Metabarcoding
The bioinformatic pipeline applied is described in Brandt et al. (2019).
Primers and leftover adapters were removed with the program Cudadapt
(Martin, 2011). The basic pipeline was then based on DADA2 using
stringed error correction (Callahan et al., 2016), including fragment
size selection with an expected length of 300-500 bp for both 16SV4 and
18SV1V2, followed by a chimera removal step. Sequences were then
clustered into operational taxonomic units (OTUs) using the program
swarm2 (Mahé, Rognes, Quince, de Vargas, & Dunthorn, 2015) with an
iterative local threshold (d) of 4 for 18SV1V2 and 1 for 16SV4. The
ribosomal sequences were taxonomically assigned against the Silva
database (release 132; Pruesse et al., 2007) with the MegaBlast
algorithm (Camacho et al., 2009).
A decontamination step was applied after clustering, using extraction
and PCR negative controls using decontam (Davis, Proctor, Holmes,
Relman, & Callahan, 2018), by the prevalence method with a threshold of
0.8. Finally, all OTU counts were adjusted using an R-based script
(Wangensteen, Palacín, Guardiola, & Turon, 2018) to renormalize
potential tag switches during library preparations (Schnell, Bohmann, &
Gilbert, 2015).