TITLE: &#34;Midnight&#34; SARS-CoV2 genome sequencing protocol using 1200bp amplicon  primer set v2 and the Nanopore Rapid library kit
AUTHORS: Nikki Freed, Lydia Murphy, Benjamin Schwessinger, Olin Silander

[DESCRIPTION]
This protocols is part of the ANU Biosecurity mini-research project #2 "An SARS-COV2 incursion scenario: Genomics, phylogenetics, and incursions." This mini-research project is modeled on the yearly Quality Assurance Program of The Royal College of Pathologists of Australia (RCPAQAP), we take part in together with ACT Pathology. 

This research project is split into two major parts, identical to how the official RCPAQAP is run every year. 

Part #1 is focusing on the 'wet- lab' by sequencing SARS-COV2 from real world RNA samples provided by ACT Pathology especially for our ANU biosecurity course (Thank YOU!). Here you will amplify and sequence five (5) RNA samples per research group. You will assess the SARS-COV2 genome sequences for their lineage assignments using online programs,  put sequences into a global context, estimate the collection date based on genetic information, and describe mutations in the spike protein.

Part #2 is focusing on the 'dry-lab' by investigating a hypothetical incursion scenario in the so-called city Fantastica. You will combine genomic surveillance of SARS-COV2 with case interview data to trace the spread into of SARS-COV2 in the community and into high risk settings. We will provide you with real publicly available SARS-COV2 genome and fantasized case interviews. You will put these two together to trace the spread and suggest potential improvements in containment strategies with a focus on high risk settings.

This protocol describes the wet-lab component of Part #1. It is an adaption (fork) of a previous protocol published by Nikki Freed and Olin Silander during the early days of the pandemic here and here. They designed the protocol to enable faster, easier sequencing of SARS-COV2 genomes with fewer steps than previous methods, we use multiplexed 1200 base pair PCR amplicons with the Oxford Nanopore RAPID barcoding kit.   

The original publication that describes this protocol is here along with important considerations is here:    https://academic.oup.com/biomethods/article/5/1/bpaa014/5873518?login=true

Primers were all designed using Primal Scheme: http://primal.zibraproject.org/, described here https://www.nature.com/articles/nprot.2017.066.

You will be using the midnight primer set v2 described here and purchased here https://sg.idtdna.com/pages/products/next-generation-sequencing/workflow/xgen-ngs-amplicon-sequencing/predesigned-amplicon-panels/sars-cov-2-midnight-amp-panel.

The final goal is to achieve the following:

Week 7:
cDNA reverse transcription of RNA into DNA. 
Pooled amplification of the SARS-COV2 genomes chunked into ~1200bp amplicons with non-overlapping pools A and B.

Week 8:
Library preparation using the Nanopore Rapid barcoding kit.
Sequencing your own library on a Nanopore flongel. 

This protocol is applicable for week 7 and 8. 



[BEFORE_START]
You must study the protocol carefully before you start. If anything is unclear post questions directly here on protocols.io.

[GUIDELINES]
You must have read, understood, and follow the health and safety instructions provided in the "Overview Mini-Research Project #2 BIOL3106/6106" provided on Wattle (ANU learning portal). 

You must have signed and returned one copy of the "Student Safety Declaration Form For Practical Class Work" before starting any laboratory work. 

You must have read and understood the Hazard Sheets (Risk assessment) of all chemicals listed bellow in the "Safety Warnings" section. These Hazard Sheets are provided on Wattle as part of the  "Overview Mini-Research Project #2 BIOL3106/6106" document.

[STEPS]
SECTION: cDNA preparation
2. You will receive a PCR strip of eight with 6 tubes containing 2uL Lunascript RT supermix. Label the tubes with 1-6 and your RG name. Note down which sample corresponds to which number.

