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Introduction to the protocol
To enable support for the rapidly expanding user requests, the team at Oxford Nanopore Technologies have put together an updated, lower cost end-to-end workflow based on the ARTIC Network protocols and analysis methods.
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 SARS-CoV-2 v3 (LoCost) 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:- Reverse transcribe your RNA samples with random hexamers
- Amplify the samples by tiled PCR using separate primer pools
- Combine the primer pools, purify and quantify the PCR products
- Prepare the DNA ends for adapter attachment
- Ligate native barcodes supplied in the kit to the DNA ends and pool the samples
- Ligate the sequencing adapters supplied in the kit to the DNA ends
- 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, which will collect raw data from the device and convert it into basecalled reads
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Before starting
This protocol outlines how to carryout PCR tiling of SARS-CoV-2 viral RNA samples on a 96-well plate using the Native Barcoding 96 Expansion (EXP-NBD196) or Native Barcoding Expansions 1-12 (EXP-NBD104) and 13-24 (EXP-NBD114).
It is required to use 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 the reduction of inhibiting compounds that can be co-extracted from samples.
Note: this is a guideline and not currently tested for COVID-19.
qPCR ct Dilution factor 18–35 none 15–18 1:10 12–15 1:100 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.