TITLE: A SARS-CoV-2 Surveillance Sequencing Protocol Optimized for Oxford Nanopore PromethION
AUTHORS: Jannatul  Ferdous, Torri Weathers, Visva Bharati  Barua, Erin Stiers, Adam France, Kevin C Lambirth, Cynthia  Gibas, Jessica A Schlueter

[DESCRIPTION]
To identify and monitor SARS-CoV-2 variant evolution, UNC Charlotte has introduced a surveillance sequencing program for the campus and surrounding county area. We have generated a step by step protocol which has been optimized from the ”High Throughput Nanopore Sequencing of SARS-CoV-2 Viral Genomes from Patient Samples”  (Pater et al 2021, https://doi.org/10.1101/2021.02.09.430478) protocol for preparing SARS-CoV 2 viral genome libraries for next generation sequencing using the Oxford Nanopore PromethION instrument. The protocol is designed to work in a 96-well format throughout. It is intended to produce sufficient sequence for genome assembly to meet standards required by GISAID and NCBI for SARS-CoV-2 sequence submission. The protocol has been modified to achieve maximum yield and includes modifications to address isolate sequencing failures arising from low viral titer in some clinical samples (Cq values below 30). The protocol provides detailed guidelines and steps for working with high Cq value clinical samples, as well as a protocol variation for working in half-reaction volumes throughout to reduce sequencing costs. 

[GUIDELINES]
Wear PPE all the time while doing this protocol. 
Clean the workbench and all the pipettes with 70% Ethanol before using. 
Be very careful while washing the samples with beads in the SPRI clean up, barcoding and adapter ligation section, as a loss of a small amount of beads can lead to a significant decrease in recovered DNA. 

[STEPS]
SECTION: cDNA Synthesis
1. Reagents : 
10x Random Primer 
25x dNTPs Mix 
10x RT Buffer
50mM MgCl2
MultiScribe  RT

Thaw everything except MultiScribe  RT at room temperature. Mix each of the tubes by vortexing. After that spin down and place on ice. 


SECTION: cDNA Synthesis
2.  Prepare master mix 1 (MM1) in a 1.5ml  Lobind tube on ice. 

 Prepare master mix for number of samples+ extra 10 samples for errors. For example, if there are 96 sample , prepare master mix for 96 + 10 = 106 samples. Here is the calculation for 96 samples: 

Components                                            Volume* Number of samples             Master mix

Nuclease free water                                4 * 106                                                     424 ul 
10x Random Primer                                2 * 106                                                     212 ul
25x dNTPs Mix                                        0.8 * 106                                                  84.8 ul 

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.

SECTION: cDNA Synthesis
3. Array 10ul RNA into a 96 well plate on an ice block. Label this plate as "CDNA synthesis".

 Add 6.8ul of MM1 into the RNA plate.



SECTION: cDNA Synthesis
5. Prepare master mix 2 (MM2) in a 1.5ml  Lobind tube on ice. 

 Prepare master mix for number of samples + extra 10 samples for errors. For example, if there are 96 sample , prepare master mix for 96 + 10 = 106 samples.

Components                        Volume* Number of samples              Master Mix

10x RT Buffer                        2 * 106                                                    212 ul 
Multiscribe RT                      1 * 106                                                    106 ul 
MgCl2                                    0.2 * 106                                                 21.2 ul

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.


SECTION: cDNA Synthesis
6.  Remove the RNA plate from the thermal cycler and take out the sealer slowly and carefully so that the solutions in the wells do not make any spill. 

Add 3.2 ul of MM2 into the RNA plate .





SECTION: cDNA Synthesis
7. Seal the plate and mix the solution by quick spin in the plate spinner.

Incubate the reactions as follows:

25 °C  for 00:10:00
37 °C  for 02:00:00
85 °C  for 00:05:00
Hold at 4 °C 


SECTION: Artic Amplification
9. Reagents : 

5X Q5 Reaction buffer
Q5 High-fidelity polymerase
V3 ARTIC primer pool 1 diluted to 10uM
V3 ARTIC primer pool 2 diluted to 10uM
2.5mM dNTP mix

Remove 5X Q5 Reaction buffer, Q5 High-fidelity polymerase, V3 Artic primer pool 1 and 2, dNTP mix from the -20 freezer and place on ice to thaw. Mix each of the tubes by vortexing. After that spin down and place on ice. 
SECTION: Artic Amplification
10. Prepare 2.5mM dNTP  in a 2ml microcentrifuge tube as below: 
50ul of dATP 
50ul of dCTP
50ul of dTTP
50ul of dGTP
1800ul of nuclease free water 
Vortex and spin 

Prepare 10uM V3 pool 1 in a 1.5 ml tube as below : 
100ul V3 pool 1
900ul Nuclease free water
Vortex and spin
 
Prepare 10uM V3 pool 2 in a 1.5 ml tube as below : 
100ul V3 pool 2
900ul Nuclease free water
Vortex and spin 


SECTION: Artic Amplification
11. Set up the two PCR plate reactions per sample . ARTIC pool 1 and ARTIC pool 2. This should be two 96 well plates for 96 samples. 

