High Throughput Semi-Automated SARS-CoV-2 Library Preparation Protocol for Ion Torrent Sequencing using Opentrons, New England Biolabs Kit, and ARTIC Primers

For each sample dispense 7µL of the extracted nucleic acids to the respective wells in the 96-well plate ( Plate 1 )

NOTE: All the calculations from here until the end of the protocol are done for 96 samples + 4 samples to account for pipetting errors

Prepare the following master mix (VILO master mix):

• 5X VILO Reaction Mix: 200µL
• 10X SuperScript Enzyme Mix: 100µL

NOTE: The 5X VILO Reaction Mix should be at room temperature 40 minutes before being used

Dispense 3µL of the VILO master mix in each well of Plate 1

NOTE: The OT-2 can perform this step using the following protocol: VILO Master Mix Dispensation for 96 Samples.json

Seal Plate 1 , perform a quick spin and place it in the thermal cycler for cDNA synthesis with the following thermal program:

• 42°C for 30 minutes
• 85°C for 5 minutes
• hold at 4°C

SAFE POINT: the cDNA samples can be stored at -20°C

Prepare two Master Mixes (one for each primer pool) containing:

• NEBNext Q5 Hot Start Master Mix (BLUE cap): 500µL
• Pool A OR B (ARTIC): 250µL

NOTE: The NEBNext Q5 Hot Start Master Mix should be at room temperature 40 minutes before being used

Dispense 7.5µL of each master mix into a new 96-well plate

NOTE: The OT-2 can perform this step using the following protocol: Pool Master Mix Dispensation for 96 Samples.json (can run the same protocol twice, once for the Master Mix of Pool A and a second time for the Master Mix of Pool B, without having to re-calibrate in-between the two runs)

Add 2.5µL of cDNA from Plate 1 to the respective wells in both plates (where one plate contains the Master Mix with Pool A and the second plate contains the Master Mix for Pool B)

Seal the 96-well plates with Pools A and B, perform a quick spin to them, and place them in the thermal cycler(s) for PCR amplification with the following program:

• 98°C for 1 minute

• 25 or 30* cycles of: - 98°C for 15 seconds

    - 65°C for 5 minutes
  

• hold at 4°C

*The number of cycles should be adjusted according to the original Ct values of the samples where 25 cycles are recommended for samples with Ct values between 15 and 28, and 30 cycles are recommended for samples with Ct values > 28 (although it is not recommended to include samples with Ct values > 30)

Mix 10µL of the PCR product from the plate of Pool A with 10µL of the PCR product from the plate of Pool B in a new 96-well plate ( Plate 2 ) (use of a multi-channel pipette is highly recommended)

NOTE: From here on, the samples must be managed in a post-PCR area

SAFE POINT: the samples can be stored at -20°C

NOTE: This entire section, minus Step 22, can be performed by OT-2 in 54 minutes using the following protocol:Post Pool Amplification Ampure Beads Purification for 20uL (96 Samples).json

Follow Steps 11 to 21 only if performing this section manually (use of a multi-channel pipette is highly recommended). Otherwise, load the above protocol in OT-2 so that it can perform these steps automatically.

Add 36µL (1.8X) of Magnetic Beads to the samples in Plate 2 and mix thoroughly

Incubate 5 minutes at room temperature

Place the plate on a magnet and wait 2 minutes for the solution to clear

Remove and discard the supernatant

Add 180µL freshly prepared 70% ethanol

Gently pipette the ethanol up and down 4 times and then remove and discard it

Leave the plate at room temperature for 2 minutes (manually remove excess ethanol without disturbing the pellet if needed)

NOTE: Do not let the pellet over-dry resulting in a dark brown-black color since this would lead to irreversible binding of the DNA and inability to elute the DNA

Remove the plate from the magnet, add 25µL of elution buffer (or TE buffer), and mix thoroughly

Incubate at room temperature for 2 minutes

Place the plate on the magnet and wait for 2 minutes for the solution to clear

Transfer 20µL of the supernatant to a new 96-well plate ( Plate 3 )

Transfer 10µL of all the sample in Plate 3 to a new Plate ( Plate 4 )

NOTE: This step needs to be performed even if the Opentrons protocol was used. For the next step, you will only be needing 10µL of the mixed and purified amplicons (the remaining 10µL can be stored at -20°C)

SAFE POINT: Both plates can be be stored at -20°C

Prepare the following End Repair Master Mix:

• Nuclease-free water: 1550µL
• NEBNext End Repair Reaction Buffer (GREEN cap): 300µL
• NEBNext End Repair Enzyme Mix (GREEN cap): 150µL

