2.3.1. Design of capture probes and in silico tests
Previously, 16S rDNA hybridization capture was successfully enforced to
describe the archaeal and bacterial diversity of soil microbial
communities. Here, we applied this approach to deep-sea samples by
adding 18S rDNA gene capture to specifically target eukaryotes. Probes
were designed to target 16S and 18S rDNA genes to ensure specificity
against the targeted genes while allowing the detection of sequence
variants not yet described in databases by selecting degenerate probes
(Table 3). Probes targeting 16S rDNA were previously published (Gasc &
Peyret, 2018). In this work, probes targeting 18S rDNA were determined
as described in a previous publication. Briefly, probes were designed
using KASpOD (Parisot, Denonfoux, Dugat-Bony, Peyret, & Peyretaillade,
2012) and PhylArray algorithms (Militon et al., 2007) and a custom
curated database derived from all 18S rDNA sequences from the EMBL and
similarly built for PhyloPDb development (Jaziri et al., 2014).
Selection of probes distributed over the entire length of the gene and
their length (31 to 50 bp) allow specific hybridization with their
target even if mismatches are present. For each set of probes, coverage
among SSU sequences from the SILVA database (release 132; Quast et al.,
2013) was predicted by mapping probes with BBDuk (k=13, copy undefined
mode, mm=f; http://jgi.doe.gov/data-and-tools/bb-tools/ ).
Finally, adaptors containing the T7 promoters “ATCGCACCAGCGTGT” and
“CACTGCGGCTCCTCA” were added to the 5’ and 3’ ends of probes,
respectively, to enable PCR amplification (Ribière et al., 2016). These
probes were designated capture probes.