Pacbio rs 2 smrt dna sequencing system

Whole-genome sequencing (WGS) was performed using the Illumina NovaSeq platform (by Berry Genomics Co., Beijing, China). Yeast cells of C. auris grown on YPD medium at 37°C for 16 h were used for genomic DNA extraction. The genome DNA of each isolate was extracted using a standard zymolyase protocol. For all 93 C. auris isolates, a paired-end library with an average insert size of 300 bp was prepared and sequenced using the Illumina NovaSeq platform with 2 × 150-bp reads. In addition, for one isolate (RICU_A1), single-molecule real-time (SMRT) sequencing was performed at Beijing Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) using the PacBio RS II SMRT DNA sequencing system (Pacific Biosciences, Menlo Park, CA, USA). Specifically, 20-kb libraries were generated with the SMRT bell TM Template Prep Kit 1.0 (Pacific Biosciences). The sequence data from the Illumina platform were used to proofread the PacBio assembly sequence.

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.

The DNAs were each amplified in triplicate using barcoded primers targeting the V4, V1–V3, V3–V5, V1–V5, V1–V6, and V1–V9 variable regions (Table 1). The primers were synthesized so that the 5′ end of the forward and reverse primers were each tagged with paired 16-nt symmetric barcodes (https://github.com/PacificBiosciences/Bioinformatics-Training/wiki/Barcoding-with-SMRT-Analysis-2.3) to allow multiplexing of samples within a single sequencing run. Methods describing PCR, amplicon cleanup, and pooling were described previously (Kozich et al., 2013 (link)). The SMRTbell adapters were ligated onto the PCR products and the libraries were sequenced by Pacific Biosciences using the P6-C4 chemistry on a PacBio RS II SMRT DNA Sequencing System. Diffusion Loading was used for regions V4, V1–V3, and V3–V5 and MagBead loading was used for regions V1–V5, V1–V6, and V1–V9. Each region was sequenced separately using movies ranging in length between 180 and 360 min. The sequences were processed using pbccs (v.3.0.1; https://github.com/PacificBiosciences/pbccs), which generates predicted error rates using a proprietary algorithm.