To continue support for SARS-CoV-2 sequencing, the team at Oxford Nanopore Technologies have put together an updated workflow based on the ARTIC Network protocols and analysis methods. The protocol uses third-party reagents and customer-ordered RT PCR primers for sample preparation; and the Oxford Nanopore Technologies' Rapid Barcoding Kit 96 V14 (SQK-RBK114.96) for barcoding and library preparation.

While this protocol is available in the Nanopore Community, we kindly ask users to ensure they are citing the members of the ARTIC network who have been behind the development of these methods.

This protocol is similar to the ARTIC amplicon sequencing protocol for MinION for SARS-CoV-2 v3 (LoCost) by Josh Quick and the method used in Freed et al., 2020. The protocol generates amplicons in a tiled fashion across the whole SARS-CoV-2 genome.

To generate tiled PCR amplicons from the SARS-CoV-2 viral cDNA for use with the Rapid Barcoding Kit 96 V14 (SQK-RBK114.96), primers were designed by Freed et al., 2020 using Primal Scheme. These primers are designed to generate 1.2 kb amplicons. Primer sequences can be found here.

As mutations in SARS-CoV-2 variants emerge, amplicon drop out may be observed. For users wishing to design their own primer spike-ins to address this, we suggest adding to the appropriate primer pool at a final concentration between 3.33 µM and 6.66 µM.

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

• Extract your RNA.
• Ensure you have your sequencing kit, the correct equipment and reagents.
• Download the software for acquiring and analysing your data.
• Check your flow cell to ensure it has enough pores for a good sequencing run.

Prepare your library

You will need to:

• Reverse transcribe your RNA samples with random hexamers.
• Amplify the samples by tiled PCR using separate primer pools.
• Combine the primer pools.
• Attach Rapid Barcodes supplied in the kit to the DNA ends, pool the samples and SPRI purify.
• Prime the flow cell and load your DNA library into the flow cell.

Sequencing and analysis

You will need to:

• Start a sequencing run using the MinKNOW software, selecting SQK-RBK114.96 in kit selection, which will collect raw data from the device and convert it into basecalled reads.
• (Optional): Perform downstream analysis of the data using the wf-artic analysis workflow integrated within the EPI2ME Labs application.

This protocol outlines how to carry out PCR tiling of SARS-CoV-2 viral RNA samples on a 96-well plate using third-party reagents, customer-ordered primers; and the Rapid Barcoding Kit 96 V14 (SQK-RBK114.96).

It is required to use total RNA extracted from samples that have been screened by a suitable qPCR assay.

When processing multiple samples at once, we recommend making master mixes with an additional 10% of the volume. We also recommend using a template-free pre-PCR hood for making up the master mixes, and a separate template pre-PCR hood for handling the samples. It is important to clean and/or UV irradiate these hoods between sample batches. Furthermore, to track and monitor cross-contamination events, it is important to run a negative control reaction at the reverse transcription stage using nuclease-free water instead of sample, and carrying this control through the rest of the prep.

All post-PCR procedures must be carried out in a separate area to the pre-PCR preparation, with dedicated equipment for liquid handling in each area.

We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within 12 weeks of purchasing your MinION/GridION Flow Cells. Oxford Nanopore Technologies will replace any unsed flow cell with fewer than the number of pores listed in the Table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.

Flow cell	Minimum number of active pores covered by warranty
MinION/GridION Flow Cell	800

Name	Acronym	Cap colour	No. of vials	Fill volume per vial (µl)
Rapid Adapter	RA	Green	2	15
Adapter Buffer	ADB	Clear	1	100
AMPure XP Beads	AXP	Amber	3	1,200
Elution Buffer	EB	Black	1	1,500
Sequencing Buffer	SB	Red	1	1,700
Library Beads	LIB	Pink	1	1,800
Library Solution	LIS	White cap, pink label	1	1,800
Flow Cell Flush	FCF	Clear	1	15,500
Flow Cell Tether	FCT	Purple	2	200
Rapid Barcodes	RB01-96	-	3 plates	8 µl per well

This product contains AMPure XP reagent manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.

For ease of use, we recommend purchasing the panel xGen™ SARS-CoV-2 Midnight Amplicon Panel (IDT, 10011644). A full list of the primers contained in the xGen™ SARS-CoV-2 Midnight Amplicon Panel (IDT, 10011644) can be found on the supplier website.

As mutations in SARS-CoV-2 variants emerge, amplicon drop out may be observed. For users wishing to design their own primer spike-ins to address this, we suggest adding to the appropriate primer pool at a final concentration between 3.33 µM and 6.66 µM.

