TITLE: Targeted NGS feline respiratory panel including SARS-CoV-2 for Ion Torrent platform
AUTHORS: Jobin J  Kattoor, Rebecca P Wilkes

[DESCRIPTION]
The current procedure is for NGS of feline respiratory pathogen panel including SARS-CoV-2 for Ion Torrent platform. Note, sensitivity of the method greatly depends on type and quality of samples. 

[BEFORE_START]
Prior to chip loading and sequencing:
Upload the reference and bed files provided below into the Ion S5 Torrent Server under Reference Sequences (.FASTA) and target files (.BED) using Import Custom Reference.

[GUIDELINES]
Prepare nucleic acid extraction in a clean extraction room to prevent contamination. 


[STEPS]
SECTION: Nucleic acid extraction
113. 
SECTION: Nucleic acid extraction
0.1. KingFisher Flex MagMAX Core extraction protocol
In the extraction room, prepare the sample as follows.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            
SECTION: Nucleic acid extraction
0.2. Add 2 mL of DMEM to 5 mL tube.

SECTION: Nucleic acid extraction
0.3. Swirl the anterior nasal or throat swab and break off the swab tip.
SECTION: Nucleic acid extraction
0.4. Vortex vigorously for 2 min or until the sample is suspended.

SECTION: Nucleic acid extraction
0.5. Use 200 µL of supernatant for the downstream process.

SECTION: Nucleic acid extraction
1. Prepare the Lysis/Binding solution. 
In the clean room, combine the following components for the required number of samples.

 
       Component         Volume per sample            MagMAX Core Lysis solution         350µL            MagMAX Core   Binding solution         350µL            Total Lysis/Binding solution*         700µL       
 *Later used in step 4.4. in the extraction room

SECTION: Nucleic acid extraction
1.1. Vortex at maximum speed for 10 s.

SECTION: Nucleic acid extraction
1.2. Store at room temperature for up to1440 min

SECTION: Nucleic acid extraction
2. Prepare the Bead/ Proteinase K mix.
In the clean room, prepare the following to be added to the lysis plate. 
SECTION: Nucleic acid extraction
2.1. Vortex the MagMAX Core Magnetic Beads thoroughly to ensure the beads are fully resuspended
 
       Component         Volume per sample            MagMAX Core Magnetic Beads         20µL            MagMAX   Core Proteinase K          10µL            Total bead mix         30µL      
 
SECTION: Nucleic acid extraction
2.2. The bead/ Proteinase K mix can be stored at 4ᵒC for up to one week. Proceed to the next step.
SECTION: Nucleic acid extraction
3. Prepare the sample plates.
In the clean room, grab four deep well plates and prepare them as follows.

       Plate position         Plate ID         Reagent         Volume per well            1         Lysis Plate         Bead mix         30µL            2         Wash Plate         MagMAX Core Wash   Solution 1         500µL            3         Wash Plate 2         MagMAX Core Wash Solution 2         500µL            4         Elution Plate         MagMAX Core   Elution Buffer         90µL      
 
SECTION: Nucleic acid extraction
3.1. Take the prepared plates to an extraction hood in the extraction room and add 200 µL of each sample in the Lysis Plate.

SECTION: Nucleic acid extraction
3.2. Mix the sample with the Bead mix by pipetting up and down several times. Let the mixture incubate for 2 minutes.
SECTION: Nucleic acid extraction
3.3. Add 700 µL of Lysis/Binding solution (from step 2) to each sample in the first plate (Lysis plate).

SECTION: Nucleic acid extraction
3.4. Turn the power on to the magnetic particle processor
SECTION: Nucleic acid extraction
3.5. Using the up and down arrow key, select the program MagMAX Core Flex.
SECTION: Nucleic acid extraction
3.6. Press the start key. When prompted, load the appropriate plate on the instrument. Properly orient the plates
SECTION: Nucleic acid extraction
3.7. Press the start key to advance the carousel to the next plate position to load.
SECTION: Nucleic acid extraction
3.8. When the final plate is loaded, place a deep well comb on the top of the first plate (Lysis Plate) and press the start key. When the program is complete, the instrument will prompt to remove elution plate. Remove the elution plate and set it in an extraction hood. Press start to advance the carousel and remove all other plates as prompted. Dispose of all the wash /lysis plates by placing in a biohazard bag.
SECTION: Nucleic acid extraction
3.9. The eluted product can be used immediately or stored in a labelled 1.5mL tube or sealed elution plate (96 well) at -10ᵒC until use.
SECTION: Reverse transcription (RT) using Ion Torrent NGS Reverse Transcription Kit
4. Reaction is set up for each sample using the following table:
 
       Component         Volume            Ion Torrent NGS 5X reaction buffer         2 µL            Ion   Torrent NGS 10X RT Enzyme Mix         1 µL            Total RNA         ≤7 µL            Nuclease   free water         To 10 µL            Total volume         10µL      

 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 
SECTION: Reverse transcription (RT) using Ion Torrent NGS Reverse Transcription Kit
4.1. Mix the reaction mixture well and seal the plate/close PCR tubes
SECTION: Reverse transcription (RT) using Ion Torrent NGS Reverse Transcription Kit
4.2. Execute the following thermocycling conditions on a thermocycler.
 
