nanopore nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon, combined RT-PCR)

Anton Pembaur, Erwan Sallard, Patrick Weil, Jennifer Ortelt, Parviz Ahmad-Nejad, Jan Postberg

Published: 2021-09-15 DOI: 10.17504/protocols.io.bx72prqe

Abstract

We established a protocol for fast, cost efficient Sars-CoV-2 sequencing with little as possible hands-on time (around 3h in total, excluding RNA extraction). The whole Sequencing can be done in one working day, including the bioinformatic pipeline. The cost per sample accumulates at around 40$, with already isolated RNA.

We adapted and simplified existing workflows using the ‘midnight’ 1,200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost all of the SARS-CoV-2 genome. Subsequently, we applied the Oxford Nanopore Rapid barcoding protocol and the portable MinION Mk1C sequencer in combination with the ARTIC bioinformatics pipeline. We tested the simplified and less time-consuming workflow on confirmed SARS-CoV-2-positive specimens from clinical routine and identified pre-analytical parameters, which may help to decrease the rate of sequencing failures. Duration of the complete pipeline was approx. 7 hrs for one specimen and approx. 11 hrs for 12 multiplexed barcoded specimens.

This protocol is a modified version of Nikki Freed and Olin Silanders protocol. To get information such as Primers, visit their protocol.

Nikki Freed, Olin Silander 2020. nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon).doi: 10.1093/biomethods/bpaa014

Steps

Sample selection, RNA isolation

For whole genome sequencing using combined RT-PCR, samples with a Ct <20 are suitable. Higher Ct values ≤26 can be suitable as well, but we strongly recommend verification of the amplicon quality utilizing gelelectrophresis and/or microcapillary electrophoresis (Agilent) after combined RT-PCR.

Isolate RNA using any suitable protocol. We tried magnetic Bead based (Nimbus/TanBead), spin collum based (QIAamp Viral RNA, Qiagen) and trizol/chloroforme extraction, with no significant differences.

Multiplex RT-PCR

For Primer dilution and setup, visit Nikki Freed and Olin Silanders protocol.

2.1.

Prepare RT-PCR reaction in a new PCR tube

Pipette Scheme for Luna Universal Probe One-Step RT-qPCR Kit (2X)

(Luna Probe One-Step RT-qPCR 4X Mix with UDG works too, adjust the volumes accordingly)

Luna Universal Probe One-Step Reaction Mix (2X) 10µL

Luna Enzyme Mix (20X) 1µL

Primer Pool1 or Pool2 1µL

Sample up to 8µL (depending on Ct Value and concentration)

Nuclease free H₂O fill up to 20µL

0,75-1$ *2 per sample

2.2.

Set-up the following program on the thermal cycler:

Step Temperature Time Cycles

Reverse Transcription 55°C 0h 30m 0s 1

Heat Activation 95°C 0h 1m 0s 1

Denaturation 95°C 0h 0m 20s 34 for Pool 1, 30 for Pool 2

Annealing and Extension 60°C 0h 3m 30s 34 for Pool 1, 30 for Pool 2

Final Extension 65°C 0h 5m 0s

Hold 4°C Indefinite 1

Citation

Final concentrations of PCR products can range from ~20-150ng/µl. We dont see unexpected products while using more than sufficient input material.

Pooling and PCR quantification/normalisation

Note

At this stage, care should be taken with amplified PCR products. Only open tubes in a designated post-PCR workspace with equipment that is separate from areas where primers and mastermixes are handled. After combining the two pools of amplified DNA, the PCR products can be used for Oxford Nanopore Sequencing, using the RAPID barcode kit RBK004, as described in this protocol.Alternatively, these amplicons can be used for Oxford Nanopore Sequencing, following Josh Quick's ligation based protocol (CoV-2019 sequencing protocol v2, dx.doi.org/10.17504/protocols.io.bdp7i5rn, at step 15) Alternatively, these amplicons can be used for Oxford Nanopore Sequencing, following Josh Quick's ligation based protocol (CoV-2019 sequencing protocol v2, dx.doi.org/10.17504/protocols.io.bdp7i5rn, at step 15) using the SQK-LSK109 kit., at step 15) using the SQK-LSK109 kit.Alternatively, these amplicons can also be used for Illumina sequencing, such as found here: Alternatively, these amplicons can also be used for Illumina sequencing, such as found here: x.doi.org/10.17504/protocols.io.betejeje Silander et al stated that performing an Ampure XP bead clean up at this stage does not improve performance. Therefore, it is not necessary to clean up the PCR reaction at this step.

