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.
Gridion instrument
Manufactured by Oxford Nanopore
Sourced in United Kingdom
The GridION instrument is a portable device designed for DNA and RNA sequencing. It functions as a multi-channel, real-time genetic analysis platform. The GridION enables the analysis of complex biological samples through parallel nanopore-based sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Nextera xt kit, by Illumina (5 mentions)
Rapid barcoding kit, by Oxford Nanopore (2 mentions)
Dneasy blood and tissue kit, by Qiagen (2 mentions)
Nextseq 550, by Illumina (2 mentions)
Direct rna sequencing kit, by Oxford Nanopore (2 mentions)
Rnadvance viral kit, by Beckman Coulter (1 mentions)
R9.4.1 flow cell, by Oxford Nanopore (1 mentions)
Novaseq instrument, by Illumina (1 mentions)
Sheep blood, by BD (1 mentions)
Native barcoding kit, by Oxford Nanopore (1 mentions)
Clc genomics workbench v20, by Qiagen (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Megaruptor system, by Diagenode (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Lsk 109 sequencing kit, by Oxford Nanopore (1 mentions)
Clc genomics workbench version 20, by Qiagen (1 mentions)
Rna clean concentrator kit, by Zymo Research (1 mentions)
Ribozero bacteria kit, by Illumina (1 mentions)
12 protocols using gridion instrument
1
Genome Sequencing of S. dysgalactiae subsp. equisimilis
2
SARS-CoV-2 Genome Sequencing and Phylogenetic Analysis
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Libraries for whole virus genome sequencing were prepared according to version 1 or 3 of the ARTIC nCoV-2019 sequencing protocol (34 ). Long reads were generated with the LSK-109 sequencing kit, 24 native barcodes (NBD104 and NBD114 kits), and a GridION instrument (Oxford Nanopore). Short reads were generated with the NexteraXT kit and a MiSeq or NextSeq 550 instrument (Illumina). Whole genome alignments of consensus virus genome sequence generated from the ARTIC nCoV-2019 bioinformatics pipeline were trimmed to the start of orf1ab and the end of orf10 and used to generate a phylogenetic tree using RAxML (https://cme.h-its.org/exelixis/web/software/raxml/index.html ). Trees were visualized and annotated with CLC Genomics Workbench v20 (Qiagen). SARS-CoV-2 clade assignment was based on procedures described elsewhere (35 ).
3
Nanopore Sequencing of Long DNA Fragments
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Library preparation and sequencing were performed at the GeT-PlaGe core facility, INRAe Toulouse, according to the manufacturer’s instructions “1D gDNA selecting for long reads (SQK-LSK109)”. At each step, DNA was quantified using the Qubit dsDNA HS Assay Kit (Life Technologies). DNA purity was assessed using the nanodrop (Thermofisher) and size distribution and degradation were assessed using the fragment analyser (AATI) High Sensitivity DNA Fragment Analysis Kit. Purification steps were performed using AMPure XP beads (Beckman Coulter).
Flow cell was run with 7 µg of DNA previously purified and sheared to 25 kb using the megaruptor system (diagenode). Calibration was performed using the Short Read Eliminator Family: SRE size XS kit (Circulomics) to deplete short fragments. A one step DNA damage repair + END-repair + dA tail of double stranded DNA fragments was performed on 2 µg of sample. Adapters were then ligated to the library. The library was loaded onto R9.4.1 revD flow cells and sequenced on the GridION instrument (Oxford Nanopore Technologies) at 0.025pmol within 72 h.
Flow cell was run with 7 µg of DNA previously purified and sheared to 25 kb using the megaruptor system (diagenode). Calibration was performed using the Short Read Eliminator Family: SRE size XS kit (Circulomics) to deplete short fragments. A one step DNA damage repair + END-repair + dA tail of double stranded DNA fragments was performed on 2 µg of sample. Adapters were then ligated to the library. The library was loaded onto R9.4.1 revD flow cells and sequenced on the GridION instrument (Oxford Nanopore Technologies) at 0.025pmol within 72 h.
4
Genomic Analysis of GAS Strains
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GAS strains of emm11 (n=11), emm75 (n=17), emm77 (n=22) and emm92 (n=11) were grown on SBA and inspected for purity prior to DNA extraction. DNA was extracted using the Qiagen DNeasy blood and tissue kit (Qiagen) with modifications for Gram-positive bacteria as we have previously described [11 (link)]. DNA quantity and quality were measured using a NanoDrop instrument and Qubit. Short-read sequencing libraries were prepared using the Illumina Nextera flex sequencing kit as per the manufacturer’s instructions and 300 bp paired-end sequence obtained using an Illumina MiSeq instrument. Long-read sequencing libraries were prepared using the Oxford Nanopore rapid barcoding kit as per the manufacturer’s instructions and sequence obtained using an Oxford Nanopore GridION instrument. Complete genome assembly was performed using both long- and short-read sequences for TSPY155 (emm11), TSPY208 (emm75), TSPY165 (emm77), TSPY453 (emm77) and TSPY556 (emm92). Details of bioinformatic methods used for genome assembly, polymorphism discovery and phylogenetics are provided in the Supplementary Material.
