Lake Towuti LTP6 amplicon seq
Using stringent precautions to prevent contamination, environmental DNA was extracted using the DNeasy PowerMax Soil Kit (Qiagen) from up to 10 g sediments from Lake Towuti. The trnLP6 loop (Taberlet et al., 2007) was amplified using the modified eco-PCR tested primers trnL-G2 and trnL-H2 after Riaz et al. (2011). Each initial round of PCR included triplicate reactions of extraction- and background blanks. One microliter of these trnL-P6 amplicons and the amplified background and extraction blanks served as a template for a nested PCR using the same primers carrying the required Illumina adapters. In addition, the reverse primers in the nested PCR carried 12 bp Golay barcodes after Caporaso et al. (2012) to support the pooling of samples and contamination controls for subsequent MiSeq Illumina sequencing. Identical barcodes were used for triplicate reactions of each sample and background vs extraction blanks. Equal amounts of each barcoded amplicon were mixed and gel-purified, and the resulting library was sequenced at the Australian Genome Research Facility (AGRF) in Melbourne; paired-end; 300 cycles. The sequence run was processed using Obtiools.