Label these plates as "Artic Pool 1" and "Artic Pool 2 ".

  

SECTION: Artic Amplification
12. Prepare master mix 1 (MM1) into  a Falcon tube on ice for ARTIC Pool 1.

Prepare master mix for number of samples + extra 10 samples for errors. For example, if there are 96 sample , prepare master mix for 96 + 10 = 106 samples. Calculations are provided below : 

Components                                    Volume* Number of samples          Master Mix

Nuclease free water                        8.75 * 106                                              927.5 ul 
Reaction buffer                                5 * 106                                                     530 ul
V3 Pool 1                                          4 * 106                                                     424 ul 
2.5mM dNTP mix                            2 * 106                                                     212 ul
Q5 High fidelity polymerase          0.25 * 106                                               26.5 ul

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.



SECTION: Artic Amplification
13.  Add 20 ul of MM1 into the ARTIC Pool 1 96 well PCR plate.

Add 5 ul of cDNA into the ARTIC Pool 1 plate.

Seal the plate and mix the solution by quick spin. 

SECTION: Artic Amplification
14. Prepare master mix 2 (MM2) into  a Falcon tube on ice for ARTIC Pool 2.

Prepare master mix for number of samples + extra 10 samples for errors. For example, if there are 96 sample , prepare master mix for 96 + 10 = 106 samples. Calculations are provided below : 

Components                                    Volume* Number of samples          Master Mix

Nuclease free water                        8.75 * 106                                              927.5 ul 
Reaction buffer                                5 * 106                                                     530 ul
V3 Pool 2                                         4 * 106                                                     424 ul 
2.5mM dNTP mix                            2 * 106                                                     212 ul
Q5 High fidelity polymerase          0.25 * 106                                               26.5 ul

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.

SECTION: Artic Amplification
15. Add 20 ul of MM2 into the ARTIC Pool 2 96 well PCR plate. 

Add 5 ul of cDNA into the ARTIC Pool 2 plate .

Seal the plate and mix the solution by quick spin. 

SECTION: Artic Amplification
16. Set up the following program on the thermal cycler and put both the ARTIC Pool plates in the cycler .

98 °C      00:00:30    1 cycle
94 °C      00:00:16    20 cycle
65 °C     00:05:00    20 cycle
94 °C     00:00:16    15 cycle
63 °C      00:05:00    15 cycle
Hold at 4 °C        Indefinite

SECTION: Artic Amplification
17. This plate can be stored at -20 °C  for long term and at 4 °C for short term. 

SECTION: cDNA Synthesis
4. Seal the plate and mix the solution by quick spin in the plate spinner. 

Incubate at 65 °C  for 5 min in the thermal cycler .
SECTION: cDNA Synthesis
8. This plate can be stored at -20 °C  for long term and at 4 °C for short term. 

SECTION: SPRI Clean up
18. Reagents : 

AMPure XP Beads
80% ethanol fresh
Omega EB 
SECTION: SPRI Clean up
20. Spin down each PCR product plate of pool 1 and pool 2 at 2,000 RPM for 30 seconds.  

 Remove the sealer slowly and carefully to avoid contamination. 
SECTION: SPRI Clean up
21. Transfer  25ul from column 1 of  pool 1 to column 1 of the pool 2 plate. Now column 1 of pool 2 should have a total solution of 50ul. Repeat this until all of the columns of pool 1 have been transferred to pool 2.

Resuspend the stock AMPure XP Beads by vortexing for 15- 20 seconds.

Put 5 mL of AMPure beads into a trough and add 50ul of AMPure beads to each well of the pool 2 plate with an 8-channel pipette. Mix  by pipetting up and down 5 times while adding.


  
From now on this plate will be called the washing plate.

SECTION: SPRI Clean up
22. Cover the 96-well washing  plate using  a tip box lid.  Incubate this plate at room temperature for 15 minutes.

 
SECTION: SPRI Clean up
23. After 15 mins, seal the plate with film, spin it for 15seconds and  place it on the magnet for 7  minutes.  

Check if the solution became clear or not. If not, continue to keep the plate on the  magnet.  

If clear, carefully discard the supernatant using a P200 multichannel pipette without disturbing  the beads.  

  

SECTION: SPRI Clean up
24. While keeping the plate on the magnet, add 200 μL of 80% ethanol to the plate without disturbing the beads.  

Wait for 3 minutes with the plate on magnet.

Carefully discard the ethanol using a multichannel pipette while the plate stays on the  magnet. This ends the first ethanol wash.  

Repeat this step  (80% ethanol wash) for a second ethanol wash.  

SECTION: SPRI Clean up
25. Seal the plate with plate film and spin down the 96-well clean-up plate at 2,000 RPM for 30 seconds.

Place the 96-well clean-up plate on the magnet and remove the plate film.

Use a P10 single channel pipette to remove any residual ethanol .  

Remove the plate from the magnet and place onto the benchtop.  

SECTION: SPRI Clean up
26.  Add 33 μL of Omega EB to each of the wells to elute the DNA and resuspend the beads. Mix by pipetting up and down 5 times.  

Remember to change tips and continue until all of the beads in the 96-well clean-up plate have been  resuspended.  

