abundant
ASVs for the samples analysed. These two genera, from the order Poales,
averaged 48~\% of the total relative abundance in samples.

\subsection*{Beta diversity}

{\label{beta-diversity}}

According to the two-way PERMANOVA comparing Bray-Curtis dissimilarity
indexes and considering the eight gut IDs and the four blocking primer
combinations as factors, most of the variance was explained by
differences between gut IDs (R\textsuperscript{2} = 0.85; p-value = 3.0
x 10\textsuperscript{-5}) and a moderate part of the variance was
explained by differences between blocking primer groups
(R\textsuperscript{2}~=~0.07;~p-value~=~1.0~x~10\textsuperscript{-4}).
Furthermore, the \emph{M-primers} was by far the combination of blocking
primers with the most influence on the sample's beta diversity in the
Pairwise Adonis test (For \emph{M-primers} , R\textsuperscript{2} =
0.09; p-value = 0.007).

In a PCoA based on Bray-Curtis's dissimilarity indexes, there was a
significant shift in beta diversity in the four samples which contained
a high ratio of host DNA to gut content (Gut IDs: 5 to 8), but no
important variation in beta diversity for the four gut samples with a
low abundance of host DNA contamination (Gut IDs: 1 to 4) {[}Fig. 3{]}.
This result was very similar when producing a PCoA based on weighted
Unifrac distances {[}Fig. S4{]}. For samples 5 to 8, the usage
of\emph{M-primers} led to a shift in beta diversity, reducing the
dissimilarity between gut IDs 5 to 8 and 1 to 4 {[}Fig.~3{]}.
Consequently, when computing a pairwise Adonis test using a dataset
omitting the class Actinopterygii, \emph{M-Primers} did not have a
significant effect on the beta diversity of samples when compared to the
control group (R\textsuperscript{2}~=~0.03;~p-value~=~0.07). Again,
the\emph{F-Primers} had little influence on the beta-diversity in
samples and showed inconsistent patterns that did not support their
effectiveness at blocking host DNA amplification.

\subsection*{Blocking primers for parasite
detection}

{\label{blocking-primers-for-parasite-detection}}

The usage of \emph{M. primers} enhanced the detection of low abundance
parasitic taxonomic classes in gut IDs 5 to 8 {[}Fig.~4{]}. For
instance, we detected 12 different taxonomic classes in
the\emph{M-primers} group against 9 in the group without blocking
primers in the top 10\% ASVs barplots {[}Fig. 4{]}. From these,
\emph{M-Primers}enhanced the detectability of Trematode in gut ID 6 and
Arcellinida in gut ID 7. Also, these classes were detected in higher
relative abundance in the \emph{M-Primers} group. Also, a parasitic
Ciliophora from the genus \emph{Nyctotherus sp.} was detected in high
relative abundance in two gut samples (Gut IDs 2 and 7) and in lower
abundance in one (Gut ID 4) {[}Fig. 4{]}. Finally, we detected the
presence of a Tubilinea in gut ID 7 and a very low relative abundance of
microsporidia in gut ID 4.

\section*{Discussion}

{\label{discussion}}

\subsection*{Blocking primer
development}

{\label{blocking-primer-development}}

From the three unique genomic regions discovered in \emph{M.
festivus}V4-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 \emph{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 \emph{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 (\emph{F-primers} ) {[}635F-C3 and 1062R-C3{]}
was binding to DNA regions afar from the amplification primers annealing
site {[}Fig. 1{]}. The \emph{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 (\emph{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.

\subsection*{Blocking primer
evaluation}

{\label{blocking-primer-evaluation-2}}

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 \emph{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 \emph{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 \emph{F-primers} , confirming that there was no blockage of
the polymerase during the PCR amplification. Considering the inefficacy
of the \emph{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 \emph{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, \emph{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 \emph{al} . 2019, Zhong et \emph{al} . 2021).
Furthermore, Zhong et \emph{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 \emph{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 \emph{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 \emph{al} . 2018, Su et \emph{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.

