TITLE: High Throughput Semi-Automated SARS-CoV-2 Library Preparation Protocol for Ion Torrent Sequencing using Opentrons, New England Biolabs Kit, and ARTIC Primers
AUTHORS: Elias Dahdouh, Fernando Lázaro Perona, María Rodríguez Tejedor, Rubén Cáceres Sánchez, Iván Bloise Sánchez, Jesús Mingorance

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>This protocol is for the preparation of libraries for 96 samples of SARS-CoV-2 using the New England Biolabs NEBNext</span><span style = "vertical-align:super;">®</span><span>Fast DNA Library Prep Set for Ion Torrent™ kit, ARTIC primer pools, the Ion Express Barcodes, and sequencing through Ion Torrent. The library preparation is semi-automated with the open source Opentrons robot. The volumes, concentrations, cycling conditions, and Opentrons protocols have been optimized through a series of experiments and are currently being used at our lab on a weekly basis. The samples used for this protocol were extracted after being obtained from nasopharyngeal swabs, placed in viral transport medium, and mixed 1:1 with AVL buffer (Qiagen). It is advisable to perform a screening for the samples before starting the library preparation by RT-qPCR and select the samples that have Ct values below 30. </span></div></div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
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[STEPS]
?. [Preparation of cDNA using VILO from Thermo Fisher]
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
?. [Preparation of cDNA using VILO from Thermo Fisher]
Prepare the following master mix (VILO master mix):5X VILO Reaction Mix: 200µL10X  SuperScript Enzyme Mix: 100µLNOTE: The 5X VILO Reaction Mix should be at room temperature 40 minutes before being used
?. [Preparation of cDNA using VILO from Thermo Fisher]
Dispense 3µL of the VILO master mix in each well of Plate 1NOTE: The OT-2 can perform this step using the following protocol:
?. [Preparation of cDNA using VILO from Thermo Fisher]
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 minutes85°C for 5 minuteshold at 4°C SAFE POINT: the cDNA samples can be stored at -20°C
?. [Amplification of the SARS-CoV-2 genome using the Primer Pools from ARTIC]
Prepare two Master Mixes (one for each primer pool) containing:NEBNext Q5 Hot Start Master Mix (BLUE cap): 500µLPool A OR B (ARTIC): 250µLNOTE: The NEBNext Q5 Hot Start Master Mix should be at room temperature 40 minutes before being used
?. [Amplification of the SARS-CoV-2 genome using the Primer Pools from ARTIC]
Dispense 7.5µL of each master mix into a new 96-well plateNOTE: The OT-2 can perform this step using the following protocol:   (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)
?. [Amplification of the SARS-CoV-2 genome using the Primer Pools from ARTIC]
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)
?. [Amplification of the SARS-CoV-2 genome using the Primer Pools from ARTIC]
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 minute25 or 30* cycles of:        - 98°C for 15 seconds        - 65°C for 5 minuteshold at 4°C         - 98°C for 15 seconds        - 65°C for 5 minutes*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)
?. [Amplification of the SARS-CoV-2 genome using the Primer Pools from ARTIC]
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 areaSAFE POINT: the samples can be stored at -20°C
?. [End Repair using the New England Biolabs Kit]
Prepare the following End Repair Master Mix:Nuclease-free water: 1550µLNEBNext End Repair Reaction Buffer (GREEN cap): 300µLNEBNext End Repair Enzyme Mix (GREEN cap): 150µL
?. [End Repair using the New England Biolabs Kit]
Mix 20µL of the End Repair Master Mix with the 10µL of the mixed amplicons in Plate 4NOTE: The OT-2 can perform this step using the following protocol:  (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)
?. [End Repair using the New England Biolabs Kit]
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 minutes70°C for 10 minuteshold at 4°C
?. [Ion Express Adaptor and Barcode Ligation]
Prepare the Barcode MixesNOTE: 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.
