Hiseq x

Several tissues were collected from a single male Atlantic Halibut. Muscle and spleen were snap frozen in liquid nitrogen and stored at -80°C until further use. Fresh blood was used to isolate erythrocyte nuclei and then prepare High Molecular Weight (HMW) DNA from these nuclei according to a Phenol/Chloroform protocol (Quick). Extracted DNA was either left unfragmented or sheared using either MegaRuptor (Diagenode) or by passing DNA through a 100μl pipette tip 10–20 times. From these DNA samples we produced MinION sequencing libraries with the standard adapter ligation protocol kit (LSK-108), 1D^2 sequencing kit (LSK-308) and the RAD002 and RAD003 kits (Oxford Nanopore Technologies). For libraries prepared with the RAD002 and RAD003 kits we used a protocol developed to produce ultra–long reads (Quick, protocols.io). In total we used 9 flow cells and sequenced 18.8 Gbp in 2,963,108 reads with a read N50 of 17.8 kb. Sequenced fast5 raw signal data files were basecalled using albacore v.2. The size distribution of sequenced reads is presented in S1A Fig. We also used the collected muscle samples to produce HiC libraries (Dovetail), sequenced on one lane of HiSeq X (Illumina). One of the DNA preps inferred to have HMW DNA was used to prepare a linked reads Chromium library (10x genomics) and was sequenced on one lane of HiSeqX (Illumina).

Total DNA from each sample was sent to Floodlight Genomics, LLC. Floodlight Genomics used an optimized Hi-Plex approach to amplify and barcode targets in a single multiplex PCR reaction followed by sequencing on an Illumina HiSeqX device running a 2x150bp paired-end configuration, as previously described [27 (link)]. Primers were designed to amplify three 150 bp regions to examine known SNPs previously correlated with acaricide resistance in T. urticae (S1 Table), G126S (Cytochrome b) [23 (link)], F1538I (Voltage gated sodium channel) [19 (link)], and I1017F (Chitin synthase 1) [20 (link)]. The sample-specific barcoded amplicons were sequenced on the Illumina HiSeq X platform according to the manufacturer’s directions. Floodlight Genomics delivered sample-specific raw DNA sequence reads as FASTQ files. Annotation of the raw reads was performed with Geneious Bioinformatics Software (Auckland, New Zealand). The reads were then mapped to reference sequences at 100% stringency to classify genotype. The genotype ratio was calculated for each population. Each population was designated with a wildtype or F15381/I1017Fgenotype if ≥90% of the reads corresponded to that associated genotype.

Quinolone resistant E. coli isolates were plated onto MacConkey agar with ciprofloxacin (0.06 mg/L) to confirm resistance, while WT isolates were plated onto MacConkey agar. Following incubation at 41.5°C for 21 h, bacteria were harvested directly from the agar plates and DNA was extracted with the QIAmp DNA mini kit (QIAGEN), according to the manufacturer’s instructions. The DNA concentration and purity was determined using a Qubit (QIAGEN) and NanoDrop ONE spectrophotometer (Thermo Scientific), respectively. Gel electrophoresis was used to determine the DNA integrity.
A total of 95 isolates were sequenced in this study, using Nextera DNA Flex library preparation (Illumina) followed by sequencing on HiSeq X (Illumina) spiked with PhiX. The remaining 11 isolates were previously sequenced using Nextera XT and HiSeq 2000 (n = 4) or HiSeq 2000R (n = 3), or Nextera DNA Flex and HiSeq 3000 (n = 1) or HiSeq X (n = 3). Library preparation and sequencing was done at the Norwegian Sequencing Centre.²

The assembly used multiple sequencing technologies. Long read libraries were prepared with SMRTbell Template Prep Kit 1.0 and 70 SMRT cells were sequenced on the PacBio Sequel system with v2.1 chemistry (Pacific Biosciences; 276.86 Gb data). Linked reads were sequenced from HMW DNA with Chromium libraries (10x Genomics) on an Illumina HiSeq X (2 × 150 bp; 269.75 Gb of data). Dovetail Genomics prepared three HiC libraries which were sequenced on an Illumina HiSeq X (2 × 150 bp paired-end reads; 121.47 Gb data, Supplementary Table 8).