2.2 Reference Sequencing and Assembly
We first deep-sequenced and assembled a reference draft genome from a specimen representing N. homalea (Ach.) Rundel & Bowler for identifying mycobiont loci by mapping during the processing of metagenomic RADseq data (Niebla culture #47: USA, California, Sonoma Co., Sonoma Coast State Park, Shell Beach, 38.4225, -123.114, on large rock, 25 July 2018, Coll: MH Huhndorf). DNA from this specimen was isolated using the ZR Fungal/Bacterial DNA MiniPrep Kit (Zymo Research, Irvine, CA, USA), converted into libraries with the KAPA Hyper Prep Kit (KAPA Biosciennces, Wilmington, MA, USA) and sequenced at the University of Illinois at Chicago Research Resource Center on Illumina’s NextSeq platform as before for lichen-forming fungi (Grewe et al., 2018). High-molecular weight DNA isolation, long-read sequencing on a Nanopore GridIONx5 sequencer, and assembly of N. homalea was done as described before for the lichen fungal culture of Physcia stellaris (Wilken et al., 2020). The pipeline used canu v1.8 (Koren et al., 2017) for a long-read assembly with a genome size estimation of 26 megabases. The raw contigs were corrected twice with racon v1.3.2 (Vaser et al., 2017) and subsequently polished twice with the Illumina short reads of N. homalea using Pilon v1.23 (Walker et al., 2014). Finally, we created a Bowtie2 (Langmead and Salzberg, 2012) database from the selected scaffolds for the mapping approach to filter for fungal RAD loci.