Strategies for sequencing fungal genomes on next-generation sequencing (NGS) platforms depend on the characteristics of the genome of the targeted species, quantity and quality of the genomic DNA, and cost considerations. Massively parallel sequencing with sequencing by synthesis (SBS) approach by Illumina produces terabases of short read sequences (i.e., ~300 bp) in a time and cost-effective manner, though the read length can limit the assembly particularly in repetitive regions. The single molecule, real-time (SMRT) sequencing approach by Pacific Biosciences (PacBio) produces longer reads (i.e., ~12,500 bp) which can facilitate de novo assembly of genomes that contain long repetitive sequences, though due to the lower-throughput of this platform achieving the coverage needed for assembly is more expensive than by SBS. Additionally, the Illumina SBS platforms can handle low quantity/quality of genomic DNA materials, while the SMRT system requires undamaged long DNA fragments as input to ensure that high-quality data is produced. Both platforms are discussed in this chapter including key decision-making points.
Keywords: HiSeq 2500; Illumina; Next-generation sequencing (NGS); Pacific Biosciences; RS II; Sequencing by synthesis (SBS); Single molecule real-time (SMRT) sequencing.