PacBio metabarcoding of environmental samples from pelagic sediment of lake Dagowsee
Sediment samples were thawed and aliquoted into two 2-ml tubes, each containing 200 mg. Beads (0.1 and 1.0 mm zirconium, and 3x 2.5mm glass beads) were added to 0.3 vol of the sample, and sample material was ground at 30Hz (Retsch mill) with 1.2 ml lysis buffer (0.6 ml 5% CTAB-phosphate buffer, 60 l sarkosyl, 60l SDS, 0.6 ml phenol:chloroform isoamyl) for 1.5 minutes, with short breaks for cooling on ice. Samples were then incubated for 1 hr at 65 C, centrifuged, the supernatant washed with an equal volume of chloroform-IAA, precipitated with 2 volumes of PEG/NaCl (30% PEG 6000 in 1.6M NaCl), cleaned with 70% ethanol, and eluted in 50 l nuclease free water. Subsamples were pooled to give 100 l nucleic extract per sample. RNA was removed by the addition of 0.5 l (5g) RNase A (10mg/ml DNase and protease free) to 80 l of the pooled sample, incubated at 37C for 30 minutes, and cleaned up using the PowerClean Pro DNA Clean-Up kit (MoBio). PCRs were conducted in 25-l reactions using 0.5 l Herculase II Fusion enzyme (Agilent Technologies), 5 l of 5x PCR buffer, 0.62 l each primer (10 uM), 0.25 l dNTPs (250 mM each), 0.3 l BSA (20mg/ml BSA) on a SensoQuest labcycler with 2 min denaturation at 95 C, 13-30 cycles of 94 C for 30 sec, 55 C for 30 sec and 70 C for 4 min, and a final elongation at 70 C for 10 min. Multiple PCR reactions (up to 50) were required for each environmental sample to ensure sufficient product for library preparation (1g purified PCR product).