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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) and Midnight RT PCR Expansion (EXP-MRT001) for barcoding and library preparation.
We have developed this automated protocol on the Hamilton NGS Star 96 liquid handling robot. The majority of the process 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 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 (SQK-RBK110.96), primers were designed by Freed et al., 2020 using Primal Scheme. These primers are in the Midnight RT PCR Expansion (EXP-MRT001) and are designed to generate 1.2 kb amplicons. Primer sequences can be found here.
Steps in the sequencing workflow:
Prepare for your experiment
you will need to:
Before starting - Manual steps:
- Extract your RNA
- Ensure you have your sequencing kit, the correct equipment and reagents
- Prepare your reagents, samples and labware to load on the Hamilton NGS Star 96
- 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:
Automated steps:
- 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
Manual steps:- Quantify your DNA library as a quality control
- Prime the flow cell and load your DNA library into the flow cell
Overview of library preparation workflow:
The image below is representative of the steps that take place in the automated runs for X96 samples.
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, selecting SQK-RBK110.96 and EXP-MRT001 in kit selection, which will collect raw data from the device and convert it into basecalled reads
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Timings
Note: Timings are approximate and subject to change with updates.
Process X24 samples X48 samples X96 samples Hands-on time Deck set-up ~30 minutes Process 1:
cDNA synthesis - Pre-on deck thermocycler6 minutes 8 minutes 10 minutes cDNA synthesis - On deck thermocycler 17 minutes 17 minutes 17 minutes cDNA synthesis - Post-on deck thermocycler 4 minutes 4 minutes 4 minutes Process 2:
cDNA amplification20 minutes 20 minutes 27 minutes cDNA amplification On/Off-deck thermocyler ~3 hours 30 minutes ~3 hours 30 minutes ~3 hours 30 minutes Process 3:
Rapid Barcoding - Pre-on deck thermocycler20 minutes 22 minutes 31 minutes Rapid Barcoding - On deck thermocyler 7 minutes 7 minutes 7 minutes Rapid Barcoding - Post-on deck thermocyler 1 minutes 2 minutes Process 4:
cDNA amplicon pooling/cleanup34 minutes 45 minutes 49 minutes Quantification ~10 minutes Total 5 hours 19 minutes 5 hours 35 minutes 5 hours 55 minutes ~40 minutes -
Nomenclature for automation protocol
Throughout this document, 'Protocol' is defined as the assay on the whole and 'Run' refers to the individual scripts for the automated liquid handling robot, which are specific to indicated protocol step(s).
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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) with the Midnight RT PCR Expansion (EXP-MRT001) using the Hamilton NGS Star 96 liquid handling robot.
We recommend performing a UV clean of the Hamilton NGS Star 96 between each run to minimise risk of contamination.
When processing multiple samples at once, we recommend making master mixes following the indicated volumes to account for the necessary excess. 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.