DNA extraction, PCR amplification and sequencing
We extracted DNA from each bulk subsample using the Biosprint 96 DNA
Blood Kit (Qiagen) on a Thermo KingFisher Flex automated extraction
instrument. We quantified the DNA concentration using a NanoDrop
spectrophotometer and combined the extracts of each pair of subsamples
at a 1:10 ratio (Arribas et al., 2021b). Then, we PCR amplified the 3’
end of the cytochrome c oxidase subunit I (COI) barcode region
corresponding to the 418 bp bc3’ fragment using the Ill_B_F (Shokralla et al., 2015) and Fol-degen-rev (Yu et al., 2012)
primers. Amplifications were performed following the PCR conditions
described in Arribas et al. (2016). We carried out five independent PCR
replicates per sample, visualised the PCR products on an agarose gel and
pooled the three amplicons per sample that presented the brightest
bands, which were purified using a magnetic bead-based protocol
(Agencourt AMPure XP). We included three negative controls corresponding
to different wet-lab steps (lysis, DNA extraction and PCR
amplification). The 88 metabarcoding samples and the three negative
controls were used for a dual-indexed library preparation following the
Nextera XT DNA workflow (llumina, San Diego, CA, USA) and were sequenced
on a paired-end 2x300 bp lane of an Illumina MiSeq platform at the
Earlham Institute (Norwich, UK).
We individually extracted DNA from each of the ‘voucher’ specimens as
described above. We amplified the Folmers COI barcode region (658 bp,
overlapping with the 418 bp metabarcoding fragment), using the primers Fol‐degen‐for and Fol‐degen‐rev (Yu et al., 2012) and
following the PCR conditions described in Arribas et al. (2016). The PCR
products were purified and bidirectionally Sanger sequenced (Macrogen,
South Korea). The generation of a curated ‘voucher’ reference catalogue
allowed us to improve the taxonomic assignment of the metabarcode reads
and to apply a recently developed method for read filtering
(metamate, Andújar et al., 2021, see below).