Alternatively, users can opt to order primers independently and generate the panels. Please find below a full list of the are the sequences for the primer scheme used in the Midnight RT PCR Expansion:

Pool A

Primer name	Primer Sequence	Final Concentration
SARSCoV_1200_1_LEFT	ACCAACCAACTTTCGATCTCTTGT	3.33μM
SARSCoV_1200_1_RIGHT	GGTTGCATTCATTTGGTGACGC	3.33μM
SARSCoV_1200_3_LEFT	GGCTTGAAGAGAAGTTTAAGGAAGGT	3.33μM
SARSCoV_1200_3_RIGHT	GATTGTCCTCACTGCCGTCTTG	3.33μM
SARSCoV_1200_5_LEFT	ACCTACTAAAAAGGCTGGTGGC	3.33μM
SARSCoV_1200_5_RIGHT	AGCATCTTGTAGAGCAGGTGGA	3.33μM
SARSCoV_1200_7_LEFT	ACCTGGTGTATACGTTGTCTTTGG	6.66μM
SARSCoV_1200_7_RIGHT	GCTGAAATCGGGGCCATTTGTA	6.66μM
SARSCoV_1200_9_LEFT	AGAAGTTACTGGCGATAGTTGTAATAACT	6.66μM
SARSCoV_1200_9_RIGHT	TGCTGATATGTCCAAAGCACCA	6.66μM
SARSCoV_1200_11_LEFT	AGACACCTAAGTATAAGTTTGTTCGCA	3.33μM
SARSCoV_1200_11_RIGHT	GCCCACATGGAAATGGCTTGAT	3.33μM
SARSCoV_1200_13_LEFT	ACCTCTTACAACAGCAGCCAAAC	3.33μM
SARSCoV_1200_13_RIGHT	CGTCCTTTTCTTGGAAGCGACA	3.33μM
SARSCoV_1200_15_LEFT	TTTTAAGGAATTACTTGTGTATGCTGCT	6.66μM
SARSCoV_1200_15_RIGHT	ACACACAACAGCATCGTCAGAG	6.66μM
SARSCoV_1200_17_LEFT	TCAAGCTTTTTGCAGCAGAAACG	3.33μM
SARSCoV_1200_17_RIGHT	CCAAGCAGGGTTACGTGTAAGG	3.33μM
SARSCoV_1200_19_LEFT	GGCACATGGCTTTGAGTTGACA	3.33μM
SARSCoV_1200_19_RIGHT	CCTGTTGTCCATCAAAGTGTCCC	3.33μM
SARSCoV_1200_21_LEFT	TCTGTAGTTTCTAAGGTTGTCAAAGTGA	6.66μM
SARSCoV_1200_21_RIGHT	GCAGGGGGTAATTGAGTTCTGG	6.66μM
21_right_spike	GTGTATGATTGAGTTCTGGTTGTAAG	6.66μM
SARSCoV_1200_23_LEFT	ACTTTAGAGTCCAACCAACAGAATCT	6.66μM
23_left_spike	ACTTTAGAGTTCAACCAACAGAATCT	6.66μM
SARSCoV_1200_23_RIGHT	TGACTAGCTACACTACGTGCCC	6.66μM
SARSCoV_1200_25_LEFT	TGCTGCTACTAAAATGTCAGAGTGT	3.33μM
SARSCoV_1200_25_RIGHT	CATTTCCAGCAAAGCCAAAGCC	3.33μM
SARSCoV_1200_27_LEFT	TGGATCACCGGTGGAATTGCTA	3.33μM
SARSCoV_1200_27_RIGHT	TGTTCGTTTAGGCGTGACAAGT	3.33μM
SARSCoV_1200_29_LEFT	TGAGGGAGCCTTGAATACACCA	3.33μM
SARSCoV_1200_29_RIGHT	TAGGCAGCTCTCCCTAGCATTG	3.33μM