Mix 20µL of the End Repair Master Mix with the 10µL of the mixed amplicons in Plate 4

• NOTE: The OT-2 can perform this step using the following protocol: End Repair Master Mix Dispensation for 96 Samples.json ( IMPORTANT: For this OT-2 protocol, the total volume of the Master Mix has to be divided into 2 tubes where each tube will have a final volume of 1000µL)

Seal Plate 4 , perform a quick spin and place it in the thermal cycler for end-repair with the following program:

• 25°C for 20 minutes
• 70°C for 10 minutes
• hold at 4°C

Prepare the Barcode Mixes

NOTE: This section considers that the barcodes 1 to 96 are being used. However, it can be adjusted to use barcodes 1 to 48 for the first 48 samples in the 96-well plate and re-use the same barcodes for the second half of the plate, as long as the samples are sequenced separately. Also, barcodes 1 to 24 can be used for four sets of 24 samples in the 96-well plates as long as the samples are to be sequenced separately.

Prepare the following P1 Adapter Master Mix:

• Nuclease-free water: 400µL
• P1 adapter: 200µL

Dispense 6µL of the P1 Adapter Master Mix in a new 96-well plate

NOTE: The OT-2 can perform this step using the following protocol: Adapter P1 Master Mix Dispensation for 96 Samples.json

Add 2µL of each barcode to a well in the 96-well plate where each well should contain a different barcode

NOTE: the plate contains Barcode Mixes for three runs. They can be stored at -20°C

Prepare the following Barcode Ligation Master Mix:

• Nuclease-free water: 900µL
• T4 DNA Ligase Buffer for Ion Torrent (RED cap): 500µL
• Bst 2.0 WarmStart DNA Polymerase (RED cap): 50µL

Add 14.5µL of the Barcode Ligation Master Mix to each well of Plate 4

NOTE: The OT-2 can perform this step using the following protocol: Barcode Ligation Master Mix Dispensation for 96 Samples.json ( IMPORTANT: For this OT-2 protocol, the total volume of the Master Mix has to be divided into 2 tubes where each tube will have a final volume of 725µL)

Add 2.5µL of the Barcode Mix from the Barcode-Containing 96-well plate to the respective wells of Plate 4 (use of a multi-channel pipette is highly recommended)

Add 3µL of the T4 DNA Ligase (RED cap) to each well of Plate 4

NOTE: It is important to add the reagents in this specific order (Master Mix first, then barcodes, and lastly the DNA ligase)

Seal Plate 4 , perform a quick spin and place it in the thermal cycler to perform ligation with the following program:

• 25°C for 15 minutes
• 65°C for 5 minutes
• hold at 4°C

SAFE POINT: the samples can be stored at -20°C

NOTE: This entire section can be performed by OT-2 in 54 minutes using the following protocol: FIRST Ampure Beads Purification for 96 Samples.json

Follow Steps 33 to 43 only if performing this section manually (use of a multi-channel pipette is highly recommended). Otherwise, load the above protocol in OT-2 so that it can perform these steps automatically.

Add 90µL (1.8X) of Magnetic Beads to the samples in Plate 4 and mix thoroughly

Incubate 5 minutes at room temperature

Place the plate on a magnet and wait 2 minutes for the solution to clear

Remove and discard the supernatant

Add 180µL freshly prepared 70% ethanol

Gently pipette the ethanol up and down 4 times and then remove and discard it

Leave the plate at room temperature for 2 minutes (manually remove excess ethanol without disturbing the pellet if needed)

NOTE: Do not let the pellet over-dry resulting in a dark brown-black color since this would lead to irreversible binding of the DNA and inability to elute the DNA

Remove the plate from the magnet, add 25µL of elution buffer (or TE buffer), and mix thoroughly

Incubate at room temperature for 2 minutes

Place the plate on the magnet and wait for 2 minutes for the solution to clear

Transfer 20µL of the supernatant to a new 96-well plate ( Plate 5 )

SAFE POINT: the samples can be stored at -20°C

Prepare the following Master Mix:

• NEBNext Q5 Hot Start Master Mix (BLUE cap): 2000µL
• NEBNext DNA Library Primers (BLUE cap): 180µL

NOTE: The NEBNext Q5 Hot Start Master Mix should be at room temperature 40 minutes before being used

Add 20µL of this Master Mix to each sample in Plate 5

NOTE: The OT-2 can perform this step using the following protocol: Library Amplification Master Mix Dispensation for 96 Samples.json ( IMPORTANT: For this OT-2 protocol, the total volume has to be divided into 2 tubes where each tube will have a final volume of 1090µL)