It is essential to move quickly as the AMPure beads should not dry out, so try to keep everything well set up in the bench before you start resuspending. 

  

SECTION: SPRI Clean up
27.  Cover the plate using a  tip box lid and incubate for 2 minutes at room temperature.  

Seal the plate with a plate sealer and spin down the plate containing the resuspended DNA at 2,000 RPM for 15 seconds.  

Place the plate on the magnet for 7 minutes.  
SECTION: SPRI Clean up
28. After 7 mins carefully remove and retain 30 μL of the eluate containing the DNA library per well into a new 96-well PCR Plate. Elution with a smaller volume prevents the beads from being eluted as well.
SECTION: SPRI Clean up
29. Seal and label the plate containing the eluate as "Clean up plate". This plate will be used for qubit quantification and end-prep reaction.

This plate can be stored at -20 °C  for long term and at 4 °C for short term. 
 


SECTION: Qubit Quantification
30. Reagents :
The Qubit quantification kit - comes with 2 standards (standard 1 and standard 2), reagent (dye) and dsDNA HS buffer. 

Equilibrate all of the kit components at room temperature. 

Note :  The qubit quantification kit can also come with prepared qubit  solution (dye already added in the HS buffer). In that case no need to prepare the  qubit solution separately and move onto step 32.
SECTION: Qubit Quantification
31. For qubit flex flurometer  prepare enough qubit working solution for number of samples and 16 standards ( 8 standard 1 and 8 standard 2). 

For examples if you have 96 samples , prepare solution for - 96 samples + 16 standard + 10 extra = 122 samples. 

Prepare Qubit solution in a 50ml tube as follow : 

Components                                     Volume * Number of samples       Solution 

dsDNA HS buffer                              199 * 122                                          24278 ul
Qubit dye                                              1 * 122                                             122 ul 

Mix by solution by vortexing and spin.  




SECTION: Qubit Quantification
32. Set up the qubit assay 8 tube strip tubes .  One 8-tube strip tube for standard 1, one 8-tube strip tube for standard 2 and  twelve 8-tube strip tube for the samples (if you are doing 96 samples).

For standard 1,  add 190 ul of qubit working solution to each tube and 10ul of standard 1. 
For standard 2, add 190 ul of qubit working solution to each tube and 10ul of standard 2.
For samples, add 199 ul of qubit working solution to each tube and 1ul of sample.

Incubate the standard and samples at room temperature for 2 minutes. 
SECTION: Qubit Quantification
33. On the home screen select 1x dsDNA High sensitivity and then select Read new standards and samples. 

Insert standard strip tube/single tube in the chamber and close the lid . Remember to put the strip tube in a way so that the left most tubes represent first of the standard 1. Press read standard. 

  

Take the tube out and insert standard 2 strip tubes/single tube. Press read standard. This completes the calibration. 

Take the reading of the samples the same way as standards. The data of the samples can be retrieved with the qubit  flash drive from the section data and then the folder of respective dates. 


SECTION: Library End Prep
34. Reagents : 

NEB Ultra ll end prep reaction buffer 
NEB Ultra ll end prep reaction enzyme

Thaw the reagents at room temperature. Mix each of the tubes by vortexing. After that spin down and place on ice. 
SECTION: Library End Prep
35. Prepare Master mix in a 1.5ul Lobind tube. If you are doing 96 samples then prepare master mix for number of samples + 10 extra samples. If you are doing 96 samples prepare master mix for 106 samples . 

Prepare master mix in a 1.5 tube as follow : 

Components                                                          Volume * Number of samples       Master Mix 

NEB Ultra ll end prep reaction buffer                            2.015 * 106                                       213.59ul
NEB Ultra ll end prep reaction enzyme                         0.8625 * 106                                     91.425 ul 

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.
SECTION: Library End Prep
36. Insert the sample DNA concentration  from Qubit in the template_sc2_end prep.xlsx spreadsheet , in the DNA (ng/ul) column. This will generate the amount if sample and water needed to be added to normalize all the samples. 

Add water according to the mentioned spreadsheet to a new PCR plate labeled as "End prep plate". 

Thaw and spin down the cleaned up PCR plate from SPRI clean up step and add sample according to the spreadsheet from this plate to the new End prep plate. 

Add 2.5ul of Mastermix to the End prep plate. 

Seal the plate with a plate sealer and spin it down for 15 seconds. 

SECTION: Library End Prep
37. Set up the following program on the thermal cycler and put  the End prep plate in the cycler.

20 °C      00:10:00    
25 °C      00:05:00    
65 °C     00:15:00    
Hold at 4 °C        Indefinite
SECTION: Library End Prep
38. Left over clean up plate and finished end prep plate both can be stored at -20 °C for long term and at 4 °C  for short term.

SECTION: Sample Barcoding
39. Reagents : 

Native barcoding Expansion (EXP - NBD 196)
NEB Ultra ll ligation master mix 
NEB Ultra ll ligation Enhancer  
ONT Short Fragment Buffer (SFB)
Elution buffer (EB)


Thaw all the reagents at room temperature . Mix each of the tubes by vortexing. After that spin down and place on ice.  
SECTION: Sample Barcoding
40. Prepare the master mix in a 2ml lobind tube. If you are doing 96 samples then prepare master mix for number of samples + 10 extra samples. If you are doing 96 samples prepare master mix for 106 samples . 

