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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 workflow based on the ARTIC Network protocols and analysis methods. The protocol uses Oxford Nanopore Technologies' Rapid Barcoding Kit 96 (SQK-RBK110.96) 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. The protocol generates 1200 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 the Twist Synthetic SARS-CoV-2 RNA controls to show what would be expected when running this protocol with SARS-CoV-2 samples.
To generate tiled PCR amplicons from the SARS-CoV-2 viral cDNA for use with the Rapid Barcoding Kit 96, primers were designed by Freed et al., 2020 using Primal Scheme. These primers are designed to generate 1.2 kb amplicons that overlap by approximately 20 bp. Primer sequences can be found here.
Primers can be ordered pre-pooled directly from IDT under the name SARS-Cov2-Midnight-1200, 500 rxn at a concentration of 100 μM per pool.
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
- 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, 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 carry out PCR tiling of SARS-CoV-2 viral RNA samples on a 96-well plate using the Rapid Barcoding Kit 96 (SQK-RBK110.96).
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.