?. [Ion Express Adaptor and Barcode Ligation]
Prepare the following P1 Adapter Master Mix:Nuclease-free water: 400µLP1 adapter: 200µL
?. [Ion Express Adaptor and Barcode Ligation]
Dispense 6µL of the P1 Adapter Master Mix in a new 96-well plateNOTE: The OT-2 can perform this step using the following protocol:
?. [Ion Express Adaptor and Barcode Ligation]
Add 2µL of each barcode to a well in the 96-well plate where each well should contain a different barcodeNOTE: the plate contains Barcode Mixes for three runs. They can be stored at -20°C
?. [Ion Express Adaptor and Barcode Ligation]
Prepare the following Barcode Ligation Master Mix:Nuclease-free water: 900µLT4 DNA Ligase Buffer for Ion Torrent (RED cap): 500µLBst 2.0 WarmStart DNA Polymerase (RED cap): 50µL
?. [Ion Express Adaptor and Barcode Ligation]
Add 14.5µL of the Barcode Ligation Master Mix to each well of Plate 4NOTE: The OT-2 can perform this step using the following protocol:   (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)
?. [Ion Express Adaptor and Barcode Ligation]
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)
?. [Ion Express Adaptor and Barcode Ligation]
Add 3µL of the T4 DNA Ligase (RED cap) to each well of Plate 4NOTE: It is important to add the reagents in this specific order (Master Mix first, then barcodes, and lastly the DNA ligase)
?. [Ion Express Adaptor and Barcode Ligation]
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 minutes65°C for 5 minuteshold at 4°CSAFE POINT: the samples can be stored at -20°C
?. [Post- Barcode Purification with Magnetic Beads]
NOTE: This entire section can be performed by OT-2 in 54 minutes using the following protocol: 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.
?. [Post- Barcode Purification with Magnetic Beads]
Add 90µL (1.8X) of Magnetic Beads to the samples in Plate 4 and mix thoroughly
?. [Post- Barcode Purification with Magnetic Beads]
Incubate 5 minutes at room temperature
?. [Post- Barcode Purification with Magnetic Beads]
Place the plate on a magnet and wait 2 minutes for the solution to clear
?. [Post- Barcode Purification with Magnetic Beads]
Remove and discard the supernatant
?. [Post- Barcode Purification with Magnetic Beads]
Add 180µL freshly prepared 70% ethanol
?. [Post- Barcode Purification with Magnetic Beads]
Gently pipette the ethanol up and down 4 times and then remove and discard it
?. [Post- Barcode Purification with Magnetic Beads]
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
?. [Post- Barcode Purification with Magnetic Beads]
Remove the plate from the magnet, add 25µL of elution buffer (or TE buffer), and mix thoroughly
?. [Post- Barcode Purification with Magnetic Beads]
Incubate at room temperature for 2 minutes
?. [Post- Barcode Purification with Magnetic Beads]
Place the plate on the magnet and wait for 2 minutes for the solution to clear
?. [Post- Barcode Purification with Magnetic Beads]
Transfer 20µL of the supernatant to a new 96-well plate (Plate 5)SAFE POINT: the samples can be stored at -20°C
?. [Library Amplification]
Prepare the following Master Mix:NEBNext Q5 Hot Start Master Mix (BLUE cap): 2000µLNEBNext DNA Library Primers (BLUE cap): 180µLNOTE: The NEBNext Q5 Hot Start Master Mix should be at room temperature 40 minutes before being used
?. [Library Amplification]
Add 20µL of this Master Mix to each sample in Plate 5 NOTE: The OT-2 can perform this step using the following protocol:  (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)
?. [Library Amplification]
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 seconds7 cycles of:        - 98°C for 10 seconds        - 58°C for 30 seconds        - 72°C for 30 seconds72°C for 5 minuteshold at 4°C         - 98°C for 10 seconds        - 58°C for 30 seconds        - 72°C for 30 seconds72°C for 5 minuteshold at 4°C SAFE POINT: the samples can be stored at -20°C
?. [Post Library Amplification Purification with Magnetic Beads]
NOTE: This entire section can be performed by OT-2 in 54 minutes using the following protocol:  (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.
