Dna flex library prep kit

Genomic DNA was purified from the phage as previously described with the Promega Wizard DNA clean-up system (Summer, 2009 (link)) after PEG precipitation, prepared as Illumina TruSeq Nano low-throughput libraries with 550-bp inserts using a Nextera DNA Flex Library Prep kit, and sequenced in paired-end 250-bp reads via Illumina MiSeq v2 300-cycle chemistry. The 396,576 sequence reads from the index containing the phage genome were quality controlled with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The phage genome was assembled into a single raw contig via SPAdes v.3.5.0 with 636.7-fold coverage after trimming with the FASTX-Toolkit 0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/) (Bankevich et al., 2012 (link)). PCR amplification across the raw contig ends (forward primer 5′-ctcgttaccagcgcagaaa-3′ and reverse primer 5′-caggtgctaaccaaggtttagg-3′) accompanied by Sanger sequencing of the DNA product verified that the contig sequence was complete. Analyses with PhageTerm indicate a novel ends type (Garneau et al., 2017 (link)).

Short- and long-read WGS was conducted in four CHDL-producing isolates to determine their complete chromosome and plasmid sequences with high accuracy. DNA was extracted with the NucleoSpin tissue kit (Macherey-Nagel, Düren, Germany) for short-read sequencing; in one of the isolates the DNA library was prepared with the Nextera DNA Flex library prep kit and sequenced with Illumina Miseq (Illumina, San Diego, CA, United States); GIEasy FS PCR-Free DNA Library Prep Set was used for the remaining three isolates and sequenced with MGI DNBSEQ (MGI Tech Co., Shenzhen, China). Nanopore MinION (Oxford Nanopore Technologies, Oxford, United Kingdom) was used for long-read sequencing; DNA was extracted with the MagAttract HMW DNA Kit (Qiagen, Hildon, Germany); multiplexed DNA libraries were prepared for all four isolates using the Native Barcoding Kit EXP-NBD104 and the ligation sequence Kit SQK-LSK109; and sequencing was performed on a single R9.4.1 flow cell. Low-quality reads (MinION Q < 10; DNBSEQ Q < 32; and MiSeq Q < 30) and short reads (MinION length <1,000 bp; DNBSEQ and Miseq length <10 bp) were filtered out.

Library preparation was performed using the tagmentation-based Illumina DNAFlex Library Prep kit (Illumina, San Diego, CA, USA), according to the manufacturer’s protocol. Paired-end 300 bp sequencing was executed on an Illumina MiSeq instrument. The raw sequencing reads were aligned to the C. auris B8441 reference genome using the Burrows–Wheeler Aligner algorithm. The genetic variants (single-nucleotide polymorphisms, mutations and indel variants) were determined using the GATK algorithm. The library preparations, sequencing and data analysis were performed at the Genomic Medicine and Bioinformatics Core Facility of the University of Debrecen, Hungary [23 (link)]. Two isolates (28 and 208) from the South Asian clade harbored mutations (S639Y and S639P, respectively) in hot-spot 1 of the FKS1 gene region, as determined by whole genome sequencing. In the case of isolate 20 (South Asian clade), a mutation (R1354H) was found in hot-spot 2 of the FKS1 gene. The remaining 12 isolates showed wild-type genotypes (Table 1).