Nanopore Flongle barcoding of Chilean plants in Bosque de Fray Jorge National Park
Per sample, we amplified 6 universal plant barcodes in either single-plex or multi-plex PCR reactions: trnL, rbcL, ITS-2, matK, psbA, and ITS-1. We verified PCR success by running 3 l of PCR product on a 1.5% agarose gel. Amplicons were pooled and library-prepped with the Oxford Nanopore Ligation Sequencing Kit v14 and Flongle Expansion. Final libraries were loaded onto Flongle flow cells and sequenced for 24 hours. Raw data outputs were basecalled with Dorado software and demultiplexed with ngsfilter in obitools.