Native barcoding kit

DNA extraction for long-read sequencing was performed using the MagAttract kit (Qiagen Cat. No. 67653) and the Wizard Genomic Purification kit (Promega Cat. No. A2920) for the DNA isolation 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, UK) 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 22.10.10 and the Guppy basecaller (Oxford Nanopore Technologies).
Plasmid content of isolates HUC-90, HUC-99 and HUC-Aba1527 was investigated in detail by hybrid assembly combining the MiSeq short-reads with MinION long-reads using Unicycler v0.5.0.

Samples were selected for sequencing based on the qPCR cycle threshold (Ct) value (≤30). SARS-CoV-2 genomic libraries were prepared using the Oxford Nanopore MinION technology. The SuperScript IV Reverse Transcriptase kit (Thermo Fisher Scientific, San Jose, CA, USA) was initially used for cDNA synthesis following the manufacturer’s instructions. The cDNA generated was subjected to multiplex PCR sequencing using Q5 High Fidelity Hot-Start DNA Polymerase (New England Biolabs, Ipswich, MA, USA) and a set of specific primers designed by the ARTIC Network for complete sequencing of the SARS-CoV-2 genome (version 3) [17 (link)]. PCR conditions were previously reported [17 (link)]. All experiments were performed in a biosafety level-2 cabinet. Amplicons were purified using 1 × AMPure XP Beads (Beckman Coulter, Brea, CA, USA) and quantified on a Qubit 3.0 fluorimeter (Thermo Fisher Scientific) using the Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific). DNA library preparation was performed using the Ligation Sequencing Kit LSK109 (Oxford Nanopore Technologies, Oxford, UK) and the Native Barcoding Kit (NBD104 and NBD114, Oxford Nanopore Technologies, Oxford, UK). Sequencing libraries were loaded into an R9.4 flow cell (Oxford Nanopore Technologies, Oxford, UK). In each sequencing run, negative controls were used to monitor potential contamination with less than 2% mean coverage.

Amplicons were purified using 1× AMPure XP Beads, and cleaned-up PCR products concentrations were measured using Qubit™ dsDNA HS Assay Kit on a Qubit 3.0 fluorimeter (Thermo Fisher Scientific, Waltham, MA, USA). DNA library preparation was carried out using the Ligation Sequencing Kit and the Native Barcoding Kit (NBD104, Oxford Nanopore Technologies, Oxford, UK) [37 (link)]. Purified PCR products pools were pooled together before barcoding reactions (taking in consideration each amplicon pool DNA concentrations), and one barcode was used per sample in order to maximize the number of samples per flow cell. Sequencing library was loaded onto a R9.4 flow cell, and data were collected for up to 6 h, but generally less.

Sequencing libraries were prepared from 32 amplicon samples using the Native Barcoding Kit (Oxford Nanopore Technologies; cat. no. EXP-NBD104) paired with the Ligation Sequencing Kit (Oxford Nanopore Technologies; cat. no. SQK-LSK109) according to the manufacturer’s directions. Between five and eight sequencing libraries per flow cell were pooled for sequencing on five GridION flow cells (Oxford Nanopore Technologies; R9.4.1) for 72 hours on a GridION Mk1 device (Oxford Nanopore Technologies; Oxford, England, UK). Raw data was live basecalled using the high accuracy model in Guppy (v.3.2.10).

Sequencing libraries were generated using the Genomic DNA Sequencing Kit SQK-LSK108 (Oxford Nanopore Technologies). Tiling PCR amplicons were generated as described above. Ligation was carried out using UltraII End Prep Reaction Mix and UltraII End Prep Enzyme Mix and barcoded using the Native Barcoding Kit (Oxford Nanopore Technologies, Oxford, UK). The libraries were cleaned using AmpureXP purification beads (Beckman Coulter, High Wycombe, UK) in a 1:1 ratio and eluted in 15 μl of elution buffer. DNA quantification was performed using the Qubit dsDNA High Sensitivity assay on the Qubit 4.0. Sequencing libraries were loaded onto an R9.4 flow cell, run on an Oxford Nanopore GridION instrument, and data were collected for up to 21 hours.

RNA samples from 14 positive feathers of confirmed positive blackbirds were selected for sequencing. These included 9 RNA samples which were extracted from feather calami at day 0 and 5 RNA samples extracted from feather calami at day 43 after storage at room temperature. Selection among samples from Day 0 and Day 43 was based on them having a C_T value below 32. USUV genomes were generated using a specific USUV multiplex PCR on the Oxford Nanopore MinION platform as previously described [49 (link)]. The libraries were generated using a Native Barcoding Kit from Oxford Nanopore Technologies (SQK-LSK109) and sequenced on an R9.4 flow cell.

The DNA library was prepared using a Ligation Sequencing Kit (SQK-LSK109) with a Native Barcoding Kit (EXP-NBD104; Oxford Nanopore Technologies) according to the manufacturer’s instructions. The libraries were barcoded, pooled, and ligated to sequencing adapters. Purified libraries were loaded onto FLO-MIN106 (R9.4; Oxford Nanopore Technologies) and sequenced using the MinION device (Oxford Nanopore Technologies). Basecalling was performed by Guppy (v3.0.3) embedded in the MinIT system (Oxford Nanopore Technologies). Raw data were demultiplexed and the adaptor sequences were trimmed using MinKNOW software (Oxford Nanopore Technologies). The filtered reads were assembled into a single file using Porechop v.9.0. Viral reads were mapped to the reference genome sequences of SFTSV SPL114A, and consensus sequences were extracted by CLC Genomics Workbench (v7.5.2; Qiagen, Hilden, Germany). Manual polishing was performed using the indel error-correction method described previously [30 (link)].