C4v2 chemistry

Details of the strains used in this study are presented in Table 1 and additional details were previously reported^{2 (link),18 (link),19 (link)}. Isolates were grown on Sabouard Dextrose media supplemented with chloramphenicol and gentamycin and incubated for 24–48 h at 37 °C. For Illumina sequencing, genomic DNA was extracted using the Quick-DNA™ (ZR) Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA, USA). Genomic libraries were constructed and barcoded using the NEBNext Ultra DNA Library Prep kit (New England Biolabs, Ipswich, MA, USA) by following manufacturer’s instructions. Genomic libraries were sequenced using either Illumina HiSeq 2500 with HiSeq Rapid SBS Kit v2 or Illumina MiSeq platform using MiSeq Reagent Kit v2 (Illumina, San Diego, CA, USA). For PacBio sequencing, DNA was extracted using MasterPure™ Yeast DNA Purification Kit (Epicenter, Madison, WI, USA). Single-molecule real-time (SMRT) sequencing was done using the PacBio RS II SMRT DNA sequencing system (Pacific Biosciences, Menlo Park, CA, USA). Specifically, 20-kb libraries were generated with the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences). Libraries were bound to polymerase using the DNA/Polymerase Binding Kit P6v2 (Pacific Biosciences), loaded on two SMRTcells (Pacific Biosciences), and sequenced with C4v2 chemistry (Pacific Biosciences) for 360 min movies.

Mmyc_Sud5 isolate from a patient from El Gaziera State was selected for long read sequencing using PacBio RS II Sequel system sequencing system (Pacific Biosciences, Menlo Park, CA, USA) to improve assembly. This isolate was selected because most patients in our study were from this state. Twenty kb libraries were generated with the SMRTbell template prep kit 1.0 (Pacific Biosciences). Libraries were bound to polymerase using the DNA/polymerase binding kit P6 v2 (Pacific Biosciences), loaded on two SMRT cells (Pacific Biosciences), and sequenced with C4 v2 chemistry (Pacific Biosciences).