Mix the following components in an 0.2mL 8-strip tube on ice; 

Component                                        Volume
LunaScript®RT SuperMix Kit (5x)           2 µL       
Template RNA                                           8 µL

                                             Total               10 µL 


This means add 8uL of sample to each tube separately. Gently mix by pipetting up and down during the adding of each sample. Change the tip between each sample addition. Once you added all samples close the lid.
SECTION: cDNA preparation
3. Pulse spin the tube to collect liquid at the bottom of the tube.
SECTION: cDNA preparation
4. Incubate the reaction in a PCR machine:

25 °C 2 min 
55 °C 10 min 
95 °C 1 min
Hold at 20 °C 
SECTION: PCR amplification with two primer pools
5. You will have to prepare two PCR master mixes one for each primer pool A and B. For this, you will have two tubes of 81.25ul Q5 hot start master mix provided. Label each tube with pool A or B and your RG name.

Component and volume per sample            Pool A                          Pool B

Q5® Hot Start HF 2x Master Mix             12.5 µL             12.5 µL
Primer Pool A (10µM)                                1.1 µL                0 µL
Primer Pool B (10µM)                                0 µL                   1.1 µL   
Nuclease-free water                                   8.9 µL               9.0 µL

                        Total                                      22.5 µL             22.5 µL 

Considering you have five samples and one negative control, generate the following two master mixes.




 

Once you added all ingredients, mix by vortexing and spin down to collect the liquid. Place on ice.

SECTION: PCR amplification with two primer pools
7. Collect your cDNA containing strip tubes (the samples will have a blue color). Spin them down briefly.
 
add 2.5 µL each cDNA sample [1-6] to a tube containing 22.5 µL  Pool A that is labelled accordingly. Mix well by pipetting up and down. Change tip after each addition.

Add 2.5 µL each cDNA sample [1-6] to a tube containing 22.5 µL  Pool B that is labelled accordingly. Mix well by pipetting up and down. Change tip after each addition.

Leave the strip tubes on ice. Close the tubes with a lid when done adding all samples.
SECTION: PCR amplification with two primer pools
8. Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
SECTION: PCR amplification with two primer pools
9. Set-up the following program on the thermal cycler:  

Step                                            Temperature     Time                    Cycles

Heat Activation                         98 °C        30 s    1
Denaturation                             98 °C        15 s     35 
Annealing and Extension        65 °C        5 min     35
Hold                                           20 °C            Indefinite              1



 

Hand over your PCR reactions to demonstrators as they will run them till next week.
SECTION: Pooling and PCR quantification
11. Label a  1.5 mL Eppendorf tube with sample name and RG name. In total this will be six tubes. 

For each sample combine the PCR reactions (pool A and B) by pipetting all 25ul of each pool into one tube. This means each 1.5 ml eppendorf tube will contain 50ul in total. 

Mix by vortexing and spin down samples briefly.

SECTION: Pooling and PCR quantification
11.1. Quantify DNA using a Qubit. 

You will be provided with 6 tubes of 198ul Qubit HS solution. 
Label the tubes with the numbers 1-6. 
Add 2uL of each combined PCR reactions into one Qubit tube. 
Vortex briefly, hand centrifuge, incubate for 2 mins, and measure with the Qubit machine.
Note down your concentration in ng/ul for each sample.




SECTION: Normalisation
12. Label a 1.5 eppendorf tube for each sample so you can generate a stock PCR solution of 13.33 ng/ul the final volume being 20uL.

Each tube must be labelled as stock 13.33 ng/ul, sample name, and RG name.

The important formula here is: 

n = c*V with n being the amount in ng, c the concentration in ng/ul, and V the volume.


You want V = 20ul and c =13.33 ng/ul. This means you will have 267ng in 20 ul.


The question is how much volume of the original PCR do you need to obtain 267ngs?


Here now n = 267g and c equals your measured concentration. Let's assume your measured c = 56 ng/ul for an assumed sample. Than we can resolve the equation as follows.


n = c*V -> V = n/c -> 267ng/56ng/ul =  4.7 ul


So you need to use 4.7ul in 20ul total volume to obtain 267ng. This means you can combine 13.7ul H20 with 4.7ul of your combined PCR sample. Vortex it and spin down.

