Amplicon library of bDNA of 10 invertebrate species mock  community number 2
A COI region was amplified with the BF2/BR2 primer set  (Elbrecht et al, 2017) with Illumina tails. PCR reactions were performed in a  total volume of 25 L with 1X of PCR buffer (including 3mM MgCl2 and 400 M  each dNTP, QIAGEN Multiplex PCR kit, Qiagen, Germany), 0.5 M of each primer  and 10 ng of template DNA for bulk DNA or 5 L of template DNA for etDNA. The  amplification consisted in an initial denaturation at 95C for 5 min,  followed by 35 cycles of denaturation at 95C for 30 s, annealing at 50C for  30 s and extension at 72C for 2 min, with a final extension at 72C for 10  min. PCR products were purified with Agentcourt AMPure XP purification beads  (Beckman Coulter, USA) and quantified using QuantiFluor dsDNA System  (Promega, USA). Ten ng of each purified PCR products were then used in a  second PCR to dual index each sample with a unique tag combination and to add  Illumina adapters. PCR reactions were performed in a total volume of 25 L  with 1X of PCR buffer (BIOAmp Blend Mix, Biofidal, France), 2,5 nM of MgCL2,  200 M of each dNTP, 0,25 M of Illumina primer and 0,02 U/L of HOT BioAmp  Taq (Biofidal, France). PCR products were purified with Agentcourt AMPure XP  purification beads (Beckman Coulter, USA), quantified using QuantiFluor dsDNA  System (Promega, USA).