DOI: 10.5061/dryad.f7m0cfxxz
title: OTUs Table and fastq sequences from environmental DNA applied to trematode communities

description: This OTUs table and fastq
 sequences underlie the main results of the study
 "Make visible the invisible: Optimized development of an
 environmental DNA metabarcoding tool for the characterization of trematode
 parasitic communities". In this study, our aim was to
 develop an optimized eDNA-based metabarcoding approach to detect
 trematodes and characterize their communities, most of which associated to
 aquatic environments. We thus assessed the ability of our
 eDNA-based metabarcoding approach to reconstruct trematode communities
 compared to a classical trematode monitoring method over four freshwater
 aquatic ecosystems.  We focused on 4 natural sites from Occitanie
 Region (Southern France) that differ in terms of habitats, and in which
 the trematode communities were previously at least partially
 characterized. At each of these sites, we sampled the water-sediment
 interface from which the eDNA was extracted and sequenced with a MiSeq
 amplicons sequencing approach.   Over the four natural ecosystems
 screened in nature, 33 OTUs were generated from the eDNA-based approach,
 from which 11 trematode species were identified. In comparison, we
 identified five trematode species using the classical monitoring method,
 three of which were also detected by the eDNA-based approach.
description: A total of 50 individual metabarcoding libraries were prepared
 following the Illumina two-step PCR protocol. These 50 libraries include
 triplicates of each water-sediment interface sample collected (i.e., 3 x 5
 = 15), four negative controls (one per sampling site) and six positive
 controls, the PCRs of each of these samples being duplicated (i.e., 25 x 2
 = 50). The positive controls consisted into two categories of mock
 communities. The first category of mock community consisted in equimolar
 pools of 28 DNA extracts (set at a 3.5×10-3ng/µL final concentration) from
 different trematode species from internal collections. The second mock
 community consisted in equimolar pools of PCR products independently
 obtained from the same 28 trematodes species. For the
 OTUs table, The resulting amplicon sequence dataset was processed using
 the Find Rapidly OTUs with Galaxy Solution (FROGS) pipeline implemented in
 Galaxy (Escudié et al., 2018) available from the Genotoul platform
 (Toulouse, France). (i) The amplicon dataset was first pre-processed by
 filtering out the sequences so as to keep amplicon sizes from 150 to 400
 nucleotides. (ii) The sequences kept were next clustered into operational
 taxonomic units (OTUs) using the swarm algorithm and using denoising and
 an aggregation distance of three nucleotides (Mahé et al., 2014). (iii)
 The dataset was filtered out for chimeras using VSEARCH (Rognes et al.,
 2016). (iv) Singletons and underrepresented clusters (i.e., clusters whose
 number of sequences were &lt;0.1% of the total number of sequences)
 were removed. Each OTU was next assigned to a species through a two-step
 BLAST affiliation procedure. The first BLAST analysis was computed using
 the standalone blastn program contained in the BLAST+ package and a custom
 trematode sequence database containing a total of 88 sequences including
 the sequences obtained from the amplicons generated by the in silico
 ecoPCR (i.e., 50 species; see Section 2.1 of the article; Table S1), the
 sequences generated by the in vitro Sanger sequencing (i.e., 26 species
 over the 34 species sequenced; see Section 2.2; Table S3), and 12
 sequences retrieved from the GenBank database (Table S4). The second BLAST
 analysis was performed using the online MEGABLAST tool without restricting
 parameters to achieve affiliation of OTUs that could not be assigned in
 the first BLAST analysis. The obtained OTUs were filtered for presenting
 minimal blast coverage of 97% and a pairwise identity above 97% with the
 affiliated sequence. The remaining OTUs were considered as “unassigned”.
 Lastly, we considered that a given OTU was present in a sub sample (i.e.,
 one of the three replicates of a single environmental sample; see section
 2.4) if its number of sequences was &gt;0.1% of the total number of
 sequences in each of the two library assigned to this sub sample and if
 this OTU was present in both libraries (i.e., the two PCR replicates
 performed on the single subsample; see section 2.4). This 0.1% threshold
 was determined as being the most stringent while allowing the retention of
 the necessary sequences to detect all the 28 species from the control mock
 communities.
description: fastq file name definitions : 
 Mo111 to Mo 132 : Mock communities (pool of DNA). The
 second number indicates the technical replicate and the final number
 indicates the PCR replicates.  Mo211 to Mo232 : Mock
 communities (pool of PCR products). The second number indicates the
 technical replicate and the final number indicates the PCR
 replicates. NS1 to NN2 : Field negative controls
 (first letter: N) of Salses (S), St Génis (G), Villelongue (V) and Néfiach
 (N). The number indicates the PCR replicates. S11 to
 S32 : Salses in May  TS11 to TS32 : Salses in
 March  G11 to G32 : St Génis 
 V11 to V32 : Villelongue  N11 to N32 :
 Néfiach For field samples, the
 first number indicates the sub-sample and the final number indicates the
 PCR replicates.