trnL barcoding of lake sediment
DNA extraction was performed with the PowerMax Soil DNA Isolation Kit (MoBio, Carlsbad, USA). The extracts were cleaned with the PowerClean DNA Clean-Up Kit (MoBio, Carlsbad, USA). We PCR-amplified short fragments of the P6 loop of the chloroplast trnL intron with the primers g-h (Taberlet et al., 2007). PCR conditions: denat 97 C for 240 sec; 60 cycles: denat. at 94 C, 45 sec, anneal. 55 C, 45 sec, elong. 68 C, 45 sec; final elong.72 C, 600 sec. 3 amplicon/sample, negative control. The post-PCR cleanup was performed with Sartobind D anion exchanger membranes (Sartorius AG, Gttingen, Germany). PCR repetitions were pooled for each sample, the samples were labeled in a second PCR with modified trnL g-h primers that contained sample multiplexing labels. The post-PCR cleanup was performed with Sartobind D anion exchanger membranes (Sartorius AG, Gttingen, Germany). The labeled samples were normalized according to their estimated DNA concentration and pooled, and the DNA fragment length distribution in the pool was measured with an Agilent High Sensitivity DNA Assay (Agilent Technologies GmBH, Waldbronn, Germany). Paired-end sequencing (2x150 bp) was performed on an Illumina MiSeq sequencer at the Biomedical Genomics Center of the University of Minnesota, U.S.A. DNA sequences were filtered using OBITOOLS (Boyer et al., 2016) along the filtering and taxonomic assignment steps described by Pansu et al., 2015.