Prepare master mix in a 1.5 tube as follow :

Components                                                  Volume * Number of samples                      Master Mix

Water                                                                          5.7 * 106                                                       604.2 ul
NEB Ultra ll ligation master mix                              10 * 106                                                         1060 ul
NEB Ultra ll ligation Enhancer                                  0.3 * 106                                                        31.8 ul

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.
SECTION: Sample Barcoding
41. Add 1.5 ul of end prepped DNA in a new 96 well PCR plate. Label this plate as " Barcoding plate". 

Add 2.5 ul of barcode in the new barcoding plate. 

Add 16 ul of the master mix to the new barcoding plate

Mix all the components by pipetting up and down. 

Seal the plate with a plate sealer. 

Spin down for 15 seconds in a plate spinner. 

Put the leftover end prep plate in -20 °C 

SECTION: Sample Barcoding
42. Set up the following program on the thermal cycler and put  the barcoding plate in the cycler .

20 °C      00:20:00    
25 °C      00:05:00    
65 °C     00:15:00    
Hold at 4 °C        Indefinite
SECTION: Sample Barcoding
43. After the barcoding reaction, take out the plate, remove the seal and pool all the reactions into two 1.5 mL microcentrifuge tube.  Split the reaction equally into 2 microcentrifuge tubes. 

Mix the contents well and spin down briefly.  

SECTION: Sample Barcoding
44. Resuspend the AMPure XP beads by vortexing for 30 seconds.  

Check the volume of each microcentrifugre tube  and  add 0.5x the volume of AMPure XP  beads to the each of the tubes.  

Mix the beads with sample by pipetting up and down.
  
Close the tube tightly and flick the sample several times.  

Incubate the tube at room temperature for 10 minutes on a Hula mixer .
SECTION: Sample Barcoding
45. Take the tubes and spin down for 15 seconds. 

Put the tubes in magnetic rack for 7 mins. 

  

Check if the solution became clear or not. If not, continue to keep the plate on the  magnet.  

If clear, carefully discard the supernatant using a P200 multichannel pipette without disturbing  the beads.  

  

Remove the tube from the magnet and place it on the benchtop. 

SECTION: Sample Barcoding
46. Add 500 µL of Short Fragment Buffer (SFB) to each of the tubes, pipette up and down  and flick the beads to resuspend.  

Pulse centrifuge the tubes and place on the magnetic stand for 5 minutes. 

Check whether the solution became clear. If not, continue to keep the plate on the  magnet.  

Keeping the tubes on the magnet, carefully  discard the supernatant  without disturbing the beads. This ends the first SFB wash.  

Repeat this whole step for a second SFB wash. 
SECTION: Sample Barcoding
47. Pulse centrifuge the tubes and place on the magnet.  

Use a P10 single channel pipette to remove any residual SFB you observe.  
 
Keeping the tube on the magnet, add 100 μL of freshly made 80% ethanol.  

Remove the ethanol, spin down, place on magnet and use a P10 single channel pipette to remove any  residual ethanol.  

Allow the beads to air dry for ~30 sec-1 min. 
SECTION: Sample Barcoding
48. Remove the tubes from the magnetic stand and place onto the benchtop. 

Add 18 μL of the Elution Buffer (EB) to the tube to elute  the DNA and resuspend the beads  and mix by pipetting up and down.

Incubate at room temperature for 5 minutes.  

Flick the tubes several times and pulse centrifuge.  

Place the tubes on magnetic stand for 5 minutes.  

Take out 15ul  clear supernatent from each of the tube. 

Pool both of the elutions into a newly labeled 1.5 mL microcentrifuge  tube. You should now have 30 μL of pooled barcoded sample.

SECTION: Sample Barcoding
49. Use this pooled sample to do qubit quantification according to the Qubit quantification section. Follow the qubit quantification with qubit flurometer step case. 

SECTION: Adapter Ligation
51. Reagents : 

Adapter Mix ll
NEB next quick ligation reaction buffer 
NEB Quick T4 DNA ligase
Short Fragment Buffer (SFB)
Elution buffer (EB)

Thaw all the reagents at room temperature. Mix each of the tubes by vortexing. After that spin down and place on ice. 
SECTION: Adapter Ligation
52. Thaw the pooled barcoded 30ul sample. 

Add 5ul AMll , 5ul Quick T4 DNA ligase and 1oul NEB reaction buffer to the pooled sample. 

Mix all the components by pipetting up and down. 

Incubate at room temperature for 25 minutes. 
SECTION: Adapter Ligation
53. Resuspend the AMPure XP beads by vortexing for 30 seconds.  

Add 30ul  AMPure XP  beads to the pooled sample.  

Mix the beads with sample by pipetting up and down.
  
Close the tube tightly and flick the sample several times.  

Incubate the tube at room temperature for 10 minutes on a Hula mixer .
SECTION: Adapter Ligation
54. Take the tube and spin down for 15 seconds. 