\subsubsection*{Alpha diversity}

{\label{alpha-diversity-1}}

There was a reduction in Faith's phylogenetic alpha diversity in samples
from the \emph{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 \emph{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 \emph{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 \emph{M-primers} and control
groups were mainly caused by the presence of many ASVs from this class.
According to these alpha diversity results, \emph{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
the\emph{M-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 \emph{M. festivus} diet should
not include vertebrates. Indeed, the species has a generalist diet
mostly composed of detritus and periphyton (Pires et \emph{al.} 2015).
For this reason, most of these Vertebrata ASVs probably originate from
host DNA present in samples. They could also come from \emph{M.
festivus} picking on other fish, as \emph{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.

\subsubsection*{Beta diversity}

{\label{beta-diversity-1}}

\emph{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,
the\emph{M-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 \emph{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.

\subsubsection*{Blocking primers for parasite
detection}

{\label{blocking-primers-for-parasite-detection-1}}

The use of \emph{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 \emph{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 \emph{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 \emph{M.
festivus} . For instance, we detected the presence of a Ciliophora from
the genus \emph{Nyctotherus sp.} , a parasite known to cause diarrhea
and digestive problems in pet turtles (Satbige et \emph{al} . 2017,
Suzuki et\emph{al} . 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 \emph{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.

\section*{Conclusion}

{\label{conclusion}}

We developed and tested a pair of elongation arrest blocking primers, a
method which could potentially be replicated for other Teleosteans to
study their parasitic communities. Our elongation arrest blocking
primers led to a 66~\% reduction in host DNA abundance in samples. This
ultimately enhanced the detection of parasitic taxa and significantly
reduced the amount of wasted sequencing. The combination of a
metataxonomic approach and blocking primers helped us detecting a
parasitic infection by the ciliate \emph{Nyctotherus sp.} for one of the
first time in fish, while also facilitating the detection of four other
parasitic taxa in samples. However, other studies using annealing
inhibiting blocking primers and CRISPR-Cas Selective Amplicon Sequencing
(CCSAS) obtained a near complete inhibition of host DNA amplification.
Notably, the newly developed CCSAS method looks promising and should be
considered for future studies. When compared to our results, it seems
like CCSAS leads to a higher inhibition of amplification while requiring
a similar development path as elongation arrest blocking primers. Still,
using this approach on a larger amount of gut samples could lead to very
interesting results about the Eukaryotic microbiome of Amazonian
cichlids.

\section*{ACKNOWLEDGEMENTS}

{\label{acknowledgements}}

We gratefully acknowledge help from all members of the Derome and Val
laboratories. Thank you to the \emph{IBIS Plateforme d'Analyse
Génomique} for the sequencing and to ICMBIO/Instituto Chico Mendes de
Conservação da Biodiversidade for in situ support for fish collection
and for issuing the permit to transport biological samples (permit
number 29837/18). This research was part of the ADAPTA project at INPA
and was supported by the INCT ADAPTA (CNPq/FAPEAM) and INPA/MCTI grants
to ALV (ORCID: 0000\selectlanguage{english}-0002\selectlanguage{english}-3823\selectlanguage{english}-3868), and the Natural Sciences and
Engineering Research Council of Canada (NSERC) Discovery grant (grant
\#6333) to ND (ORCID: 0000\selectlanguage{english}-0002\selectlanguage{english}-2509\selectlanguage{english}-6104), the Canada Graduate
Scholarship (BESC M) Alexander-Graham-Bell to NL, the FRQNT Master
research scholarship to NL and the MITACS GlobaLink grant to NL. Also,
Ressources Aquatiques Quebec (RAQ) contributed to this research both by
monetary and specialized support.

\section*{AUTHOR CONTRIBUTIONS}

{\label{author-contributions}}

N.L., S.B. and N.D. designed the experiment. F.S., N.L., A.V. and N.D.
organized sampling expeditions. F.S. and N.L. sampled fish during field
expeditions. N.L. processed samples in the laboratory (fish dissections
and DNA extractions). N.L. and S.B. performed bioinformatic analyses.
N.L. wrote the manuscript. All authors reviewed the manuscript.