3.1.

Quantify each PCR reaction using a Qubit or other method. Quantification using Nanodrop is not recommended.

DNA quantification using the Qubit fluorometer 0.35$ *2 per sample

3.2.

Pool the two PCR reactions for each sample in a new PCR tube using 200 ng for each sample to a total amount of 400ng . Add nuclease free water to a total volume of 7.5µL .

Rapid barocoding using the SQK RBK004

Multiple samples can be run on the same flow cell by barcoding. Up to 12 samples can be multiplexed in this approach. Amplicons from each sample will be individually barcoded in the following steps. These follow the RBK004 protocol from Oxford Nanopore. Tip: aliquot the Rapid barcodes into a PCR strip to enable multichannelling.

4.1.

Set up the following reaction for each sample:

Component Volume

DNA amplicons from step 15 (100ng total) 7.5µL

Fragmentation Mix RB01-12 (one for each sample, included in kit) 2.5µL

Total 10µL

8.12$ per sample For higher number of samples it may be more efficient, to use SQK-RBK110.96 with 96 Barcodes, to come to a cost of 3$ per sample.

4.2.

Mix gently by flicking the tube, and spin down.

4.3.

Incubate the reaction in a PCR machine:

30°C for 0h 1m 0s

80°C for 0h 1m 0s

4°C for 0h 0m 30s

4.4.

Pool all barcoded samples, noting the total volume.

Ampure XP Bead Cleanup. Use a 1:1 ratio of sample to beads.

Amplicon clean-up using SPRI beads for RAPID nanopore kit RBK004

Add 1µLof RAP (from the RBK004 kit) to 10µLcleaned, barcoded DNA from previous step . Mix gently by flicking the tube, and spin down.

Incubate the reaction for 0h 5m 0s at room temperature.

The prepared library is used for loading into the MinION flow cell according to Oxford Nanopore Rapid Barcoding (RBK004) protocol. Please refer to the Oxford Nanopore Rapid Barcoding RBK004 protocol at this stage. Store the library on ice until ready to load .

MinION sequencing

Start the sequencing run using MinKNOW.

For real-time surveillance of basecalling and demultiplexing for each of the 12 multiplexed samples per run, enable these options in the MinKNOW software or use RAMPART (https://github.com/artic-network/rampart) on a dedicated LINUX environment.

Equipment

Value	Label
MinION	NAME
Sequencer	TYPE
Oxford Nanopore Technologies	BRAND
MinION 1B / MinION 1C	SKU
https://nanoporetech.com/	LINK

Equipment

Value	Label
MinION Flow Cell	NAME
Sequencer	TYPE
Oxford Nanopore Technologies	BRAND
FLO-MIN106D	SKU
https://nanoporetech.com/	LINK

24$ per sample if using the flowcell three times with 12 Barcodes each

10.

Depending on the variation in coverage of each amplicon, generally, you will need approx 10,000 to 20,000 reads or 10-20Mb per sample to confidently assemble and call variants. This can typically be achieved on a minION flow cell in around four hours when runnning 12 samples. Shorter, if running fewer samples.

11.

For a simple bioinformatic pipeline optimized on Sars-CoV-2 genome assembly and analysis, we recommend using the interARTIC tool, wich provides a GUI to ether medaka or nanopolish workflow.

If needed, for more accurate variant calling, we wrote a python script which combines the consensus sequence from medaka and nanopolish pipeline. After downloading this toolconsensus_merging.py , direct to the save path of this tool in the command line of the console via cd command. Follow the example below, which, after python command, uses the arguments 'medaka_consesensus_sequence_path', 'nanopolish_consesensus_sequence_path', 'reference_genome_path' and 'output_path':

python consensus_merging.py '/home/documents/interartic_medaka/nCoV_2019_midnight_V1_sample1_medaka_single_medaka/medaka.consensus.fasta' '/home/documents/interartic_nanopolish/nCoV_2019_midnight_V1_sample1_nanopolish_single_nanopolish/param2.consensus.fasta' '/home/documents/sarscov2reference.fasta' '/home/documents/merge_output'

All data from our SEQs is available: https://t1p.de/seqdata