Put the tubes in magnetic rack for 5 mins. 

Check if the solution became clear or not. If not, continue to keep the plate on the  magnet.  

if clear, carefully discard the supernatant using a P200 multichannel pipette without disturbing  the beads.  

Remove the tube from the magnet and place it on the benchtop. 

SECTION: Adapter Ligation
55. Add 200 µL of Short Fragment Buffer (SFB) to each of the tubes, pipette up and down  and flick the beads to resuspend.  

Pulse centrifuge the tubes and place on the magnetic stand for 5 minutes. 

Check whether the solution became clear. If not, continue to keep the plate on the  magnet.  

Keeping the tubes on the magnet, carefully  discard the supernatant  without disturbing the beads. This ends the first SFB wash.  

Repeat this whole process for a second SFB wash. 
SECTION: Adapter Ligation
56. Pulse centrifuge the tubes and place on the magnet.  

Use a P10 single channel pipette to remove any residual SFB you observe.  

Remove the tubes from the magnetic stand and place onto the benchtop. 
SECTION: Adapter Ligation
57. Add 18 μL of the Elution Buffer (EB) to the tube to elute  the DNA and resuspend the beads  and mix by pipetting up and down.

Incubate at room temperature for 5 minutes.  

Flick the tubes several times and pulse centrifuge.  

Place the tubes on magnetic stand for 5 minutes.  

Take out 15ul of clear supernatent and put it into a newly labeled 1.5ml tube . This is the final library. 
SECTION: Adapter Ligation
58. Use this pooled sample to do qubit quantification according to the Qubit quantification section. Follow the qubit quantification with qubit flurometer step case. 
SECTION: SPRI Clean up
19. Prepare 80% ethanol fresh in a 50ml tube as below:   
  
    Pour 40 mL 100% ethanol.  
    Add 10 mL of ddH2O.  
    Mix by inversion

SECTION: Sample Barcoding
50.  The rest of the pool can be stored at 4 °C  for short term and at -20 °C  for long term.

SECTION: Adapter Ligation
59. The rest of the pool can be stored at 4 °C  and at -20 °C  for long term. 

SECTION: cDNA Synthesis
2.1. For High Cq samples :

Prepare master mix 1 (MM1) in a 1.5ml  Lobind tube on ice. 


Prepare master mix for (number of samples * 2 ) + extra 10 samples for errors. For example, if there are 96 sample , prepare master mix for (96*2) + 10 = 202 samples. Here is the calculation for 96 samples: 

Components                                            Volume* Number of samples             Master mix

Nuclease free water                                4 * 202                                                    808 ul 
10x Random Primer                                2 *  202                                                   404 ul
25x dNTPs Mix                                        0.8 * 202                                                  161.6 ul 

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.
SECTION: cDNA Synthesis
3.1. For High Cq samples :

Array 20ul RNA into a 96 well plate on an ice block. Label this plate as "CDNA synthesis".

Add 13.6ul of MM1 into the RNA plate.
SECTION: cDNA Synthesis
5.1. For High Cq samples :

Prepare master mix 2 (MM2) in a 1.5ml  Lobind tube on ice. 

 Prepare master mix for (number of samples * 2 ) + extra 10 samples for errors. For example, if there are 96 sample , prepare master mix for 96*2 + 10 = 202samples.

Components                        Volume* Number of samples              Master Mix

10x RT Buffer                        2 * 202                                                    404 ul 
Multiscribe RT                      1 * 202                                                     202 ul 
MgCl2                                    0.2 * 202                                                 40.4 ul

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.
SECTION: cDNA Synthesis
6.1. For High Cq samples :

Remove the RNA plate from the thermal cycler and take out the sealer slowly and carefully so that the solutions in the wells do not make any spill. 

Add 6.4 ul of MM2 into the RNA plate .
SECTION: Artic Amplification
12.1. For High Cq samples :

Prepare master mix 1 (MM1) into  a Falcon tube on ice for ARTIC Pool 1.

Prepare master mix for (number of samples*2) + extra 10 samples for errors. For example, if there are 96 sample , prepare master mix for 96*2 + 10 = 202 samples. Calculations are provided below : 

Components                                    Volume* Number of samples          Master Mix

Nuclease free water                        8.75 * 202                                             1767.5 ul 
Reaction buffer                                5 * 202                                                     1010 ul
V3 Pool 1                                          4 * 202                                                     808 ul 
2.5mM dNTP mix                            2 * 202                                                     404 ul
Q5 High fidelity polymerase          0.25 * 202                                               50.5 ul

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.
SECTION: Artic Amplification
13.1. 
For High Cq samples :

Add 40 ul of MM1 into the ARTIC Pool 1 96 well PCR plate.

Add 10 ul of cDNA into the ARTIC Pool 1 plate.

