To enable the support for the rapidly expanding user requests, the team at Oxford Nanopore Technologies have put together a partially automated end-to-end workflow based on the ARTIC Network protocols and analysis methods.

We have developed this automated protocol on the Hamilton NGS Star 96 liquid handling robot. The protocol begins with manual preparation of the RNA samples, which includes reverse transcription and tiled PCR. The remainder of the library preparation is automated with minimal hands-on time which is required for sample quantification and deck re-loading.

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 based on the ARTIC amplicon sequencing protocol for MinION for nCoV-2019 by Josh Quick. The protocol generates 400 bp amplicons in a tiled fashion across the whole SARS-CoV-2 genome. Some example data is shown in the Downstream analysis and expected results section, this is generated using human coronavirus 229E to show what would be expected when running this protocol with SARS-CoV-2 samples.

Primers were designed by Josh Quick using Primal Scheme; the primer sequences can be found here.

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 third-party 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:

Off-deck:

• Reverse transcribe your RNA samples with random hexamers
• Amplify the samples by tiled PCR using separate primer pools
• Combine the primer pools

Note: On the Hamilton software, each step of the protocol is set up as a process. Currently, the off-deck section comprises Processes 1-2.

On-deck:

• Process 3: Purify and quantify the PCR products
• Processes 4-8: Prepare the DNA ends for adapter attachment and ligate the native barcodes supplied in the kit to the DNA ends and pool the samples
• Processes 10-11: Ligate the sequencing adapters supplied in the kit to the DNA ends

Off-deck:

• Prime the flow cell and load your DNA library into the flow cell

Note: Timings are dependent on number of samples and include hands on time, such as deck loading and sample quantification

Sequencing and analysis You will need to:

• Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads
• Start the EPI2ME software and select the barcoding workflow

Off-deck library preparation:

On-deck library preparation:

This protocol requires total RNA extracted from samples that have been screened by a suitable qPCR assay. Here we demonstrate the level of sensitivity and specificity by titrating total RNA extracted from cell culture infected with Human coronavirus 229E spiked into 100 ng human RNA extracted from GM12878 to give approximate figures.

Although not tested here, work performed by Josh Quick et al. on the Zika virus gives approximate dilution factors that may help reduction of inhibiting compounds that can be co-extracted from samples.

Note: this is a guideline and not currently tested for SARS-CoV-2.

When processing multiple samples at once, we recommend making master mixes with an additional 10% of the volume. We also recommend using pre- and post-PCR hoods when handling master mixes and 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.

To minimise the chance of pipetting errors when preparing primer mixes, we recommend ordering the tiling primers from IDT in a lab-ready format at 100 µM.

Where sample RNA is added to the below reaction, it is likely advantageous to follow the dilution guidelines proposed by Josh Quick:

If the sample has a low copy number (ct 18–35) use up to 16 µl of sample. Use nuclease-free water to make up any remaining volume. Take note to be aware that co-extracted compounds may inhibit reverse transcription and PCR.

Two worklist input excel files are required prior to running the protocol on the Hamilton NGS Star: one for processes03 to 08 and another for processes10 to 11. These will contain information regarding the appropriate number of samples, well identifiers and source concentration. In the first worklist, the target well is the well for pooled samples at the end of Process07. A maximum of 24 samples should be combined to make each pool, so for 96 samples, there will be 4 pools in target wells A1 to D1. Samples should be split evenly, so that the pools contain equivalent volumes. This worklist must be updated after the automated library preparation sections with the relevant plate information. Note that the pools are combined at the end of process08 into well A2, so the Source_Well for the second worklist will be A2.

Example:

This method has been tested and validated using the Hamilton NGS Star 96 (with 8 channels and MPH96), including an on-deck thermal cycler (ODTC), a Hamilton Heater Shaker (HHS) and the Inheco Cold Plate Air Cooled (CPAC) Modules. All modules are used at different stages of the protocol. This protocol may require some fine tuning for the specific NGS Star set-up and the temperature/humidity of the customer laboratory.

Please contact your Hamilton representative for further details.

