Pursuing Advances in DNA Sequencing Technology to Solve a Complex Genomic Jigsaw Puzzle: The Agglutinin-Like Sequence () Genes of .

The agglutinin-like sequence () gene family encodes cell-surface adhesins that interact with host and abiotic surfaces, promoting colonization by opportunistic fungal pathogens such as . Studies of Als protein contribution to adhesion would benefit from an accurate catalog of gene sequences as well as insight into relative gene expression levels. Even in the genomics era, this information has been elusive: genome assemblies are often broken within genes because of their extensive regions of highly conserved, repeated DNA sequences and because there are many similar genes at different chromosomal locations. Here, we describe the benefit of long-read DNA sequencing technology to facilitate characterization of loci. Thirteen loci in strain MYA-3404 were deduced from a genome assembly constructed from Illumina MiSeq and Oxford Nanopore MinION data. Although the MinION data were valuable, PCR amplification and Sanger sequencing of loci were still required to complete and verify the gene sequences. Each predicted Als protein featured an N-terminal binding domain, a central domain of tandemly repeated sequences, and a C-terminal domain rich in Ser and Thr. The presence of a secretory signal peptide and consensus sequence for addition of a glycosylphosphatidylinositol (GPI) anchor was consistent with predicted protein localization to the cell surface. TaqMan assays were designed to recognize each gene, as well as both alleles at the divergent locus. cells grown in five different conditions showed differential expression of various genes. To place the data into a larger context, TaqMan assays were also designed and validated for analysis of gene expression in and . These comparisons identified the subset of highly expressed genes that were predicted to encode proteins with the most abundant cell-surface presence, prioritizing them for subsequent functional analysis. Data presented here provide a solid foundation for future experimentation to deduce family contributions to adhesion and pathogenesis.