Whole genome sequencing of Candida albicans SC5314 CRISPR-competent wildtype and tlo-null derivatives

Whole genome sequencing of Candida albicans SC5314 strain MAY1376 (a Chr5AB disomic isolate from the CRISPR-competent Chr5ABB trisomic AHY940 (doi:10.1128/mSphereDirect.00149-17)) and two independent tlo-null mutants MAY1739 and MAY1740 (all TLO paralogs deleted in MAY1376 via targeted TLO34 CRISPR-Cas9 mutagenesis, then pan-TLO CRISPR-Cas9 mutagenesis). The primary purpose was to genotype both the parental MAY1376 and the mutant MAY1739 and MAY1740 to verify lack of genomic rearrangements and lack of altered ploidy in the tlo-null strains. The secondary purpose was to generate a de novo, telomere-to-telomere, haplotype-phased assembly for MAY1376/wildtype SC5314. Briefly, for short-read sequencing the DNA was extracted from overnight cultures grown at 30C (approx. 10^8 cells) using the Zymogen Quick-DNA Fungal/Bacterial Miniprep kit (Zymogen, cat# D6005). gDNA concentration was quantified using the Qubit dsDNA Broad Range Assay kit (Thermo Fisher, cat# Q32853). Following quantification, the samples were sent to the Applied Microbiology Services Laboratory (AMSL, now IDI-GEMS) at The Ohio State University for processing. Libraries were constructed via tagmentation and dual-index barcoding using a modified protocol for the Illumina (L) Tagmentation kit (Illumina, cat# 20040537) to produce average final fragment sizes of approximately 450-500 bp. The libraries were sequenced for 2x150 paired-end reads on an Illumina NextSeq 2000. Reads were demultiplexed and Illumina adaptors were trimmed by AMSL. Briefly, for long-read sequencing intact cell pellets of MAY1376, MAY1739, and MAY1740 were sent to the University of Wisconsin-Madison's Biotechnology Center (UWBC). UWBC performed high molecular weight DNA extraction on the samples to prepare Oxford Nanopore barcoded libraries, with DNA fragment lengths being on average ~20 kb and some fragments reaching 70+ kb. Libraries were then sequenced on a PromethION 24 using a FLO-PRO114M (R10.4.1) flow cell and an SQK-NBD114-24 (V14) kit for 12 hours. Prior to data delivery, basecalling was performed by UWBC using Guppy 6.4.6 (high-accuracy model, 400 bps) and reads were demultiplexed.