Deck layout

• MIDI plates: Abgene™ 96 Well 0.8mL Polypropylene Deepwell Storage Plate (ThermoFisher, Cat # AB0859)
• HSP plates: Bio-Rad Hard-Shell® 96-Well PCR Plates (Cat# HSP9601)
• ODTC: On-Deck Thermal Cycler Module
• HHS: Hamilton Heater Shaker Module
• CPAC: Inheco Cold Plate Air Cooled Module
• 20 ml reagent troughs: X150 Reagent tub MagNA Pure LC medium 20 plastic (zigzag troughs, Roche #03004058001
• 60 ml self-standing large troughs (Hamilton, 56694-01)

Note: These quantities are for one run of the protocol for the selected number of samples.

Kits in batches NBD196.10.0007 onwards have barcodes ordered in columns on the plate:

Kits in batches prior to NBD196.10.0007 have barcodes ordered in rows:

Sequencing on a MinION Mk1B requires a high-spec computer or laptop to keep up with the rate of data acquisition. For more information, refer to the MinION Mk1B IT requirements document.

The MinION Mk1C contains fully-integrated compute and screen, removing the need for any accessories to generate and analyse nanopore data. For more information refer to the MinION Mk1C IT requirements document.

Sequencing on a MinION Mk1D requires a high-spec computer or laptop to keep up with the rate of data acquisition. For more information, refer to the MinION Mk1D IT requirements document.

MinKNOW

The MinKNOW software controls the nanopore sequencing device, collects sequencing data and basecalls in real time. You will be using MinKNOW for every sequencing experiment to sequence, basecall and demultiplex if your samples were barcoded.

For instructions on how to run the MinKNOW software, please refer to the MinKNOW protocol.

EPI2ME (optional)

The EPI2ME cloud-based platform performs further analysis of basecalled data, for example alignment to the Lambda genome, barcoding, or taxonomic classification. You will use the EPI2ME platform only if you would like further analysis of your data post-basecalling.

For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to this link.

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 for MinION/GridION/PromethION or within four weeks of purchasing Flongle Flow Cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores 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.

To generate tiled PCR amplicons from the SARS-CoV-2 viral cDNA, primers were designed by Josh Quick using Primal Scheme. These primers are designed to generate 400 bp amplicons that overlap by approximately 20 bp. These primer sequences can be found here. Where we show example data outputs in this protocol, the same parameters were used to design primers to the human coronavirus 229E to provide guideline statistics.

For a full overview of nanopore data analysis, which includes options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.

The sequencing device control, data acquisition and real-time basecalling are carried out by the MinKNOW software. Please ensure MinKNOW is installed on your computer or device. There are multiple options for how to carry out sequencing:

1. Data acquisition and basecalling in real-time using MinKNOW on a computer

Follow the instructions in the MinKNOW protocol beginning from the "Starting a sequencing run" section until the end of the "Completing a MinKNOW run" section.

2. Data acquisition and basecalling in real-time using the MinION Mk1B/Mk1D device

Follow the instructions in the MinION Mk1B user manual or the MinION Mk1D user manual.

3. Data acquisition and basecalling in real-time using the MinION Mk1C device

Follow the instructions in the MinION Mk1C user manual.

4. Data acquisition and basecalling in real-time using the GridION device

Follow the instructions in the GridION user manual.

5. Data acquisition and basecalling in real-time using the PromethION device

Follow the instructions in the PromethION user manual or the PromethION 2 Solo user manual.

6. Data acquisition using MinKNOW on a computer and basecalling at a later time using MinKNOW

Follow the instructions in the MinKNOW protocol beginning from the "Starting a sequencing run" section until the end of the "Completing a MinKNOW run" section. When setting your experiment parameters, set the Basecalling tab to OFF. After the sequencing experiment has completed, follow the instructions in the Post-run analysis section of the MinKNOW protocol.

The recommended workflows for the bioinformatics analyses are provided by the ARTIC network and are documented on their web pages at https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html.

The reference guided genome assembly and variant calling are also performed according to the bioinformatics protocol provided by the ARTIC network. Their best practices guide uses the software contained within the FieldBioinformatics project on GitHub.

