Nanopore MinION sequencing of Ca. Odinarchaeum LCB_4: long-range PCR gap-closing fragments
Four regions were targeted for long-range PCR amplification: two assembly gaps spanning estimated lengths of 5-10 kb, and two positive control regions spanning approximately 5 (surrounding the rRNA operon) and 10 (surrounding multiple ribosomal protein genes) kilobases. MDA-amplified DNA from the Lower Culex Basin at Yellowstone National Park (USA) was then amplified with primers for these four regions using Herculase polymerase. Products were separated in agarose gel, purified, pooled, and ligated for library construction. Sequencing was performed on an Oxford Nanopore MinION Mk1C sequencer using an R9.4.1 flow cell, and basecalling was performed using Guppy v4.2.2 (High Accuracy)