5
SARS-CoV-2 Whole-Genome Sequencing Protocol
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Libraries for whole viral genome sequencing were prepared according to version 1 ARTIC nCoV-2019 sequencing protocol (https://artic.network/ncov-2019 , last accessed May 6, 2020). Long reads were generated with the LSK-109 sequencing kit, 24 native barcodes (NBD104 and NBD114 kits), and a GridION instrument (Oxford Nanopore, Oxford, UK). Short reads were generated with the NexteraXT kit and a MiSeq or NextSeq 550 instrument (Illumina, San Diego, CA). Whole genome alignments of consensus viral genome sequence generated from the ARTIC nCoV-2019 bioinformatics pipeline were trimmed to the start of orf1ab and the end of orf10 and used to generate a phylogenetic tree using RAxML version 8.2 (https://cme.h-its.org/exelixis/web/software/raxml/index.html , last accessed May 3, 2020)26 to determine viral clade. Trees were visualized and annotated with CLC Genomics Workbench version 20 (Qiagen).
6
Nanopore Sequencing of Bacterial Transcriptome
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Total RNA was purified from bacterial cells as described above (Lasa et al. 2011 (link)). Ribosomal RNAs were removed from 10 µg of total RNA with the RiboZero Bacteria kit (Illumina, CA). Enriched mRNA was treated with E. coli Poly(A) Polymerase (NEB) in the presence of 10 mM ATP for 30 min at 37ºC to add a polyA tail. PolyAdenylated mRNAs were cleaned using the RNA Clean & Concentrator kit (Zymo, CA). Purified mRNAs were then converted into a nanopore-compatible RNA library using the Direct RNA Sequencing Kit from Oxford Nanopore (SQC-RNA002) (Pust et al. 2021 (link)). The library was sequenced on an R9.4 flowcell in a GridION instrument (Oxford Nanopore) for 24 h and base called in real-time in the instrument with the software Guppy. Data obtained by Oxford Nanopore are available at NCBI under Bioproject PRJNA922758 (S. aureus RN10359) and Bioproject PRJNA860339 (S. aureus MW2). The resulting reads were mapped using the Geneious Prime tool (Biomatters) using the S. aureus NCTC8325 genome as a reference (accession number: NC_007795.1) (Baba et al. 2008 (link)). The transcriptome mapping information is available on our website (https://staph.unavarra.es:10443 ).
7
SARS-CoV-2 Genome Sequencing Protocols
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SARS-CoV-2 genome sequencing was performed as previously described. Briefly, viral RNA was extracted from 200 μL of nasopharyngeal swab fluid using the EZ1 Virus Mini kit v2.0 on an EZ1 Advanced XL instrument (Qiagen, Courtaboeuf, France) or using the MagMax Viral/Pathogen Nucleic Acid Isolation kit on the KingFisher Flex system (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. SARS-CoV-2 genome sequences were obtained by next-generation sequencing with various procedures with the Illumina COVIDSeq protocol on a NovaSeq 6000 instrument (Illumina Inc., San Diego, CA, USA), or by multiplex PCR with ARTIC nCoV-2019 V3 Panel primers (IDT, Coralville, IA, USA) were combined with the Oxford Nanopore technology (ONT) on a GridION instrument (Oxford Nanopore Technologies Ltd., Oxford, UK), as previously described [14 (link),27 (link)]. After its extraction, viral RNA was reverse-transcribed according to the COVIDSeq protocol (Illumina Inc., San Diego, CA, USA) or by using the LunaScript RT SuperMix kit (New England Biolabs, Ipswich, MA, USA) when performing next-generation sequencing with the Nanopore technology, following the manufacturer’s recommendations.
8
SARS-CoV-2 Whole-Genome Sequencing Protocol
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Libraries for whole-virus genome sequencing were prepared according to version 1 or version 3 of the ARTIC nCoV-2019 sequencing protocol (https://artic.network/ncov-2019 ). Long reads were generated with the LSK-109 sequencing kit, 24 native barcodes (NBD104 and NBD114 kits), and a GridION instrument (Oxford Nanopore). Short reads were generated with a NexteraXT kit and a NextSeq 550 instrument (Illumina).
9
SARS-CoV-2 Whole Genome Sequencing
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Libraries for whole virus genome sequencing were prepared according to version 1 or version 3 of the ARTIC nCoV-2019 sequencing protocol (https://artic.network/ncov-2019 ). Long reads were generated with the LSK-109 sequencing kit, 24 native barcodes (NBD104 and NBD114 kits), and a GridION instrument (Oxford Nanopore). Short reads were generated with the NexteraXT kit and NextSeq 550 instrument (Illumina).
10
HPV Transcriptome Profiling via Nanopore
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Cell line RNA (500ng Poly-A+) was sequenced using the Direct RNA kit (DCS109) and Direct RNA sequencing kit (SQK-RNA002, Oxford Nanopore Technologies). RNA libraries were loaded onto MinION R9.4 flow cells on a GridION instrument (Oxford Nanopore Technologies). Transcriptome fastq files were aligned and reads assigned to genes using the Exome workflow in EPI2ME (EPI2ME ™ :: Dashboard (nanoporetech.com)). Reads were normalized as reads per million (RPM). HPV reads were identified by alignment to a file with 13 high-risk HPV sequencing plus HPV26 and HPV30 using EPI2ME. HPV reads were counted and normalized as RPM and E6 splicing was assessed by manual counting of spliced and unspliced reads in BAM file produced in the CGC platform (https://cgc.sbgenomics.com/).
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