Seal Plate 5 , perform a quick spin and place it in the thermal cycler for PCR amplification with the following program:

• 98°C for 30 seconds

• 7 cycles of: - 98°C for 10 seconds

    - 58°C for 30 seconds
  
    - 72°C for 30 seconds
  

• 72°C for 5 minutes

• hold at 4°C

SAFE POINT: the samples can be stored at -20°C

NOTE: This entire section can be performed by OT-2 in 54 minutes using the following protocol: SECOND Ampure Beads Purification for 96 Samples.json (if this and the previous purifications with magnetic beads are performed on the same day , then the same reservoir well can be re-used for the second purification).

Follow Steps 48 to 58 only if performing this section manually (use of a multi-channel pipette is highly recommended). Otherwise, load the above protocol in OT-2 so that it can perform these steps automatically.

Add 36µL (0.9X) of Magnetic Beads to the samples in Plate 5 and mix thoroughly

Incubate 5 minutes at room temperature

Place the plate on a magnet and wait 2 minutes for the solution to clear

Remove and discard the supernatant

Add 180µL freshly prepared 70% ethanol

Gently pipette the ethanol up and down 4 times and then remove and discard it

Leave the plate at room temperature for 2 minutes (manually remove excess ethanol without disturbing the pellet if needed)

NOTE: Do not let the pellet over-dry resulting in a dark brown-black color since this would lead to irreversible binding of the DNA and inability of eluting the DNA

Remove the plate from the magnet, add 30µL of elution buffer (or TE buffer), and mix thoroughly

Incubate at room temperature for 2 minutes

Place the plate on the magnet and wait for 2 minutes for the solution to clear

Transfer 25µL of the supernatant to a new 96-well plate ( Plate 6 )

SAFE POINT: the libraries can be stored at -20°C

Quantify the samples in the Plate 6 using either the Broad Range (BR) or the High Sensitivity (HS) kit and Qubit Fluorometer (Thermo Fisher), or any other compatible kit and machine at your disposal

NOTE: Make sure that the output is in ng/mL and that it is adjusted to the original concentration in the sample

Normalize the samples' concentrations to 24ng/mL (equivalent to 100pM) by mixing the calculated sample amount with elution buffer (TE) to a final volume of 100µL in a new 96-well plate ( Plate 7 )

NOTE: The final volume of the normalized sample can be adjusted if the sample volume to be added is too large or too small, as long as the final volume of the normalized sample is more than 10µL

Create the pools for sequencing mixing 10µL from each normalized sample in a polypropylene tube. Four pools of 24 samples each may be prepared from the 96 samples.

NOTES:

1. According to our experience 20 to 30 samples may be included in a 530 Chip with good coverage and depth. If a larger or a smaller chip is to be used then the number of samples to be included in a single pool should be adjusted accordingly
2. Always be sure not to mix samples with the same barcode in the same pool

Use 25µL from each pool in order to load the Ion Chef with 530 Chips. Two pools can be simultaneously loaded in two different chips using the same Ion Chef reagents.

NOTES:

1. For Steps 62 to 64, follow the manufacturer's instructions in order to load the Ion Chef and sequence with the Ion S5 system
2. The Ion Chef Reagents need to be at room temperature 45 minutes before being used
3. Change the templating protocol to 400bp while planning the run since the average amplicon length using the ARTIC primer pools is 320bp

Initialize the Ion S5 system using the S5 solutions

NOTES:

1. The Ion S5 Reagents need to be at room temperature 45 minutes before being used
2. One initialization can be used to sequence two 530 Chips if the number of flows is selected to be 550 (without the need to wash in-between the two chips)
3. Once the initialization is complete, both chips have to be sequenced within 24 hours

Sequence the chips one after the other using the Ion S5 system

NOTES:

1. On day 1, load the Ion Chef with two pools and initialize the S5 in the afternoon
2. On day 2, load the first chip in the morning and then directly load the second chip after it (keep the second chip in the membrane protector at 4°C and remove it to room temperature 45 minutes before sequencing)
3. On day 2, load the Ion Chef with the other two pools and re-initialize the S5 in the afternoon after it has finished sequencing the first 2 chips
4. On day 3, load the first chip in the morning and then directly load the second chip after it (keep the second chip in the membrane protector at 4°C and remove it to room temperature 45 minutes before sequencing)
5. Keep the Ion S5 system on after it finishes sequencing and until it processes all the data.