Bioinformatic analysis
The sequence reads were bioinformatically processed on the Genobioinfo
computing cluster (GenoToul, Toulouse, France). Forward and reverse
reads were assembled using the program illuminapairend from theOBITools v1.2.11 package (Boyer et al., 2016) and the resulting
sequences were trimmed for primers using the program cutadapt (Martin, 2011). Strictly identical sequences were clustered together
using obiuniq (dereplication step). Amplicon Sequence Variants
(ASVs hereafter) occurring fewer than 100 times across all samples were
discarded. Additionally, sequence length filtering was applied
specifically for each primer set: 40-100 bp for Teleo ; 140-200 bp
for MiFish ; and >100 bp for MG2 . To denoise the
dataset, we used the obiclean function,keeping only sequences
that were more frequently “head” or “singleton” than “internal” in
the global dataset, with “internal” reads being potential PCR
substitutions or indel errors (Giguet-Covex et al., 2014). The MetaBar
package v1.0.0 (Zinger et al., 2021) was used to lower tag jumping and
remove contaminants through detection of ASVs whose relative abundance
was highest in negative controls. Finally, to remove false-positive
results, we discarded not-shared occurrences among technical replicates
(Robasky et al., 2014) (i.e., detected in only one of the three PCR
replicates).