Page 108 - Microbial methane cycling in a warming world From biosphere to atmosphere Michiel H in t Zandt

Chapter 5. Early Holocene carbon storage and microbial activity in North Sea peats
1 min at 10,000 xg. DNA quality was assessed by agarose gel electrophoresis, spectrophotometrically using a NanoDrop 1000 (Invitrogen, Thermo Fisher, Carlsbad, CA, USA) and fluorometrically using the Qubit dsDNA HS Assay Kit (Invitrogen, Thermo Fisher, Carlsbad, CA, USA) according to the manufacturer’s instructions. Duplicate samples with the highest yield and quality were selected for downstream application.
Amplicon sequencing, analysis and quantification
Amplification. For DNA purification, the QIAquick PCR Purification Kit was used (Qiagen, Venlo, the Netherlands). For DNA amplification, a 2-step amplicon sequencing protocol was used. In the first step, the V3-V4 region of the bacterial 16S rRNA gene was amplified using the universal primers Bac 341F (5′-CCTACGGGNGGCWGCAG-3′) (Herlemann et al. 2011) and Bac785R (5′-GACTACHVGGGTATCTAATCC-3′) (Klindworth et al. 2013) for 30 cycles. Archaeal 16S rRNA genes were amplified with the universal archaeal primers Arch349F (5′- GYGCASCAGKCGMGAAW-3′) (Takai and Horikoshi 2000) and Arch806R (5′- GGACTACVSGGGTATCTAAT-3′) (Takai and Horikoshi 2000) for 30 cycles. All primers were purchased from Biolegio (Biolegio B.V., Nijmegen, the Netherlands).
The following cycling parameters were used for the polymerase chain reaction (PCR): initial denaturation for 10 min at 98°C; 25/30 cycles of denaturation for 1 min at 95°C, annealing for 1 min at 60°C, and elongation for 2 min at 72°C; and a final elongation step for 10 min at 72°C. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Venlo, the Netherlands) in two elution steps. A 20-μl aliquot of 55°C Milli-Q water was added to the spin column and incubated for 2 min prior to centrifugation. Next, the eluate was added to the spin column, incubated at 55°C for 2 min, and centrifuged again as described in the manual. The purified PCR products were used in a second 10-cycle nested PCR performed with IonTorrent adapters using the PCR protocol described above. After purification with the QIAquick PCR Purification Kit as described earlier, the PCR products were used for library preparation and sequencing steps according to the manufacturer’s instructions (Life Technologies, Carlsbad CA, United States).
Sequencing. Samples were sequenced on an Ion 318 Chip Kit v2 (Thermo Fisher, Waltham, MA, USA). Amplicon sequences were quality checked for chimeras and clustered into OTUs
106