In case you concentration is < 13.33 ng/ul use 20ul of sample as stock without dilution.  



SECTION: Rapid barocoding using the SQK RBK110.96
13. Mulitple samples can be run on the same flow cell by barcoding. Up to 96 samples at a time can be  sequenced using the RBK110.96 kit. Amplicons from each sample will be individually barcoded in the following steps.

You will receive a PCR strip of eight with six tubes containing 2.5ul of the fragmentation mix each. Label the strip with sample number [1-6] and RG name.
SECTION: Rapid barocoding using the SQK RBK110.96
13.1. Add 7.5 µL of each diluted PCR stock [13.33 ng/ul] reaction from step 12 to the labeled PCR tube.  

Set up the following reaction for each sample:


Component                                                                                                            Volume
DNA amplicons from step 12  (100ng total)                                                         7.5 µL
Fragmentation Mix (one for each sample, already aliquoted for you)                2.5 µL
Total                                                                                                                         10 µL

Important keep samples on ice!

Mix gently by pipetting up and down briefly, flick the tubes, and spin down.

SECTION: Rapid barocoding using the SQK RBK110.96
13.2. Incubate the reaction in a PCR machine:
30 °C     for     1 min
80 °C     for      1 min
4 °C        for      30 s

SECTION: Rapid barocoding using the SQK RBK110.96
13.3. From here on out you will combine your barcoded samples with one other RG on your bench. Each final library will contain 12 samples to be sequenced together. [With the exception of RG0 who have a single flongel to themselves]. RG1+2, RG3+4 and so on will go together.

For each pair of RGs, label one 1.5 ml Eppendorf tube with BC and RG names. Combine all your barcoded samples in this tube. This tube will contain 120ul total. [RG0 will have 60uL]

Mix by vortexing and spin down.


SECTION: Rapid barocoding using the SQK RBK110.96
14. SPRI Bead Cleanup. Use a 1:1 ratio of sample to beads. This means in case of 120ul combined barcoded samples add 120ul of beads. Ask for help of a demonstrator as needed.


SECTION: Rapid barocoding using the SQK RBK110.96
15. Add 0.5 µLof RAP (from the RBK110.96 kit, a demonstrator will you provide you with this)  to 5 µLcleaned, barcoded DNA from step 14 . Mix gently by flicking the tube, and spin down.




SECTION: Rapid barocoding using the SQK RBK110.96
16. Incubate the reaction for 5 min at room temperature. Place reaction on ice.

SECTION: MinION sequencing
20. To-Do



SECTION: MinION sequencing
23. 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 under two hours when running 12 samples. Less time if running fewer samples. You can also use the Rampart pipeline for a real time assessment of the coverage, which enables you to turn off the sequencing when you have captured enough data. Details on that here:  https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html

SECTION: MinION sequencing
24. The primer scheme .bed and .tsv files necessary for the ARTIC variant calling pipeline are at Zenodo: https://zenodo.org/record/3897530#.Xv5EFpMzadY
SECTION: Week 7: cDNA synthesis and two pooled PCR reactions
1. In week 7 you will perform the cDNA synthesis and PCR reactions on five samples and one negative control. You will be provided with five samples of 10 ul each and one tube of negative control.
SECTION: PCR amplification with two primer pools
6. Label two PCR strips of eight with the PCR pool A or B, number 1-6, and your RG name. Aliquot out 22.5 ul of each master mix (Pool A or B) into the corresponding PCR strips.
SECTION: Week 8: Library preparation and sequencing
10. In week 8 you will perform your own Nanopore sequencing library preparation and sequencing. You will do this by using your PCR reactions from week 7.
SECTION: Library finalisation for Flongel sequencing
17. To-Do
SECTION: Library finalisation for Flongel sequencing
19. 
SECTION: Loading of flongel
18. To-Do
SECTION: MinION sequencing
22. 
SECTION: MinION sequencing
21. 
SECTION: Rapid barocoding using the SQK RBK110.96
14.1. Add 120uL of SPRI beads to your combined barcoded samples. Mix by flicking the tube. Hand centrifuge to collect all liquid in the bottom of the tube while the brown beads stays suspended.
SECTION: Rapid barocoding using the SQK RBK110.96
14.2. Incubate for 5 min at room temperature.