Pool B

Primer name	Primer sequences	Final concentration
SARSCoV_1200_2_LEFT	CCATAATCAAGACTATTCAACCAAGGGT	7.14μM
SARSCoV_1200_2_RIGHT	ACAGGTGACAATTTGTCCACCG	7.14μM
SARSCoV_1200_4_LEFT	GGAATTTGGTGCCACTTCTGCT	3.57μM
SARSCoV_1200_4_RIGHT	CCTGACCCGGGTAAGTGGTTAT	3.57μM
SARSCoV_1200_6_LEFT	ACTTCTATTAAATGGGCAGATAACAACTG	7.14μM
SARSCoV_1200_6_RIGHT	GATTATCCATTCCCTGCGCGTC	7.14μM
SARSCoV_1200_8_LEFT	CAATCATGCAATTGTTTTTCAGCTATTTTG	7.14μM
SARSCoV_1200_8_RIGHT	TGACTTTTTGCTACCTGCGCAT	7.14μM
SARSCoV_1200_10_LEFT	TTTACCAGGAGTTTTCTGTGGTGT	3.57μM
SARSCoV_1200_10_RIGHT	TGGGCCTCATAGCACATTGGTA	3.57μM
SARSCoV_1200_12_LEFT	ATGGTGCTAGGAGAGTGTGGAC	3.57μM
SARSCoV_1200_12_RIGHT	GGATTTCCCACAATGCTGATGC	3.57μM
SARSCoV_1200_14_LEFT	ACAGGCACTAGTACTGATGTCGT	3.57μM
SARSCoV_1200_14_RIGHT	GTGCAGCTACTGAAAAGCACGT	3.57μM
SARSCoV_1200_16_LEFT	ACAACACAGACTTTATGAGTGTCTCT	3.57μM
SARSCoV_1200_16_RIGHT	CTCTGTCAGACAGCACTTCACG	3.57μM
SARSCoV_1200_18_LEFT	GCACATAAAGACAAATCAGCTCAATGC	3.57μM
SARSCoV_1200_18_RIGHT	TGTCTGAAGCAGTGGAAAAGCA	3.57μM
SARSCoV_1200_20_LEFT	ACAATTTGATACTTATAACCTCTGGAACAC	7.14μM
SARSCoV_1200_20_RIGHT	GATTAGGCATAGCAACACCCGG	7.14μM
SARSCoV_1200_22_LEFT	GTGATGTTCTTGTTAACAACTAAACGAACA	3.57μM
SARSCoV_1200_22_RIGHT	AACAGATGCAAATCTGGTGGCG	3.57μM
22_right_spike	AACAGATGCAAATTTGGTGGCG	3.57μM
SARSCoV_1200_24_LEFT	GCTGAACATGTCAACAACTCATATGA	7.14μM
24_left_spike	GCTGAATATGTCAACAACTCATATGA	7.14μM
SARSCoV_1200_24_RIGHT	ATGAGGTGCTGACTGAGGGAAG	7.14μM
SARSCoV_1200_26_LEFT	GCCTTGAAGCCCCTTTTCTCTA	3.57μM
SARSCoV_1200_26_RIGHT	AATGACCACATGGAACGCGTAC	3.57μM
SARSCoV_1200_28_LEFT	TTTGTGCTTTTTAGCCTTTCTGCT	3.57μM
SARSCoV_1200_28_RIGHT	GTTTGGCCTTGTTGTTGTTGGC	3.57μM
SARSCoV_1200_28_LEFT_27837T	TTTGTGCTTTTTAGCCTTTCTGTT	7.14μM

To generate tiled PCR amplicons from the SARS-CoV-2 viral cDNA, primers were designed by Freed et al., 2020 using Primal Scheme. These primers are designed to generate 1200 bp amplicons that overlap by approximately 20 bp. These primer sequences can be found here.

Please note the Oxford Nanopore Technologies method uses the PCR primers at a different concentration to the manufacturers recommendation to optimise reagent usage and keep the method in-line with our legacy product.

If using the xGen™ SARS-CoV-2 Midnight Amplicon Panel (IDT, 10011644)

• Use the primers at the increased stock concentration of ≥ 100 μM provided by the manufacturer (you are not required to dilute these primers).

If you have ordered your own primers using the primer sequences and concentrations documented in the equipment and consumables section of this method

• Primer pool A should be used at the formulated concentration of ~133 μM.
• Primer pool B should be used at the formulated concentration of ~143 μM.

For most sequencing experiments, use the Library Beads (LIB) for loading your library onto the flow cell. However, for viscous libraries it may be difficult to load with the beads and may be appropriate to load using the Library Solution (LIS).

Data acquisition and real-time basecalling are carried out by the MinKNOW software. Please ensure MinKNOW is installed on your computer or device. Further instructions for setting up a sequencing run can be found in the MinKNOW protocol.

We recommend setting up your sequencing run using the basecalling and barcoding recommendations outlined below. All other parameters can be left to their default settings.

For fastest turnaround time and easiest analysis, basecalling should be performed live during the sequencing run using the High Accuracy (HAC) basecaller.

Below are the recommendations for MinKNOW settings:

Positions

Flow cell position: [user defined]

Experiment name: [user defined]

Flow cell type: FLO-MIN114

Sample ID: [user defined]

Kit

Kit selection: Rapid Barcoding Kit 96 (SQK-RBK114-96)

Run configuration

Sequencing and analysis

Basecalling: On [default]
Modified bases: Off
Model: High-accuracy basecalling (HAC) [default]

Barcoding: On [default]
Trim barcodes: Off [default]
Barcode both ends: On
Custom barcodes selection: Off [default]

Alignment: Off [default]

Adaptive sampling: Off [default]

Advanced options
Active channel selection: On [default]
Time between pore scans: 1.5 [default]
Reserve pores: On [default]

Data targets

Run limit: [user defined]*

*Sequencing time will depend on data requirements.

