2.2 Molecular laboratory processes
We cleaned up each sample (comprised of 20 pitfalls pooled) following a
Flotation-Filtration-Stereoscope protocol (FFS) that allowed us to have
a ‘clean’ extraction (see Figure S1b-g). To prevent differences of
biomass from causing biases at the DNA extraction and PCR steps
(Elbrecht & Leese,
2015), we divided the specimens by size into: small (e.g., sizeDrosophila melanogaster ), medium (e.g., size Apis
mellifera ), or large specimens (e.g., adult grasshopper). We then
divided each sample in two subsamples, one including the complete bodies
from small arthropods, and the second with the thorax (head included)
from medium-sized arthropods and two legs from large arthropods.
Subsamples (n=84) were processed independently for DNA extraction and
library construction, but using the same library barcode identifier
(thus, the final number of libraries retrieved was 42, one library per
sample). For DNA extractions, we treated each bulk-sample with an
electric homogenizer (Qiagen TissueLyser II: two times for 1 min at 30
Hz and 30 s at 30 Hz) using platinum beads with Qiagen DNeasy Blood &
Tissue Kit and BSA buffer 1x.
For metabarcoding library construction, we used the double dual tagging
method with the XT Illumina Adapter Kit as in
Arribas et al.
(2016). We amplified a 418-base pair region from the 5’ end of
mitochondrial COI gene (within the standard barcode region for metazoa)
with the primers B_F 5’ CCIGAYATRGCITTYCCICG 3’
(Shokralla et al.,
2015) and Fol-degen-R 5’ TANACYTCNGGRTGNCCRAARAAYCA 3’
(Yu et al., 2012)
modified to include Illumina overhang adaptors for subsequent nested
PCR. For each subsample (n=84), we performed and pooled together three
independent PCR replicates for each arthropod-size subsample. We
included a negative control reaction with no DNA template in all
experiments. All information regarding PCR reagents and conditions is
available in Material S1 (Supporting Materials S1). Each pool of PCR
amplicons was cleaned with Agencourt AMPure XP beads (Beckman Coulter)
to purify the COI amplicon away from remaining primers and primer
dimers. Then, we used primary amplicons as template for a limited-cycle
PCR amplification to add dual-indices barcodes (N7 index and S5 index)
and the P5 and P7 Illumina sequencing adapters using the Nextera XT
Index Kit from Illumina. We sent the 42 resulting metabarcoding
libraries and a negative control to the Cornell Institute of
Biotechnology, Cornell University, USA, for sequencing on a lane of
Illumina MiSeq 2x300 bp (Figure S2a-f).