R9.4.1 flow cell

The genomes of the S. dysgalactiae subsp. equisimilis strains were sequenced with methods described previously for S. pyogenes (17 (link), 38 (link), 54 (link), 61 (link), 62 (link)). Briefly, strains were grown at 37°C with 5% CO₂ on tryptic soy agar with 5% sheep blood (Becton, Dickinson, Franklin Lakes, NJ) or in Todd-Hewitt broth with 2% (wt/vol) yeast extract (THY; Difco Laboratories, Franklin Lakes, NJ). Chromosomal DNA for Illumina short-read sequencing was isolated with the RNAdvance viral kit (Beckman Coulter, Brea, CA) and a BioMek i7 instrument (Beckman Coulter). Libraries were prepared with the NexteraXT kit (Illumina, San Diego, CA) and sequenced with a NovaSeq instrument (Illumina) using a 2 × 250-bp protocol. Chromosomal DNA for long-read sequencing was isolated with a DNeasy blood and tissue kit (Qiagen, Germantown, MD). Libraries were prepared with either a native barcoding kit or rapid barcoding kit (Oxford Nanopore Technologies, United Kingdom) and sequenced with a GridION instrument using version R10.4 or R9.4.1 flow cells (Oxford Nanopore Technologies), respectively.

A total of 1 µg of DNA from the unprocessed spindle biopsy was subjected to library construction using the SQK-LSK109 kit and sequenced on the GridION sequencer with R9.4.1 flow cells (Oxford Nanopore Technologies, Oxford, UK). The adaptive sampling option was enabled in MinKNOW 20.10.6. For targeting, a bed-file and the human genome GRCh38 were used. The intervals were constructed manually, consisting of individual SV breakpoints with 15 kb padding as well as larger regions, e.g., the complete chr9p region. In total, 112 regions comprising 112 Mb were targeted with this approach (Table S2). Base calling was done with guppy 5.1.13 and the super-accuracy model. Reads were trimmed with Porechop 0.2.4 [54 (link)] and mapped to GRCh38 with minimap2 2.17 [55 (link)] using the -R, -Y, and the –MD parameters. SNP calling was performed with longshot 0.4.1 [59 (link)], SNPs were filtered for QUAL > 100, and reads were phased with WhatsHap [60 (link)].

Poly(A)+ RNAs were enriched from total RNAs using Dynabeads mRNA Purification Kit (Invitrogen). IVT RNAs were polyadenylated using Escherichia coli poly(A) polymerase (New England Biolabs). A total of 1,000 ng of poly(A)+ RNAs were subjected to DRS library preparation using an SQK-RNA002 kit (Oxford Nanopore Technologies, Oxford, UK). The optional reverse transcription step was included using SuperScript III Reverse Transcriptase (Invitrogen). Sequencing was performed on the MinION platform using R9.4.1 flow cells (Oxford Nanopore Technologies).
Reads were base called using the Guppy workflow (v5.0.16) with the configuration of rna_R9.4.1_70bps_hac.cfg or using the Dorado workflow (v0.3.4) with the configuration of rna002_70bps_hac@v3. The resulting FASTQ files were aligned to the SINV genome (GenBank: NC_001547.1) using Minimap2 v2.17 with parameter settings “-ax map-ont” (51 (link)). Mapping results were stored in SAM files, which were subsequently converted into bam files and sorted and indexed using SAMtools v1.17 (52 (link)). Read alignment information was extracted using a custom Python script based on Pysam. Full-length viral reads, defined as those covering ≥90% of reference lengths, and subgenomic reads were subsampled from total viral reads with a custom script.

The hexon gene sequences of the samples screened positive for adenovirus were obtained by nested PCR amplification with the Adv Hex F1 (5′-TICTTTGACATICGIGGIGTICTIGA-3′), Adv Hex F2 (5′-GGYCCYAGYTTYAARCCCTAYTC-3′), Adv Hex R1 (5′-CTGTCIACIGCCTGRTTCCACA-3′), and Adv Hex R2 (5′-GGTTCTGTCICCCAGAGARTCIAGCA-3′) as described previously (27 (link)). Briefly, the first PCR was carried out using Taq DNA polymerase (Roche Diagnostics) in a 50-μL reaction volume containing 5 μL of DNA, 5 μL of 10× buffer, 4 μL of dNTPs, 1 μL of each primer (diluted at 10 μM) forward Adv Hex F1 and the reverse Adv Hex R1, 0.25 μL enzyme, and 33.75 μL RNase-free H₂O. The amplification conditions were as follows: 94°C for 2 min, followed by 35 cycles of 94°C for 1 min, 45°C for 1 min, and 72°C for 2 min, with a final extension of 72°C for 5 min. Then 1 μL of the first PCR was added to a second PCR master mix for nested amplification using the same conditions. The PCR products were prepared for sequencing using the Native barcoding genomic DNA kit (EXP-NBD196) and the NEBNext ARTIC library prep kit according to the manufacturer’s instructions and sequenced using a R9.4.1 flowcells (Oxford Nanopore Technologies) on a GridION (Oxford Nanopore Technologies).

RNA extraction was performed on nasopharyngeal swabs taken at t_BTI, using the DEXR-15-LM96 kit for automated extraction (Diagenode, Seraing, Belgium) with 350 µL sample input. Extracted RNA was eluted from magnetic beads in 50 μL of UltraPure DNase/RNase-free distilled water. Following RNA extraction, cDNA was synthesized followed by multiplex PCR amplification using a modified version of the ARTIC V3 LoCost protocol with the Midnight primer set (1200 bp amplicons). The libraries were sequenced on a MinION using R9.4.1 flow cells (Oxford Nanopore Technologies, Oxford, UK) and MinKnow software (v21.02.1 for Windows, Oxford Nanopore technologies, Oxford, UK) The resulting fast5 reads were base called and demultiplexed using Guppy (v5.0.16 for Windows, Oxford Nanopore technologies, Oxford, UK) in super accuracy mode. Genome assembly was performed using the ARTIC bioinformatics pipeline v1.1.3, which entails adapter trimming and mapping to the reference strain Wuhan-Hu-1 (MN908947), as previously described [37 (link)].