This workflow uses only the basecalled FASTQ files to perform a high-quality reference-guided assembly of the SARS-CoV-2 genome. Sequenced reads are re-demultiplexed with the requirement that reads must contain a barcode at both ends of the sequence (this only applies to the Classic and Eco PCR tiling of SARS-CoV-2 protocols but not the Rapid Barcoding PCR tiling of SARS-CoV-2), and must not contain internal barcodes. The reads are mapped to the reference genome, primer sequences are excluded and the consensus sequence is polished. The Medaka software is used to call single-nucleotide variants while the ARTIC software reports the high-quality consensus sequence from the workflow.

The FieldBioinformatics workflow for SARS-CoV-2 sequence analysis is provided as a Jupyter notebook tutorial in the EPI2ME Labs software. The coronavirus workflow has been augmented to include additional steps that help with the quality control of individual libraries, and aid in the presentation of summary statistics and the final sets of called variants.

The FieldBioinformatics workflow for SARS-CoV-2 sequence analysis is also provided as an EPI2ME workflow – this provides a more accessible interface to a bioinformatics workflow and the provided cloud-based analysis also performs some secondary interpretation by preparing an additional report using the Nextclade software.

Here, results are shown based on human coronavirus 229E spiked into 100 ng of human RNA derived from GM12878 cell line. 10 pg–0.001 pg of viral RNA obtained from ATCC was spiked into the human RNA and human-only and reverse transcription negative controls were carried through the prep to sequencing. Every sample underwent 30 and 35 cycles of PCR to determine sensitivity and specificity guidelines, as well as the expected amplicon drop-out rate for each sample.

Note: The viral RNA from ATCC is generated from cell lines infected with human coronavirus 229E. The RNA supplied is total RNA extracted from the cell lines and includes both human and viral RNA. Therefore, the levels of sensitivity are likely to be higher than those reported here.

The graph below shows the expected sequence balancing if the protocol is followed. Here, equal masses went into the end-prep and native barcode ligation prior to pooling by equal mass for adapter ligation.

Figure 3. Number of reads per sample after native barcode demultiplexing in MinKNOW. All 14 samples were run on a single flow cell.

Sequences from each demultiplexed sample were aligned to the human coronavirus 229E genome using minimap2. The proportion of primary alignments per sample are reported below.

Figure 4. Proportion of reads for each sample aligning to the human coronavirus 229E reference genome.

After 12 hours of sequencing, the number of reads from the negative control samples aligning to the viral reference genome is shown in the graph below and is compared with the absolute number of sequences aligning to the lowest input (0.001 pg).

Figure 5. Absolute number of reads aligning to the human coronavirus 229E reference genome in the negative controls compared with the lowest input of viral RNA. Sequencing was carried out for 12 hours to pick up low levels of sequences assigned to barcodes representing these samples.

To assess the impact of PCR dropout with lowering input viral load and increasing PCR cycles, Mosdepth was used to calculate the proportion of the viral genome covered to different depth levels. These numbers were calculated after 12 hours of sequencing with 14 samples multiplexed.

Figure 6. Coverage and depth of the human coronavirus 229E genome for different input quantities of viral RNA and different cycle numbers after 12 hours of sequencing on a single flow cell.

This is unknown in real clinical samples. The graph below can be used to determine the proportion of the genome that could be covered to a given depth with different numbers of reads (30 cycles) at different input amounts in a background of 100 ng human RNA. Note: this is absolute depth.

Figure 7. Subsampled sequences to give an indication of the depth of sequencing achievable covering different amounts of the human coronavirus 229E genome. Input quantities and cycle number titrations show that high cycle numbers should be avoided where possible to minimise amplicon drop out.

This protocol provides amplification of low copy number viral genomes in a tiled method with low off-target amplification and minimal cross-contamination between samples. With <60 copies per reaction (0.001 pg viral input) in 100 ng background human RNA, under ideal circumstances, one should expect to cover >75% of the targeted genome at a depth of 200X within under 50,000 reads in the samples with the lowest viral titre and <20,000 reads in those with a higher viral titre.

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