\section*{ETHICAL APPROVAL}

{\label{ethical-approval}}

This study was carried out in accordance with the recommendations of the
Ethics Committee for the Use of Animals of the~\emph{Instituto Nacional
de Pesquisas da Amazonia} ~(INPA). The permit (number 29837-18 as of 23
March 2021) was approved by the Ethics Committee for the Use of Animals
of INPA.

\section*{DATA AVAILABILITY STATEMENT}

{\label{data-availability-statement}}

The scripts and the datasets used for the statistical analysis of this
project are freely available from the Open Science Network platform
(URL: https://osf.io/u6w3f/?view\_only=27cefb515bd345648867fc20268c2d88
). The consensus sequence used to detect unique sites in \emph{M.
festivus} 18S rRNA V4-V5 gene is also available on OSN platform.
Sequences from Sanger sequencing are available on Genbank NCBI with the
following accession numbers: MZ736888:MZ736899{[}accn{]}).

\section*{Competing Interests
Statement}

{\label{competing-interests-statement}}

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to
influence the work reported in this paper.

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\section*{Table}

{\label{table}}

\textbf{Table 1:} Relative abundances of the class Actinopterigii
in\emph{Mesonauta festivus} gut samples for each combination (n = 2) and
each concentration (Conc.) (n = 3) of blocking primer tested in the
study. \emph{M-primers} are elongation arrest blocking primers located
in the middle of the target gene (816F-C3 and 846R-C3)
and\emph{F-primers} are located at the extremities of the target gene
(635F-C3 and 1062R-C3). At a concentration higher than 2X, M-primers
completely inhibited the PCR amplification. In the table, N.a. stands
for no amplification.\selectlanguage{english}
\begin{longtable}[]{@{}llllllllll@{}}
\toprule
Blocking primer combination & Conc. & Actinopterigii relative abundance
(\%) & Actinopterigii relative abundance (\%) & Actinopterigii relative
abundance (\%) & Actinopterigii relative abundance (\%) & Actinopterigii
relative abundance (\%) & Actinopterigii relative abundance (\%) &
Actinopterigii relative abundance (\%) & Actinopterigii relative
abundance (\%)\tabularnewline
\midrule
\endhead
& & Low & Low & Low & Low & High & High & High & High\tabularnewline
& & 1 & 2 & 3 & 4 & 5 & 6 & 7 & 8\tabularnewline
Control & 0 & 0.2\% & 0.3\% & 0.7\% & 3.6\% & 39.0\% & 46.5\% & 72.0\% &
77.9\%\tabularnewline
M-primers & 2X & 0.0\% & 0.2\% & 0.2\% & 0.3\% & 9.0\% & 26.5\% & 24.0\%
& 21.5\%\tabularnewline
M-primers & 5X & N.a. & N.a. & N.a. & N.a. & N.a. & N.a. & N.a. &
N.a.\tabularnewline
M-primers & 10X & N.a. & N.a. & N.a. & N.a. & N.a. & N.a. & N.a. &
N.a.\tabularnewline
F-primers & 2X & 0.2\% & 1.0\% & 4.0\% & 1.3\% & 27.0\% & 33.0\% &
58.9\% & 70.2\%\tabularnewline
F-primers & 5X & 0.9\% & 4.9\% & 9.4\% & 0.7\% & 27.7\% & 38.8\% &
58.2\% & 66.3\%\tabularnewline
F-primers & 10X & 1.2\% & 8.9\% & 18.7\% & 0.7\% & 31.7\% & 39.5\% &
56.1\% & 73.1\%\tabularnewline
\bottomrule
\end{longtable}

\section*{Figures}

{\label{figures}}

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/447881/articles/595089-elongation-arrest-blocking-primers-enhance-parasite-detection-in-18s-rrna-metabarcoding-study-of-mesonauta-festivus-gut-eukaryotic-communities}

Figure 1: Starting position of the 4 designed blocking primers on the
consensus sequence of \emph{Mesonauta festivus} V4-V5 rRNA 18S gene. The
sequence and direction of each blocking primer is identified on the
figure. The 3 unique regions discovered are marked in red.