Seal the plate and mix the solution by quick spin. 
SECTION: Artic Amplification
14.1. For High Cq samples :

Prepare master mix for (number of samples*2) + extra 10 samples for errors. For example, if there are 96 sample , prepare master mix for 96*2 + 10 = 202 samples. Calculations are provided below : 

Components                                    Volume* Number of samples          Master Mix

Nuclease free water                        8.75 * 202                                             1767.5 ul 
Reaction buffer                                5 * 202                                                     1010 ul
V3 Pool 1                                          4 * 202                                                     808 ul 
2.5mM dNTP mix                            2 * 202                                                     404 ul
Q5 High fidelity polymerase          0.25 * 202                                               50.5 ul

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.
SECTION: Artic Amplification
15.1. For High Cq samples :

Add 40 ul of MM2 into the ARTIC Pool 2 96 well PCR plate.

Add 10 ul of cDNA into the ARTIC Pool 2 plate.

Seal the plate and mix the solution by quick spin. 
SECTION: SPRI Clean up
21.1. For High Cq samples :

Transfer  50ul from column 1 of  pool 1 to column 1 of pool 2 plate. Now column 1 of pool 2 should have a total solution of 100ul. Repeat this untill all of of the columns of pool 1 have been tranferred to pool 2.

Put 10 mL of AMPure beads into a trough and add 100ul of AMPure beads to each well of the pool 2 plate with an 8-channel pipette. Mix  by pipetting up and down 5 times while adding.

From now on this plate will be called the washing plate.
SECTION: SPRI Clean up
26.1. For High Cq samples :

Add 18 μL of Omega EB to each of the wells to  elute the DNA and resuspend the beads. Mix by pipetting up and down 5 times.  

Remember to change tips and continue until all of the beads in the 96-well clean-up plate have been  resuspended. 

It is essential to move quickly as the AMPure beads should not dry out, so try to keep everything well set up in the bench before you start resuspending. 

SECTION: SPRI Clean up
28.1. For High Cq samples :

After 7 mins carefully remove and retain 15 μL of the eluate containing the DNA library per well into a new 96-well PCR Plate. Elution with a smaller volume prevents the beads from being eluted as well.
SECTION: cDNA Synthesis
2.2. For Reduced  cost : 

 Prepare master mix 1 (MM1) in a 1.5ml  Lobind tube on ice. 

 Prepare master mix for number of samples + extra 10 samples for errors. For example, if there are 96 sample , prepare master mix for (96/2) + 10 = 58 samples. Here is the calculation for 96 samples: 

Components                                            Volume* Number of samples             Master mix

Nuclease free water                                4 * 58                                                     232 ul 
10x Random Primer                                2 * 58                                                     116 ul
25x dNTPs Mix                                        0.8 * 58                                                  46.4 ul 

Divide the Master mix into a strip tube so its easy to add to the 96-well plate

SECTION: cDNA Synthesis
3.2. For Reduced  cost : 

Array 5ul RNA into a 96 well plate on an ice block. Label this plate as "CDNA synthesis".

Add 3.4ul of MM1 into the RNA plate.
SECTION: cDNA Synthesis
5.2. For Reduced  cost : 

Prepare master mix 2 (MM2) in a 1.5ml  Lobind tube on ice. 

 Prepare master mix for number of samples+ extra 10 samples for errors. For example, if there are 96 sample , prepare master mix for 96 + 10 = 106 samples.

Components                        Volume* Number of samples              Master Mix

10x RT Buffer                        2 * 58                                                      116 ul 
Multiscribe RT                      1 * 58                                                        58 ul 
MgCl2                                    0.2 * 58                                                    11.6 ul

Divide the Master mix into a strip tube so its easy to add to the 96-well plate. 
SECTION: cDNA Synthesis
6.2. For Reduced  cost : 

Remove the RNA plate from the thermal cycler and take out the sealer slowly and carefully so that the solutions in the wells do not make any spill. 

Add 1.6 ul of MM2 into the RNA plate .
SECTION: Artic Amplification
12.2. For Reduced  cost : 

Prepare master mix 1 (MM1) into  a Falcon tube on ice for ARTIC Pool 1.

Prepare master mix for number of samples + extra 10 samples for errors. For example, if there are 96 sample , prepare master mix for 96*0.5 + 10  = 58 samples. Calculations are provided below : 

Components                                    Volume* Number of samples          Master Mix

Nuclease free water                        8.75 * 58                                              507.5 ul 
Reaction buffer                                5 * 58                                                     290 ul
V3 Pool 1                                          4 * 58                                                    232 ul 
2.5mM dNTP mix                            2 * 58                                                    116 ul
Q5 High fidelity polymerase          0.25 * 58                                              14.5 ul

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.

SECTION: Artic Amplification
14.2. For Reduced  cost : 

Prepare master mix 1 (MM1) into  a Falcon tube on ice for ARTIC Pool 1.

Prepare master mix for number of samples + extra 10 samples for errors. For example, if there are 96 sample , prepare master mix for 96*0.5 + 10  = 58 samples. Calculations are provided below : 

Components                                    Volume* Number of samples          Master Mix

Nuclease free water                        8.75 * 58                                              507.5 ul 
Reaction buffer                                5 * 58                                                     290 ul
V3 Pool 2                                          4 * 58                                                    232 ul 
2.5mM dNTP mix                            2 * 58                                                    116 ul
Q5 High fidelity polymerase          0.25 * 58                                              14.5 ul

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.

