PICO metagenome
Nucleic acids were extracted as described previously (Massana et al., 1997), with some modifications. In brief, the cells were lysed by bead-beating on ice three times for 30 s in lysis solution (0.75 M sucrose, 40mM EDTA, 50mM Tris pH 8.0), followed by consecutive incubations with lysozyme (60mgml1; 37 C) and SDS (1%; 55 C). DNA was purified by phenol-chloroform extraction, RNase treatment, isopropanol precipitation and PCR inhibitor removal (Zymo, Irvine, CA, USA). DNA concentration was measured using a NanoDrop ND-1000.