COI metabarcoding of earthworms: community DNA
Two-step PCR approach. First-round PCR was performed with a modified version of the mlCOIintF/HCO2198 primer pair tailed with the Illumina sequencing primer sequences. The purified amplicons from this first reaction were dual-indexed in a second-round PCR. Finished libraries were purified with magnetic beads and pooled in equimolar amounts for Illumina MiSeq PE300 sequencing.