Single-molecule real-time (SMRT) sequencing developed by Pacific BioSciences (PacBio) offers three major advantages compared to second-generation sequencing: long read length and high consensus accuracy, and a low degree of bias. Together with high sequencing coverage, these advantages overcome the difficulty of sequencing genomic regions such as long AT-rich islands and repeated regions (e.g., ribosomal DNA) in the genome of Trichoderma reesei QM6a. Herein, we describe a protocol for preparing high-quality, high molecular weight genomic DNA for PacBio long-read sequencing, de novo assembly and streamlined annotation of the QM6a genome.
Trichoderma reesei is an industrially important cellulolytic filamentous fungus and a new fungal model organism for studying epigenetics, meiosis and sexual development.(A) Germination of QM6a conidia on a PDA plate. (B) Two single-ascospore cultures of T. reesei env1 mutant were inoculated on a 10-cm MEA plate under a 12-hour light and 12-hour dark photoperiod. (C) T. reesei fruiting bodies and droplets of liquid forming on their surface (D) T. reesei linear asci and each asci contains 16 ascospores. The complete genome sequence of QM6a is publicly available at https://mycocosm.jgi.doe.gov/Trire_Chr/Trire_Chr.home.html