?. [Post Library Amplification Purification with Magnetic Beads]
Add 36µL (0.9X) of Magnetic Beads to the samples in Plate 5 and mix thoroughly
?. [Post Library Amplification Purification with Magnetic Beads]
Incubate 5 minutes at room temperature
?. [Post Library Amplification Purification with Magnetic Beads]
Place the plate on a magnet and wait 2 minutes for the solution to clear
?. [Post Library Amplification Purification with Magnetic Beads]
Remove and discard the supernatant
?. [Post Library Amplification Purification with Magnetic Beads]
Add 180µL freshly prepared 70% ethanol
?. [Post Library Amplification Purification with Magnetic Beads]
Gently pipette the ethanol up and down 4 times and then remove and discard it
?. [Post Library Amplification Purification with Magnetic Beads]
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
?. [Post Library Amplification Purification with Magnetic Beads]
Remove the plate from the magnet, add 30µL of elution buffer (or TE buffer), and mix thoroughly
?. [Post Library Amplification Purification with Magnetic Beads]
Incubate at room temperature for 2 minutes
?. [Post Library Amplification Purification with Magnetic Beads]
Place the plate on the magnet and wait for 2 minutes for the solution to clear
?. [Post Library Amplification Purification with Magnetic Beads]
Transfer 25µL of the supernatant to a new 96-well plate (Plate 6)SAFE POINT: the libraries can be stored at -20°C
?. [Library Quantification, Normalization and Sequencing]
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 disposalNOTE: Make sure that the output is in ng/mL and that it is adjusted to the original concentration in the sample
?. [Library Quantification, Normalization and Sequencing]
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
?. [Library Quantification, Normalization and Sequencing]
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: 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 accordinglyAlways be sure not to mix samples with the same barcode in the same pool
?. [Library Quantification, Normalization and Sequencing]
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: For Steps 62 to 64, follow the manufacturer's instructions in order to load the Ion Chef and sequence with the Ion S5 systemThe Ion Chef Reagents need to be at room temperature 45 minutes before being usedChange the templating protocol to 400bp while planning the run since the average amplicon length using the ARTIC primer pools is 320bp
?. [Library Quantification, Normalization and Sequencing]
Initialize the Ion S5 system using the S5 solutionsNOTES: The Ion S5 Reagents need to be at room temperature 45 minutes before being usedOne 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)Once the initialization is complete, both chips have to be sequenced within 24 hours
?. [Library Quantification, Normalization and Sequencing]
Sequence the chips one after the other using the Ion S5 systemNOTES: On day 1, load the Ion Chef with two pools and initialize the S5 in the afternoonOn 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)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 chipsOn 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)Keep the Ion S5 system on after it finishes sequencing and until it processes all the data.
?. [Post-PCR Purification with Magnetic Beads]
NOTE: This entire section, minus Step 22, can be performed by OT-2 in 54 minutes using the following protocol: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.
?. [Post-PCR Purification with Magnetic Beads]
Add 36µL (1.8X) of Magnetic Beads to the samples in Plate 2 and mix thoroughly
?. [Post-PCR Purification with Magnetic Beads]
Incubate 5 minutes at room temperature
?. [Post-PCR Purification with Magnetic Beads]
Place the plate on a magnet and wait 2 minutes for the solution to clear
?. [Post-PCR Purification with Magnetic Beads]
Remove and discard the supernatant
?. [Post-PCR Purification with Magnetic Beads]
Add 180µL freshly prepared 70% ethanol
?. [Post-PCR Purification with Magnetic Beads]
Gently pipette the ethanol up and down 4 times and then remove and discard it
?. [Post-PCR Purification with Magnetic Beads]
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
?. [Post-PCR Purification with Magnetic Beads]
Remove the plate from the magnet, add 25µL of elution buffer (or TE buffer), and mix thoroughly
?. [Post-PCR Purification with Magnetic Beads]
Incubate at room temperature for 2 minutes
?. [Post-PCR Purification with Magnetic Beads]
Place the plate on the magnet and wait for 2 minutes for the solution to clear
?. [Post-PCR Purification with Magnetic Beads]
Transfer 20µL of the supernatant to a new 96-well plate (Plate 3)
?. [Post-PCR Purification with Magnetic Beads]
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