SECTION: Artic Amplification
13.2. For Reduced  cost : 

Add 10 ul of MM1 into the ARTIC Pool 1 96 well PCR plate.

Add 2.5 ul of cDNA into the ARTIC Pool 1 plate.

Seal the plate and mix the solution by quick spin. 
SECTION: Artic Amplification
15.2. For Reduced  cost : 

Add 10 ul of MM1 into the ARTIC Pool 2 96 well PCR plate.

Add 2.5 ul of cDNA into the ARTIC Pool 2 plate.

Seal the plate and mix the solution by quick spin. 
SECTION: SPRI Clean up
21.2. For Reduced  cost : 

Transfer  12.5ul from column 1 of  pool 1 to column 1 of pool 2 plate. Now column 1 of pool 2 should have a total solution of 25ul. Repeat this untill all of of the columns of pool 1 have been tranferred to pool 2.

Put 3 mL of AMPure beads into a trough and add 25ul of AMPure beads to each well of the pool 2 plate with an 8-channel pipette. Mix  by pipetting up and down 5 times while adding.

From now on this plate will be called the clean-up plate.
SECTION: SPRI Clean up
26.2. For Reduced  cost : 

Add 18 μL of Omega EB to each of the wells to  elute the DNA and resuspend the beads. Mix by pipetting up and down 5 times.  

Remember to change tips and continue until all of the beads in the 96-well clean-up plate have been  resuspended. 

It is essential to move quickly as the AMPure beads should not dry out, so try to keep everything well set up in the bench before you start resuspending. 

SECTION: SPRI Clean up
28.2. For Reduced  cost : 

After 7 mins carefully remove and retain 15 μL of the eluate containing the DNA library per well into a new 96-well PCR Plate. Elution with a smaller volume prevents the beads from being eluted as well.
SECTION: Library End Prep
35.1. For Reduced  cost : 

If you are doing 96 samples then prepare master mix for number of samples*0.5 + 10 extra samples. If you are doing 96 samples prepare master mix for 58 samples . 

Prepare master mix in a 1.5 tube as follow : 

Components                                                          Volume * Number of samples       Master Mix 

NEB Ultra ll end prep reaction buffer                            2.015 * 58                                      116.87ul
NEB Ultra ll end prep reaction enzyme                         0.8625 * 58                                     50.025ul 

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.
SECTION: Library End Prep
36.1. For Reduced  cost :  

Insert the sample DNA concentration  from Qubit in the template_sc2_end prep.xlsx spreadsheet , in the DNA (ng/ul) column. This will generate the amount if sample and water needed to be added to normalize all the samples. Divide the amount of water and sample by 0.5 for each sample. This newly calculated amount of water and sample will be used for normalization. 

Add water according to the newly calculated amount to a new PCR plate that we will call end prep plate. 

Thaw and spin down the cleaned up PCR plate from SPRI clean up step and add sample according to the newly calculated amount from this plate to the new End prep plate. 

Add 1.25ul of Mastermix to the End prep plate. 

Seal the plate with a plate sealer and spin it down for 15 seconds. 
SECTION: Sample Barcoding
40.1. For Reduced  cost : 

Prepare the master mix in a 1.5ml lobind tube . If you are doing 96 samples then prepare master mix for number of samples*0.5 + 10 extra samples. If you are doing 96 samples prepare master mix for 58 samples . 

Prepare master mix in a 1.5 tube as follow :

Components                                                  Volume * Number of samples                      Master Mix

Water                                                                          5.7 * 58                                                       330.6 ul
NEB Ultra ll ligation master mix                              10 * 58                                                        580 ul
NEB Ultra ll ligation Enhancer                                  0.3 * 58                                                        17.4 ul

Divide the Master mix into a strip tube so its easy to add to the 96-well plate.
SECTION: Sample Barcoding
41.1. For Reduced  cost : 

Add 0.75 ul of end prepped DNA in a new 96 well PCR plate.  Label this plate as " Barcoding plate". 

Add 1.25 ul of barcode in the new barcoding plate. 

Add 8 ul of the master mix to the new barcoding plate

Mix all the components by pipetting up and down. 

Seal the plate with a plate sealer. 

Spin down for 15 seconds in a plate spinner. 

Put the leftover end prep plate in -20 °C 
SECTION: Sample Barcoding
43.1. For Reduced  cost : 

After the barcoding reaction, take out the plate, remove the seal and pool all the reactions into a 1.5 mL microcentrifuge tube.  

Mix the contents well and spin down briefly.  
SECTION: Sample Barcoding
48.1. For Reduced  cost : 

Remove the tube from the magnetic stand and place onto the benchtop. 

Add 18 μL of the Elution Buffer (EB) to the tube to elute  the DNA and resuspend the beads  and mix by pipetting up and down.

Incubate at room temperature for 5 minutes.  

Flick the tubes several times and pulse centrifuge.  

Place the tubes on magnetic stand for 5 minutes.  

Take out 15ul  clear supernatent from  the tube  into a newly labeled 1.5 mL microcentrifuge  tube.



