Exp nbd104 native barcoding kit

Library preparation was performed according to the “nCoV-2019 sequencing protocol” (doi.org/10.17504/protocols.io.bdp7i5rn) from the ARTICnetwork (https://artic.network/ncov-2019). Briefly, viral RNA was isolated for SARS-CoV-2 virus strains 5159, 5587, and 5588 via the QIAmp viral RNA kit (Qiagen, Hilden, Germany) according to the manufacturer’s guide. The cDNA preparation was performed using SuperScript IV (Thermo Fisher), followed by a multiplex PCR to generate overlapping 400-nucleotide (nt) amplicons using version 3 of the primer set (https://github.com/artic-network/artic-ncov2019/tree/master/primer_schemes/nCoV-2019/V3). After PCR cleanup, library preparation was performed using the Ligation sequencing kit (LSK-109; Oxford Nanopore Technologies) and the native barcoding expansion (EXP-NBD104, native barcoding kit; Oxford Nanopore Technologies). Sequencing was performed on a MinION device using an R.9.4.1 flow cell (Oxford Nanopore Technologies). Base calling and genome reconstruction were performed using poreCov v.0.2 with the default settings (https://github.com/replikation/poreCov).

Oxford Nanopore Technologies (ONT) sequencing data have been deposited in the Sequence Read Archive under BioProject PRJNA879282. DNA was extracted from an overnight culture in YPD using the QIAGEN genomic tip 100/G kit (Qiagen, #10243) according to manufacturing protocol. Long read sequencing libraries were prepared using the SQK-LSK109 Ligation Sequencing Kit with the EXP-NBD104 Native Barcoding Kit (Oxford Nanopore Technologies) from approximately 1μg of high molecular weight genomic DNA, following the manufacturer’s protocol. Long read libraries were sequenced on R9.4.1 Spot-On Flow cells (FLO-MIN106) using the GridION X5 platform set to super accurate base calling.

Frozen E. coli stocks (GoldBio #CC-101-B) were scraped and transferred to 15 mL tubes filled with 5 mL Lysogeny broth (LB) and agitated overnight at 37°C. 1 mL of E. coli culture was transferred to a 2 mL Eppendorf tube containing homogenization beads. After homogenization DNA was extracted from these samples by phenol:chloroform extraction and cleaned with the DNeasy Blood and Tissue Kit (Qiagen 69504). DNA was prepared as outlined in the “Genomic sequencing” section below. Additional DNA was prepared for Oxford Nanopore sequencing. First, DNA was cleaned again with Qiagen Genomic-tip 100/G DNA Purification (Qiagen 10243), then it was run on a 0.8% TBE gel to determine approximate fragment size. Samples were barcoded with the Oxford Nanopore EXP-NBD104 native barcoding kit and prepared with the Oxford Nanopore SQK-LSK109 sequencing kit. Prepared DNA was then run on a MinION sequencer with MinION flowcell. Long-read sequences were processed with Guppy and hybrid assembly was performed with Unicycler.^{41 (link)}

RPRD: To fully characterize samples with CNVs, XL-PCR amplicons encompassing the duplicated and/or nonduplicated gene copies were generated and subjected to single molecule sequencing using Nanopore as previously described [36 (link),39 (link)]. Primers and PCR conditions used to generate the amplicons are shown in Table S2 and are as previously described [33 (link)]. Libraries were prepared using the Ligation Sequencing DNA Kit (SQK-LSK109) and Native Barcoding Kit EXP-NBD104 (Oxford Nanopore Technologies, Oxford, UK) per manufacturer recommendations. Barcoded libraries were pooled and sequenced on the MinION Sequencing instrument with a FLO-MIN106D (R9.4.1) flow cell (Oxford Nanopore Technologies, Oxford, UK) as prescribed to a minimum read depth of 10,000×). Reads were filtered for qscore >10 and base-called with MinKNOW (v21.06.13). FASTQ files were mapped to GRCh38 (NC_000022.11) using Minimap2 [40 (link)], and variant calling was performed using Nanopolish [41 (link)]. Thresholds were set as recommended (0.2). Alignments were viewed using the Integrative Genomics Viewer v.2.4.1 (IGV, Broad 153 Institute, Boston, MA, USA) aligning to the human GRCh38 reference genome to confirm genotype calls [42 (link)].

Total DNA was extracted using the MagAttract HMW DNA Kit (Qiagen) according to manufacturer’s instructions and used for short-read sequencing. Sequencing libraries were prepared using a Nextera XT library prep kit (Illumina GmbH, Munich, Germany) for a 250 bp paired-end sequencing run on an Illumina MiSeq platform. The obtained reads were de novo assembled with the Velvet assembler integrated in the Ridom SeqSphere+ v. 7.0.4 software (Ridom GmbH, Münster, Germany).
DNA extraction for long-read sequencing was performed using the Genomic-Tips 100/G kit and Genomic DNA Buffers kit (Qiagen) according to the manufacturer’s instructions. Libraries were prepared using the 1D Ligation Sequencing Kit (SQK-LSK109) in combination with Native Barcoding Kit (EXP-NBD104; Oxford Nanopore Technologies, Oxford, United Kingdom) and were loaded onto a R9.4 flow cell (Oxford Nanopore Technologies). The run was performed on a MinION MK1b device. Collection of raw electronic signal data and live base-calling was performed using the MinKNOW software and the Guppy basecaller (Oxford Nanopore Technologies). The long-reads were assembled using ONT assembly and Illumina polishing pipeline (Oxford Nanopore Technologies), performing Canu assembly followed by polishing steps, including pilon and BWA mem mapping using the Illumina reads.³

Genomic DNA was extracted from the sputum sample using the QIAGEN DNeasy PowerSoil Pro Kit (Hilden, Germany). An aliquot of 6 ng DNA was amplified using Qiagen REPLI-g WGA kit and then debranched using T7 endonuclease I (New England Biolabs), while another aliquot of 1.7 ug DNA was directly used for PromethION library preparation. PCR-free libraries were constructed with Native Barcoding Kit EXP-NBD104 (Oxford Nanopore Technologies, Oxford, UK) according to the manufacturer’s instructions. The MinKNOW software v19.10.1 was used to collect raw sequencing data, and Guppy v3.2.4 was used for local base-calling.