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.