title: MiSeq of freshwater invertebrates 16S gene: eDNA
design: Samples were PCR amplified with a four-step library preparation protocol. The first step of four cycles used assay-specific primers (COIAF: 5AGTGTTYTKATTCGTTTRGAGCTWAGKGC3, R: 5GTWAGWTTTTGGGTTYTACCTRTC3 or COIBF: 5GGATTYGGKGGKGGTTGAAC3, R: 5ACWTCTATTGGTGTKACMAGWTTYC3) to append 21nt APEX tails (F:5GATCAGGCGTCTGTCGTGCTC3, R:5CTCGACGACAGACGCCTGATC3) to the target regions, which aimed to reduce PCR bias by retaining the relative ASV proportions of the sample throughout library preparation (Krjutkov et al., 2008). PCRs contained 1X PCR buffer, 0.2mM dNTPs, 0.5M of each primer, 1U AmpliTaq, 2.5l template DNA, and the necessary volume of ddH20 to constitute 25l. Conditions were: 30 sec initial denaturation at 95C, followed by four cycles of 95C for 30 sec, 62C for 30 sec, and 72C for 1 min, with 2min final extension at 72C. Next, the APEX primers were used to amplify targets adhered with the APEX tail, using the same PCR conditions for 35 cycles. The third step amplified previous products using APEX primers appended with 717nt (termed spacers) and a 3334nt sequencing primer on the 5 end. The spacer region increased nt diversity of sequence reads and enhanced cluster formation, improving sequencing quality (Fadrosh et al., 2014; Wu et al., 2015). Four primer sets were used, which differed in their spacer combinations (see Klymus et al. 2017). PCR conditions were identical, except that the cycle number was reduced to eight. The final (fourth) PCR incorporated Nextera paired-end indices (Illumina, kit FC-121-1011), as well as the P5 and P7 adaptor sequences, allowing the sample to bind onto the Illumina MiSeq flowcell. The 25l PCR reactions contained a variable volume of ddH20, 1X PCR buffer, 0.2mM dNTPs, 2.5l of each primer, 1.57U NEB Hotstart Taq polymerase (New England Biolabs Inc.), and 2.5 L of the previous PCR cleanup. Conditions were: 30sec initial denaturation at 95C, followed by 8 cycles at 95C for 30 sec, 55C for 30sec, and 68C for 1min, with a final 2min 68C extension. Four samples were run with the Mol16S primer set followed the library preparation, which was published in Klymus et al. (2017). Each library was normalised and samples subsequently pooled. Libraries were run at a 6 pM concentration on an Illumina MiSeq using the 2 x 300 bp V3 chemistry. In order improve clustering during the initial sequencing cycles 50% of PhiX genomic library was added.