Minion sequencing device

Five tumor DNA samples were sequenced on a MinION sequencing device (Oxford Nanopore Technologies, ONT). CNVs in tumor DNA were assessed using the SMURF-seq protocol as described previously [45 (link)]. More specifically, tumor DNA was fragmented into short fragments with Anza 64 SaqAI restriction enzyme (Thermo Fisher, Waltham, MA, USA) then randomly ligated to form a long DNA fragment using Anza T4 DNA Ligase Master Mix (Thermo Fisher, Waltham, MA, USA). Then, a rapid barcoding kit (SQK-RBK004, ONT) was used for library preparation according to the manufacturer’s protocol. Sequencing of the tumor DNA was performed on a single R9.4/FLO-MIN106 flow cell (ONT) on a MinION Mk1B.
Raw data from sequencing was generated with MinKNOW software version 1.7.14 (ONT) and converted to FAST5 files that were used for base calling with filter quality read score > 8; data was then de-multiplex barcoded with Guppy version 2.3.4 software (ONT) into FASTQ files. Then, reads were mapped to a human reference genome (GRCh37) with Minimap2 version 2.17 software [56 (link)], and BAM files were created with Samtools version 1.10 [57 (link)]. BAM files were sorted and converted to BED format using bamtobed from Bedtools package version 2.25. The BED files were used as an input file for Gingko [54 (link)] to perform CNV analysis for each sample.

Total genomic DNA from the isolates was extracted from an overnight culture (14–16 h) grown at 37°C on blood agar using the fully automated QIAsymphony instrument (Qiagen, Germany) according to the manufacturer’s instructions. The extracted DNA was quantified using NanoDrop One spectrophotometry (Thermo Fisher Scientific, MA, USA) and Qubit 3.0 Fluorometry (Life Technologies, CA, USA) and stored at −20°C until further use.
The genomic DNA samples were subjected to whole genome sequencing using the Ion Torrent PGM platform with Ion 316™ chip v2 for 400bp chemistry sequencing (Life Technologies, Carlsbad, CA). For this, DNA library was prepared using 1 g of the genomic DNA using Ion Xpress Plus Fragment Library Kit (Life Technologies) following the protocol recommended for 400 bp fragment library preparation. For long read sequencing, Oxford Nanopore MinION sequencing device was used with FLO-MIN106 R9 MinION flow cells. Long read DNA library was prepared using the SQK-LSK108 ligation sequencing kit (v.R9) along with ONT EXP-NBD103 Native Barcode Expansion kit following the manufacturer’s protocol (Oxford Nanopore Technologies, Oxford, UK). The library was loaded onto the flow cells, run for nearly 48 h using the standard MinKNOW software. The Fast5 files generated from MinION sequencing were subjected to base calling with Albacore software (v.2.0.1).

Two DNA samples putatively positive for F. tularensis by microfluidic qPCR were whole genome amplified (RepliG midi kit, Qiagen, Hilden, Germany) before being subjected to further characterization using two sequencing technologies. Non-enriched samples were prepared using TruSeq PCR-free library kits (Illumina, San Diego, CA, USA) and sequenced as 2 × 150 base pairs (bp) in one lane on an S4 flow cell in a NovaSeq 6000 sequencing instrument (Illumina) at the SNP&SEQ platform at NGI Uppsala (Sweden). Enriched samples were prepared using Nextera library kits (Illumina) and sequenced as 2 × 150 bp on a 300-cycle sequencing flow cell using a NextSeq instrument (Illumina) at NAU (Northern Arizona University). Non-enriched samples were also prepared using LSK-109 library kits (Oxford Nanopore Technologies, Oxford, UK) and sequenced in MinION R9.4.1 flow cells using a MinION sequencing device (Oxford Nanopore Technologies).

All samples were submitted to total DNA extraction using Quick-DNA™ HMW MagBead Kit (D6060; Zymo Research, Irving, USA) according to the manufacturer’s recommendation. We used NanoVue™ Plus (28956058; Biochrom™, Holliston, USA) to obtain quantitative and qualitative data from our DNA samples. Metagenomic libraries were prepared using Rapid Barcoding Kit (SQK-RBK004; Oxford Nanopore Technologies, Oxford, UK) with a DNA input of 400 ng per sample. All the steps were performed following the manufacturer’s recommendations. For each run that was performed during 24 h, we used 12 samples with different barcodes each. All flow cells used were FLO-MIN106D (R9.4.1; Oxford Nanopore Technologies, Oxford, UK) model, and we used a MinION sequencing device (Oxford Nanopore Technologies, Oxford, UK).

Metagenomic libraries were prepared using the Nextera Library Preparation kit (Illumina Inc.), while metatranscriptomic libraries were prepared using the TruSeq Stranded total RNA Library Prep Kit (Illumina Inc.). Libraries concentrations were measured through quantitative qPCR using the KAPA Library Quantification Kit (Roche Inc.) and assayed for quality using an Agilent Bioanalyzer 2100 system (Agilent Technologies). MG and MT libraries were paired-end sequenced in two runs (2 × 100 bp) on the Illumina HiSeq 2500 platform at the NGS sequencing facility at LNBR/CNPEM (Campinas, Brazil). Furthermore, the cecal and rectal gDNA were homogenized into a single sample and sequenced on a MinION sequencing device (Oxford Nanopore Technologies Inc.) to obtain long reads. About 1 µg of ultra-long high-molecular weight gDNA from the homogenized samples was used for library preparation using the SQK-LSK109 Kit (Oxford Nanopore Technologies Inc.). The MinION run was performed on a Flow cell R9 version, generating around 3 Gb of long reads.

Purification of mRNA from total RNA samples was carried out using the Dynabeads mRNA Purification Kit (Thermo Fisher Scientific, Carlsbad, USA). The subsequent reverse transcription reaction was performed using SuperScript IV reverse transcriptase (Thermo Fisher Scientific, Carlsbad, USA). RNA sequencing preparation was carried out using, the Low Input by PCR Barcoding Kit and the cDNA-PCR Sequencing Kit (Oxford Nanopore Technologies, Oxford, United Kingdom), using the MinION Sequencing Device, the SpotON Flow Cell (R9.4.1), and MinKNOW software (Oxford Nanopore Technologies, Oxford, United Kingdom) according to the manufacturer’s instructions. Samples were sequenced for 48 h on two flow cells. Base-calling was performed by Albacore implemented in the nanopore software. Only D²-Reads with a quality score above 8 were used for further alignment.

Total DNA from resistant mutants was isolated using the MasterPure complete DNA and RNA purification kit (Lucigen, Middleton, WI) according to the manufacturer’s instructions, except that tubes were mixed by inversion rather than vortexing to preserve high-molecular-weight DNA. Isolated DNA was suspended in nuclease-free water and sequenced using the Oxford Nanopore MinION sequencing device with a Rapid Barcoding kit on a 9.4.1 flow cell (Oxford Nanopore Technologies, Oxford Science Park, UK) according to the manufacturer’s instructions. Sequencing output was as follows: MtzR(A), 150 Mb of passed bases, 170× coverage, N₅₀ of 12.0 kb; MtzR(B), 75 Mb of passed bases, 125× coverage, N₅₀ of 16.1 kb; MtzR(E), 24 Mb of passed reads, 78× coverage, and N₅₀ of 7.9 kb; and TdzR(A), 49 Mb of passed reads, 93× coverage, and N₅₀ of 7.9 kb. Read files were processed using SAMtools (43 (link)), aligned to the G37 reference genome using GraphMap (44 (link)), and then manually examined for mutations using the Integrative Genomics Viewer (45 (link)).