SECTION: Adapter Ligation
52.1. For Reduced  cost : 

Thaw the pooled barcoded 15ul sample. 

Add 2.5ul AMll , 2.5ul Quick T4 DNA ligase and 5ul NEB reaction buffer to the pooled sample. 

Mix all the components by pipetting up and down. 

Incubate at room temperature for 25 minutes. 
SECTION: Adapter Ligation
53.1. For Reduced  cost : 

Resuspend the AMPure XP beads by vortexing for 30 seconds.  

Add 25ul  AMPure XP  beads to the pooled sample.  

Mix the beads with sample by pipetting up and down.
  
Close the tube tightly and flick the sample several times.  

Incubate the tube at room temperature for 10 minutes on a Hula mixer .
SECTION: Adapter Ligation
57.1. For Reduced  cost : 

Add 10 μL of the Elution Buffer (EB) to the tube to elute  the DNA and resuspend the beads  and mix by pipetting up and down.

Incubate at room temperature for 5 minutes.  

Flick the tubes several times and pulse centrifuge.  

Place the tubes on magnetic stand for 5 minutes.  

Take out 7.5ul of clear supernatent and put it into a newly labeled 1.5ml tube . This is the final library. 
SECTION: Loading Library Preperation
60. Reagents : 

Sequencing Buffer (SQB)
Loading buffer (LB)
Flush buffer (FB)
Flush Tether (FLT)


Thaw all the reagents at room temperature . Mix each of the tubes by vortexing. After that spin down and place on ice.  
SECTION: Loading Library Preperation
62. Go to the Biomath calculator. ( https://www.promega.com/resources/tools/biomath/) . Insert 400 as DNA length and the calculated DNA amount in previous step in the DNA amount box. Click on calculate. This will give us the pmols of DNA in the sample. 

For example, if you give 400 and 0.45 as the input of DNA length and DNA amount in Biomath calculator, it will show that the sample has 1.7pmols of DNA in the sample. 
SECTION: Loading Library Preperation
63. Convert the pmol into fmol by multiplying it by 1000.  Now divide this fmol by the elution volume to get DNA amount per ul.

For example, the pmol from previous step is 1.7. From there the calculated DNA fmol/ug is  (1.7*1000)/15 = 113.33

SECTION: Loading Library Preperation
64. To prepare the final loading library, find out how much sample is needed to be added in the final library by multiplying it different numbers till it makes the final fmol to 100-105. Make sure not to load less than 100fmol/ul and more than 105fmol/ul. Take the calculated ul of sample in a 1.5ul lobind tube.

After that add water to the sample to make a total solution of 24ul. 

Vortex the LB to mix it properly.  Add 75ul of SQB and 51ul of LB. Mix by pipetting. 

For example : To make it approximately 100fmol/ug , we need to take [(113.33* 0.9) = 101.99 fmol]   0.9 ul of the library. 
Add (24-0.9) =23.1 ul water, 75ul SQB and 51 ul of the LB. 

Mix by pipetting. 
SECTION: Loading Library Preperation
61. Take the last qubit value and multiply by the elution volume of Adapter ligation . Divide the result by 1000. This will be the DNA amount in ug

For example , if the qubit value is 30 then, the DNA amount is (30*15) /1000 = 0.45ug . 
SECTION: Loading Library Preperation
61.1. For Reduced cost : 

Take the last qubit value and multiply by the elution volume of Adapter ligation . Divide the result by 1000. This will be the DNA amount in ug

For example , if the qubit value is 30 then, the DNA amount is (30*7.5) /1000 = 0.225ug . 
SECTION: Loading Library Preperation
62.1. For Reduced cost : 

Go to the Biomath calculator. ( https://www.promega.com/resources/tools/biomath/) . Insert 400 as DNA length and the calculated DNA amount in previous step in the DNA amount box. Click on calculate. This will give us the pmols of DNA in the sample. 

For example, if you give 400 and 0.225 as the input of DNA length and DNA amount in Biomath calculator, it will show that the sample has 0.85pmols of DNA in the sample. 
SECTION: Loading Library Preperation
63.1. For Reduced cost :

Convert the pmol into fmol by multiplying it by 1000.  Now divide this fmol by the elution volume to get DNA amount per ul.

For example, the pmol from previous step is 0.85. From there the calculated DNA fmol/ug is  (0.85*1000)/7.5 = 113.33
SECTION: Loading Library Preperation
65. Take out the flow cell from 4 °C and keep in the room temperature for 25-30 mins and then load them into the docking port and do the flow cell check. Make sure the flow cells click into the port. If the flow cells pass , move on to priming the flow cell. If not, try a different one. 

SECTION: Loading Library Preperation
66. Prepare the priming mix by adding 30ul of FLT to a tube if FB. Mix by vortex and spin. 

Open the inlet port of the flow cell and draw back a small volume(20-50ul) to remove any air bubble . 

  

Flush 500ul of the priming mix into the inlet port and close the port. Wait for 15 mins. 

After 15mins, repeat the priming step with 200ul of priming mix. This ends the priming step .

Add 150ul of the final loading sample library into the inlet port. Close the port. 

Wait 10 mins to initiate any experiment in the PromethION.