Mockcommunity DNA metabarcode
All 20 DNA extracts were pooled as one mock sample, and Elbrecht and Steinke's (2019) fusion primers (BF2 and BR2) for freshwater arthropods were used to construct amplicon libraries following a one-step polymerase chain reaction (PCR) protocol. The PCR master mix consists of 0.25 l Phusion, 0.75 l DMSO, one l dNTPs, 1.25 l each of the fusion forward and reverse primers (10 M), five l HF Buffer (New England Biolabs), and 15.5 l of PCR-grade water. PCR cycling conditions were 30 seconds of initial denaturation at 98C, followed by 20 cycles of 10 seconds denaturation at 98C, 30 seconds annealing at 55C, 30 seconds extension at 72 C, and a final extension step of 5 minutes at 72C. The same PCR protocol was done for eleven other cycles from 24 to 64 (increments of 4) in triplicate PCR amplification, including negative controls. The resulting 72 PCR amplicons were pooled according to replicates and size selected via solid-phase reversible immobilization (SPRI) beads. Each replicate was quantified via qPCR and quality assessed via BioAnlyzer DNA1000 Assay before sequencing. The replicates were normalized to 2nM and pooled. Paired-end sequencing of the 2nM pooled library (diluted to 6 pM) spiked with 20% PhiX was performed in paired-end 300 cycles sequencing performed on the Illumina MiSeq system.