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/447881/articles/595089-elongation-arrest-blocking-primers-enhance-parasite-detection-in-18s-rrna-metabarcoding-study-of-mesonauta-festivus-gut-eukaryotic-communities}

Figure 2: Mean relative abundance of ASVs from the class Actinopterygii
detected in \emph{Mesonauta festivus} gut samples based on the
amplification of the 18S rRNA V4-V5 gene (n~=~8). Samples have been
grouped according to the blocking primers used during their
amplification. The percentage above each bar plot represent the residual
relative abundance of the class Actinopterygii when compared to the
control group (colored in blue).

\textbf{Hosted file}

\verb`image4.emf`  available at \url{https://authorea.com/users/447881/articles/595089-elongation-arrest-blocking-primers-enhance-parasite-detection-in-18s-rrna-metabarcoding-study-of-mesonauta-festivus-gut-eukaryotic-communities}

Figure 3: Principal coordinate analysis (PCoA) based on the Bray-Curtis
distances between \emph{Mesonauta festivus} gut samples Eukaryotic
communities (n~=~40). Samples were colored according to their gut ID
(n~=~8) and the pinch types represent the blocking primers used during
their amplification (n~=~5). The fraction of the total variance
explained by each axis is identified.

\textbf{Hosted file}

\verb`image5.emf`  available at \url{https://authorea.com/users/447881/articles/595089-elongation-arrest-blocking-primers-enhance-parasite-detection-in-18s-rrna-metabarcoding-study-of-mesonauta-festivus-gut-eukaryotic-communities}

Figure 4: Relative abundance bar plots of the 43 most abundant ASVs
present in \emph{Mesonauta festivus} gut samples with a high relative
abundance of host DNA (n =~4). We omitted the Phylum Streptophyta from
the bar plots since its high relative abundance hindered the
detectability of other taxa. At the left, all samples are from the
control group, which have not been affected by any blocking primers. At
the right, samples have been amplified using \emph{M-primers} at a
concentration of 2X.

\section*{Appendix A}

{\label{appendix-a}}

\textbf{Hosted file}

\verb`image6.emf`  available at \url{https://authorea.com/users/447881/articles/595089-elongation-arrest-blocking-primers-enhance-parasite-detection-in-18s-rrna-metabarcoding-study-of-mesonauta-festivus-gut-eukaryotic-communities}

\textbf{Figure S1:} Rarefaction analysis produced representing the total
number of ASVs detected in \emph{Mesonauta festivus} gut samples in
function of the sampling depth (n = 40). We coloured lines according to
the gut ID of samples. While the sequencing depth is not equal for every
sample, it seems like all the diversity was unravelled for every sample.

\textbf{Hosted file}

\verb`image7.emf`  available at \url{https://authorea.com/users/447881/articles/595089-elongation-arrest-blocking-primers-enhance-parasite-detection-in-18s-rrna-metabarcoding-study-of-mesonauta-festivus-gut-eukaryotic-communities}

\textbf{Figure S2:} Observed, Faith's phylogenetic, Shannon and Simpson
alpha diversity indexes from \emph{M. festivus} gut samples (n = 40)
amplified using different blocking primer combinations (n = 5). We
clustered and colored samples according to their blocking primer group.
The plot was produced using the function \emph{plot\_richness}
from\emph{Phyloseq} . There was a significant difference between the
phylogenetic diversity from the M\_2X group and the control (p-value
\textless{} 0.05), but there was no significant difference in in Shannon
and Simpson diversity indexes (p-value \textgreater{} 0.9).

\textbf{Hosted file}

\verb`image8.emf`  available at \url{https://authorea.com/users/447881/articles/595089-elongation-arrest-blocking-primers-enhance-parasite-detection-in-18s-rrna-metabarcoding-study-of-mesonauta-festivus-gut-eukaryotic-communities}

\textbf{Figure S3:} When omitting the class Actinopterygii, observed,
Faith's phylogenetic, Shannon and Simpson alpha diversity indexes
from\emph{M. festivus} gut samples (n = 40) amplified using different
blocking primer combinations (n = 5). We clustered and colored samples
according to their blocking primer group. The plot was produced using
the function \emph{plot\_richness} from \emph{Phyloseq} . There was a
significant difference between the phylogenetic diversity from the M\_2X
group and the control (p-value \textless{} 0.05), but there was no
significant difference in Shannon and Simpson diversity indexes (p-value
\textgreater{} 0.9).