SECTION: Rapid barocoding using the SQK RBK110.96
14.3. Place tube on magnetic rack and incubate for 3 min until the beads have pelleted towards the magnet and the supernatant is completely clear. 

SECTION: Rapid barocoding using the SQK RBK110.96
14.6.   and repeat one more ethanol wash.

SECTION: Rapid barocoding using the SQK RBK110.96
14.5. Add 1ml of freshly prepared room-temperature 80 % volume ethanol to wash the pellet.

SECTION: Rapid barocoding using the SQK RBK110.96
14.4. Leave tube on the magnet and pipette off the supernatant, being careful not to touch the bead pellet. The DNA is now bound to the beads.
SECTION: Rapid barocoding using the SQK RBK110.96
14.5. 
SECTION: Rapid barocoding using the SQK RBK110.96
14.6. Keeping the magnetic rack on the benchtop, rotate the bead-containing tube by 180°. Wait for the beads to migrate towards the magnet and re-form a pellet. Remove the ethanol using a pipette and discard.  
SECTION: Rapid barocoding using the SQK RBK110.96
14.7. 
SECTION: Rapid barocoding using the SQK RBK110.96
14.7. Leave tube on the magnet and pipette off the supernatant, being careful not to touch the bead pellet. The DNA is now bound to the beads.
SECTION: Rapid barocoding using the SQK RBK110.96
14.8. Pulse centrifuge to collect all liquid at the bottom of the tube, place tube back to the magnet, and carefully remove as much residual ethanol as possible using a P10 pipette.
SECTION: Rapid barocoding using the SQK RBK110.96
14.9. With the tube lid open incubate for 3 min to let the pellet try.

SECTION: Rapid barocoding using the SQK RBK110.96
14.10. Remove the tube from the magnetic rack. Resuspend pellet in 15 µL Elution Buffer (EB). Incubate for 3 min at RT. with flicking the tube.

Label a new 1.5ml tube with BCD and your RG names.
SECTION: Rapid barocoding using the SQK RBK110.96
14.11. Place on magnet, incubate for 3 min  so that all beads are on the side of the tube towards the magnet. Transfer 10 µL  to a clean labelled 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.

SECTION: Rapid barocoding using the SQK RBK110.96
15. Label a new 1.5ml Eppendorf tube with L and your RG name.

Transfer 5 µL of your cleaned combined barcoded samples into this tube.

Ask a demonstrator to add 0.5 µL of Rapid Adapter F (RAP-F) to 5 μl of barcoded DNA.

Mix gently by flicking the tube, and spin down.

Incubate the reaction for 5 min at RT. 


SECTION: Rapid barocoding using the SQK RBK110.96
16. Now prepare the final library to be loaded by combining the following a 1.5ml tube labelled FL and your RG name.

15 µL Sequencing Buffer II (SBII)
10 µLLoading Beads II (LBII) mixed immediately before use
2.5 µL EB buffer
2.5 µLof RAP-F adapted barcoded DNA from step 15.

Place library to the side till you are ready to load in step 23.

19. Plug in the MinION contain the flongle adapter in the computer.

  

18. Place the Flongle adapter into the MinION.

  
  

The adapter should sit evenly and flat on the MinION. This ensures the flow cell assembly is flat during the next stage.