Output

Output format
.POD5: On [default]
.FASTQ: On [default]
.BAM: On

Filtering: On [default]
Qscore: 9 [default]
Minimum read length: 200 bp [default]

The wf-artic is a bioinformatics workflow for the analysis of ARTIC sequencing data prepared using the Midnight protocol. The bioinformatics workflow is orchestrated by the Nextflow software. Nextflow is a publicly available and open-source project that enables the execution of scientific workflows in a scalable and reproducible way. The use of the Nextflow software has been integrated into the EPI2ME Labs software that we recommend for running our downstream analysis methods.

Alternative methods for downstream analysis are available using your device terminal or command line, however we only suggest this for experienced users.

Demultiplexed sequence reads are processed using the ARTIC Field Bioinformatics software that has been modified for the analysis of FASTQ sequences prepared using Oxford Nanopore Rapid Sequencing kits. The other modification to the ARTIC workflow is the use of a primer scheme that defines the sequencing primers used by the Midnight protocol and their genomic locations on the SARS-CoV-2 genome.

The wf-artic workflow includes other analytical steps that include cladistic analysis using Nextclade and strain assignment using Pangolin. The data facets included in the report are parameterised and additional information such as plots of depth-of-coverage across the reference genome is optional.

The complete source for wf-artic is linked, and the Nextflow software will download the scripts and logic flow from this location.

The wf-artic workflow needs to be started manually as outlined below in 'Running a Midnight analysis using EPI2ME Labs'.

The EPI2ME application provides a clean interface to accessing bioinformatics workflows, and is our recommended method in performing your post-sequencing analysis.

Follow the instructions in the EPI2ME Installation guide to install the application on your device.

For more information on how to use EPI2ME, refer to the EPI2ME Quick Start guide.

Ensure you have installed the wf-artic workflow prior to the first analysis set-up.

In the EPI2ME Labs home page, scroll down to the "Install workflows" section and click on epi2me-labs/wf-artic:

If you have already installed the wf-artic workflow, ensure you are using the latest version.

Updating the workflow can be done directly through EPI2ME Labs by navigating to the wf-artic workflow page and clicking Update Workflow:

The wf-artic analysis requires FASTQ sequence data that has already been demultiplexed.

Reads will be demultiplexed during sequencing if you are following the recommended "Required settings in MinKNOW". However, demultiplexing can also be done post-sequencing using the MinKNOW software.

For more information and guides on demultiplexing using MinKNOW, refer to the "Post-run analysis" section in our MinKNOW Protocol.

The expected input for wf-artic is a folder of folders as shown below. Each of the barcode folders should contain the FASTQ sequence data and files may either be uncompressed or gzipped.

$ tree -d MidnightFastq/

MidnightFastq/

├── barcode01

├── barcode02

├── barcode03

├── barcode04

├── barcode05

├── barcode06

└── unclassified

The wf-artic analysis outputs will be written to the Working Directory folder specified in the EPI2ME Labs Settings tab. The location of this folder is specified in the wf-artic run Instance parameters preceeded by out_dir.

However, these files can also be accessed directly in the EPI2ME Labs application from the completed analysis page for your run:

These outputs include:

• all_consensus.fasta A multi-FASTA format sequence file containing the consensus sequence for each of the samples investigated. This consensus sequence has been prepared for the whole SARS-CoV-2 genome, not just the spike protein region. The consensus sequence masks the non-spike regions and regions of low sequence coverage with N residues.

• all_variants.vcf.gz A gzipped VCF file that describes all high-quality genetic variants called by medaka from the sequenced samples.

• all_variants.vcf.gz.tbi An index file for the gzipped VCF file.

• consensus_status.txt A tab delimited file that reports whether a consensus sequence has been successfully prepared for a sample, or not.

• wf-artic-report.html A report summarising these data. This HTML format report also includes the output of the Nextclade software that can be used for a visual inspection of, for example, primer drop out or other qualitative consensus sequence aspects.

Other files are included in the work-directory. This includes per sample VCF files of all genetic variants prior to filtering and other sequences.

The "Working Directory" can be specified in the EPI2ME Labs "Settings" tab and defines where the workflow intermediate files and outputs are stored.

This folder will accumulate a significant number of files that correspond to raw BAM files, other larger intermediates and analysis results files. We recommend this folder to be routinely cleared.