\textbf{Hosted file}

\verb`image9.emf`  available at \url{https://authorea.com/users/447881/articles/595089-elongation-arrest-blocking-primers-enhance-parasite-detection-in-18s-rrna-metabarcoding-study-of-mesonauta-festivus-gut-eukaryotic-communities}

\textbf{Figure S4:} Principal coordinate analysis (PCoA) based on the
Weighted Unifrac distances between \emph{Mesonauta festivus} gut sample
18S rRNA V4-V5 communities (n~=~40). Samples were colored according to
their gut ID (n~=~8) and the pinch types represent the blocking primers
used during their amplification (n~=~5). The percentage of the total
variance explained by each axis is identified.

\textbf{Hosted file}

\verb`image10.emf`  available at \url{https://authorea.com/users/447881/articles/595089-elongation-arrest-blocking-primers-enhance-parasite-detection-in-18s-rrna-metabarcoding-study-of-mesonauta-festivus-gut-eukaryotic-communities}

\textbf{Figure S5:} Principal coordinate analysis (PCoA) based on the
Bray-Curtis distances between \emph{Mesonauta festivus} gut sample 18S
rRNA V4-V5 communities (n~=~40) without considering the Class
Actinopterygii in the \emph{Phyloseq} object. Samples were colored
according to their gut ID (n~=~8) and the pinch types represent the
blocking primers used during their amplification (n~=~5). The percentage
of the total variance explained by each axis is identified.

\section*{Appendix B}

{\label{appendix-b}}

\subsection*{Primer list:}

{\label{primer-list}}\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
& \textbf{Sequence (5'-\textgreater{}3')} & \textbf{Template strand} &
\textbf{Length} & \textbf{Start} & \textbf{Stop} & \textbf{Tm} &
\textbf{GC\%} & \textbf{Citation}\tabularnewline
\midrule
\endhead
\textbf{635F-C3} & CGCGAGGGGAGCCACCGTCTG & Plus & 21 & 69 & 89 & 69.58 &
76.19 & Na\tabularnewline
\textbf{846R-C3} & ATCATGGCCCCAGTTCAGAGAGAG & Minus & 24 & 280 & 257 &
63.80 & 54.17 & Na\tabularnewline
\textbf{1062R-C3} & TGCCCGGCGGGTCATGGGAAT & Minus & 21 & 496 & 476 &
68.99 & 66.67 & Na\tabularnewline
\textbf{816F-C3} & GGGTTTTCTCTCTCTGAACTGGGGC & Plus & 25 & 250 & 274 &
65.02 & 56.00 & Na\tabularnewline
\textbf{574*F} & CGGTAAYTCCAGCTCYV & Plus & 17 & 574 & 591 & 58.8 & 56.9
& (Kounosu et \emph{al}. 2019)\tabularnewline
\textbf{616*F} & TTAAARVGYTCGTAGTYG & Plus & 18 & 616 & 634 & 53.8 &
39.8 & (Kounosu et \emph{al}. 2019)\tabularnewline
\textbf{1132R} & CCGTCAATTHCTTYAART & Minus & 19 & 1132 & 1113 & 53.9 &
35.2 & (Kounosu et \emph{al}. 2019)\tabularnewline
\textbf{566F} & CAGCAGCCGCGGTAATTCC & Plus & 19 & 566 & 585 & 70.2 &
63.2 & (Hadziavdic et \emph{al}. 2014)\tabularnewline
\textbf{1289R} & ACTAAGAACGGCCATGCACC & Minus & 20 & 1289 & 1269 & 57.9
& 55 & (Hadziavdic et \emph{al}. 2014)\tabularnewline
\bottomrule
\end{longtable}

\selectlanguage{english}
\FloatBarrier
\end{document}

