Genomic sequencing of Histoplasma ohiense G217B isolate UCSF2
Growth and DNA extractionHistoplasma growth conditions were performed as described previously unless otherwise indicated (Assa et al https://doi.org/10.1101/2023.04.21.537729).  For extraction of genomic DNA to be used for long read Nanopore sequencing and associated short read sequencing of these strains, 2-10 ml of a 3 day liquid culture was pelleted (2500 rpm 5 minutes), washed once in TE buffer (spin at 13000 rpm for 1 min), then stored at -80 C until DNA extraction. DNA extraction was then performed using components of the Qiagen Gentra Puregene Yeast/Bacteria kit (158567) with a slightly modified protocol. Wide-mouth pipette tips were used when pipetting cell lysate or DNA, and vortexing was avoided other than as noted. Histoplasma cell pellets were resuspended in 300 l Cell Suspension buffer, approximately 100 l 0.5 mm glass beads were added, the mixture was thrice vortexed for 10 seconds and put on ice for 10 seconds between each vortex. 4 l of the Lytic enzyme was then added, this mix was inverted 25 times, then incubated for 2 hours at 37 C with inversion 3 times an hour. This mixture was then pelleted at max speed for 1 minute at 4 C, supernatant was discarded, 300 l Cell Lysis Solution was added, then the mix was slowly pipetted several times and incubated at ambient temperature for 10-20 minutes. 100 l Protein Precipitation Solution was then added, the mixture was immediately vortexed for 5 seconds, put on ice, vortexed for 1 second, then put on ice for 5 minutes. This mix was pelleted at max speed for 3 minutes at 4 C. Supernatant was then transferred to a tube containing 300 l isopropanol and inverted 50 times to mix. This sample was pelleted at max speed for 2 minutes at 4 C. Supernatant was removed leaving approximately 50 l so as not to disturb the DNA pellet. 500 l 70% Ethanol wash was added, then DNA was pelleted at max speed for 1 minute. Supernatant was fully removed, pellet was air dried for 15 minutes, then 70 l DNA Hydration Solution was added along with 1.5 l RNase A Solution. This was gently mixed by slowly pipetting up and down 10 times, then incubated at 37 C for 5 minutes, 65 C for 10 minutes, then overnight at 4 C to dissolve the pellet. Nanodrop and TapeStation (Agilent Genomic DNA reagents) were used to assess quality and concentration prior to library preparation and sequencing at SeqCenter.Illumina sequencing (SeqCenter)Illumina sequencing libraries were prepared using the tagmentation-based and PCR-based Illumina DNA Prep kit and custom IDT 10bp unique dual indices (UDI) with a target insert size of 280 bp. No additional DNA fragmentation or size selection steps were performed. Illumina sequencing was performed on an Illumina NovaSeq X Plus sequencer in one or more multiplexed shared-flow-cell runs, producing 2x151bp paired-end reads. Demultiplexing, quality control and adapter trimming was performed with bcl-convert1 (v4.2.4).