MesocosmExpt2_2013
Amplification of the V3 and V4 hypervariable regions of the 16S rRNA gene was performed  using oligonucleotide primers (Klindworth et al., 2013) modified by the addition of Illumina Tags  (underlined): S-D-Bact-0341-b-S-17 (5′-ACGACGCTCTTCCGATCTCCTAC  GGGNGGCWGCAG-3′) and S-D-Bact-0785-a-A-21 (5′-CGTGTGCTCTTCCGATCTGACT  ACHVGGGTATCTA-3′), resulting in amplicons that contained a 16S rRNA gene region of ~440  bp. The HiFi HotStart PCR kit (KAPA Biosystems, Boston, MA, USA) was used with the  following protocol: 0.2 mM dNTPs, 40 ng of DNA template, 0.02 U/μl KAPA HiFi Taq, 0.2 μM of  each primer in a total volume of 50 μl. PCR conditions were: initial denaturation at 95°C for 5 min;  followed by 27 cycles of 95°C for 1 min, 56°C (1 min) and 72°C (1 min); final extension at 72°C (5  min). Amplified products were purified using Agencourt AMPure XP purification kit (protocol  000387v001; Beckman Coulter, Brea, CA, USA) and quantified using Qubit dsDNA High  Sensitivity Assay (Agilent Technologies, Palo Alto, CA, USA). Then a second round of PCR was  performed using TruSeq® RNA PCR Primers (RPI1-33; Illumina, San Diego, CA, USA) to attach  6-bp multiplexing indices and Illumina sequencing adapters using the following protocol: 0.2 mM  dNTPs, 2 ng of the purified PCR product, 0.02 U/μl KAPA HiFi Taq, 0.2 μM of each primer in a  total volume of 50 μl. PCR conditions were: initial denaturation at 95°C for 20 sec; 10 cycles of  95°C (20 sec), 55°C (20 sec) and 68°C (20 sec); final extension at 68°C (40 sec). Amplicons were  purified and quantified as above and run on the MiSeq Illumina sequencing platform, producing 2 ×  250 bp reads (Illumina, San Diego, CA, USA).
University of Otago
DCD_Expt2_C90