2.3.2. Capture and sequencing
Illumina
libraries were constructed from eDNA samples using the Nextera XT (for 9
samples) or TruSeq (for 1A) Kits (Illumina). Biotinylated RNA capture
probes were obtained by in vitro transcription following the
protocol described by Ribière et al. (2016). Hybridization capture was
carried out independently for each sample with 16S and 18S rDNA probes
at a ratio of 50/50, as described by Gasc and Peyret (2018). In brief,
500 ng of sequencing library mixed with 2.5 μg of salmon sperm DNA was
incubated with 500 ng of biotinylated probes in a hybridization buffer
(10 × SSPE, Denhardt’s 10X solution, 10 mM EDTA and 0.2% SDS) for 24
hours at 65°C. After hybridization, the probe/target heteroduplexes were
captured with 500 µg of paramagnetic beads coated with streptavidin
(Dynabeads M-280 Streptavidin, Invitrogen). Beads were collected using a
magnetic stand (Ambion), washed once with 500 μl of 1x SSC/0.1% SDS
buffer, and then three times with 500 μl of 0.1 × SSC/0.1% SDS buffer
preheated at 65°C. The captured DNA fragments were eluted with 50 µl of
0.1 M NaOH and transferred to a sterile tube containing 70 µl of 1 M
Tris-HCl pH 7.5 buffer. Captured libraries were finally PCR-amplified
with 25 cycles using primers fully complementary to Illumina adapters.
To increase the enrichment efficiency, the second round of capture was
performed from the first-round capture products. Captured libraries were
then sequenced with two Illumina MiSeq 2x300 bp runs.
2.4. Sequencing data processing and analysis