SECTION: Loading the library onto a Flongel
17. Please familiarize yourself with how to load a flongel in this video https://nanoporetech.com/resource-centre/video/ncm22/how-to-load-a-flongle-flow-cell [0-12 minutes only]. Ask a demonstrator to help with the loading!
19. Plug the MinION containing the Flongle adapter into the laptop.

  

21. 
20. Place the flow cell into the Flongle adapter, and press the flow cell down until you hear a click.

The flow cell should sit evenly and flat inside the adapter, to avoid any bubbles forming inside the fluidic compartments.

  

21. Now label take a 1.5 ml Eppendorf tube labelled FL that contains 117 μl of Flush Buffer (FB) with 3 μl of Flush Tether (FLT). Mix by flicking and collect liquid at the bottom of the tube with a spin.

Peel back the seal tab from the Flongle flow cell, up to a point where the sample port is exposed, as follows:

1. Lift up the seal tab:

  

2. Pull the seal tab to open access to the sample port:

  
3. Hold the seal tab open by using adhesive on the tab to stick to the MinION lid:

  
To prime your flow cell with the mix of Flush Buffer (FB) and Flush Tether (FLT) that was prepared earlier, ensure that there is no air gap in the sample port or the pipette tip. Place the P200 pipette tip inside the sample port and slowly dispense the priming fluid into the Flongle flow cell by slowly pipetting down. We also recommend twisting the pipette plunger down to avoid flushing the flow cell too vigorously.

  

23. 
22. Now you will load the final library from step 16. Make sure to resuspend the library and beads by flicking followed by a quick hand centrifuge. 

To add the 30 µL of your final library to the flow cell, ensure that there is no air gap in the sample port or the pipette tip. Place the P200 tip inside the sample port and slowly dispense the Sequencing Mix into the flow cell by slowly pipetting down. We also recommend twisting the pipette plunger down to avoid flushing the flow cell too vigorously.

  

24. 
23. Seal the Flongle flow cell using the adhesive on the seal tab, as follows:

1. Stick the transparent adhesive tape to the sample port.


2. Replace the top (Wheel icon section) of the seal tab to its original position.

Close the MinION li lid.
21. Take the tube labelled with FL that contains 117 μl of Flush Buffer (FB) with 3 μl of Flush Tether (FLT). Mix it by flicking an collect it at the bottom of the tube.

Peel back the seal tab from the Flongle flow cell, up to a point where the sample port is exposed, as follows:

1. Lift up the seal tab:

  
2. Pull the seal tab to open access to the sample port:

  

3. Hold the seal tab open by using adhesive on the tab to stick to the MinION Mk 1B lid:

  

20.  
Place the flow cell into the Flongle adapter, and press the flow cell down until you hear a click.
The flow cell should sit evenly and flat inside the adapter, to avoid any bubbles forming inside the fluidic compartments. 

23. Take the final library from step 16 and mix by flicking so it is milky again. Collect by a quick hand centrifuge. Take the 30 ul and load.

To add the final library to the flow cell, ensure that there is no air gap in the sample port or the pipette tip. Place the P200 tip inside the sample port and slowly dispense the Sequencing Mix into the flow cell by slowly pipetting down. We also recommend twisting the pipette plunger down to avoid flushing the flow cell too vigorously.

  

22. Take the 120ul of FL. To prime your flow cell with the mix of Flush Buffer (FB) and Flush Tether (FLT) that was prepared earlier, ensure that there is no air gap in the sample port or the pipette tip. Place the P200 pipette tip inside the sample port and slowly dispense the priming fluid into the Flongle flow cell by slowly pipetting down. We also recommend twisting the pipette plunger down to avoid flushing the flow cell too vigorously.

  

24. Seal the Flongle flow cell using the adhesive on the seal tab, as follows:
1. Stick the transparent adhesive tape to the sample port.

  
2. Replace the top (Wheel icon section) of the seal tab to its original position.
  

Close the MinION lid.
SECTION: Data acquisition and basecalling
25. Open the MinKNOW  software on the laptop and get a demonstrator to help you get the sequencing run started.