       Temperature         Time            25ᵒC         10 minutes            50ᵒC         10 minutes            85ᵒC         5 minutes            10ᵒC         Hold      

SECTION: Reverse transcription (RT) using Ion Torrent NGS Reverse Transcription Kit
4.3. Short spin the PCR tube/plate to bring down the contents (cDNA) to the bottom of the tubes.
SECTION: Reverse transcription (RT) using Ion Torrent NGS Reverse Transcription Kit
4.4. Proceed to the section Option 1 or section Option 2 or store at -30 ᵒC to -10 

SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
5. Preparing cDNA target amplification reaction
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
5.1. A master mix is prepared according to the following table
 
       Component         Volume            5X Ion Ampliseq HiFI mix (red cap)         5 µL            cDNA         7.5 µL            Nuclease free water         to 12.5 µL            Total   volume          12.5 µL      
 
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
5.2. Aliquot 5 µL of master mix into two PCR tubes.

SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
5.3. Add 5 µL of primer pool 1 to first tube and primer pool 2 to the second tube. Mix the reaction using pipette.

Note: For SARS CoV-2 two different primer pools are used (WGAG19041_PRD_CFPv01 CFP_with_SARS_2 pool 1 and WGAG19041_PRD_CFPv01 CFP_with_SARS_2pool 2)
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
5.4. Seal the tubes and proceed with the thermocycling shown in the following table
 
 
       Stage         Temperature         Time            Hold         99ᵒC         2 minutes            Denature*         99ᵒC         15 seconds            Anneal and extend*         60ᵒC         4 minutes            Hold         10ᵒC         Hold      
 *The cyclic condition is repeated 30 times
 
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
6. Combine the target amplification reaction and partial digestion of the targets
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
6.1. Short spin the reaction tubes after thermocycling to collect the contents to the bottom of the wells.
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
6.2. Combine the 10 µL target reactions into a single tube and proceed for partial digestion using FuPa reagent.

SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
6.3. Add 2 µL of FuPa reagent to the combined targets to make the volume to 22 µL. Mix well with pipette and proceed for thermocycling as shown in the following table.
 
 
       Temperature         Time             50ᵒC         10 minutes            55ᵒC         10 minutes            60ᵒC         20 minutes            10ᵒC         Hold (max to 1   hour)      
 
* Partially digested targets can be stored -20ᵒC for longer periods
 
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
7. Ligate adaptors to the amplicons and purification.
          
 
                                                                          
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
7.1. Following reaction is set up for each sample
 
 
       Component         Volume            Switch solution (yellow cap)         4 µL            Ion P1   adapter         0.5 µL            IonXpress barcode*         0.5 µL            Nuclease   free water         1 µL            DNA Ligase (blue cap)         2 µL            Target   from the partial digestion step         22 µL            Total volume         ~30 µL      
 
*IonXpress barcodes are available from 001-096. Separate barcodes are used to differentiate libraries from individual samples.
 
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
7.2. The mixture was then incubated in the following conditions.
 
       Temperature         Time            22ᵒC         30 minutes            68ᵒC         5 minutes            72ᵒC         5 minutes            10ᵒC         Hold (maximum 24   hours)      
 
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
7.3. The contents in the tube were briefly centrifuged after the incubation to collect it to the bottom of the wells.
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
7.4. Add 45 µL of Agencourt™ AMPure™ XP Reagent to each tube. Pipet up and down to mix it properly.

SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
7.5. Incubate the mixture for 3-5 minutes at room temperature and keep the magnetic rack. Incubate for 2 minutes or until solution becomes clear. 
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
7.6. Remove the supernatant and wash the pellet with 150 µL of fresh 70% ethanol. Rotate the tubes or move the position of the tubes so that there will be maximum contact of magnetic beads with the wash solution.

SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
7.7. Repeat the washing step one more time and air dry the beads in the tube by keeping it open at room temperature for 2-3 minutes.
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
7.8. Add 20-25 µL of low ionic buffer/TE buffer/ nuclease free water to the dried magnetic beads and mix it well.

SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
7.9. Keep the mixture in room temperature for approx. 2 minutes.
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
7.10. After the incubation keep the tubes back to the magnetic rack.
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
7.11. Remove the clear solution to a new 1.5 mL microcentrifuge tube and proceed with the next steps.
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
8. Quantify and equalize the library.
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
8.1. Quantification of the prepared libraries can be done using Qubit fluorometer according to manufacturer’s protocols.
SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
8.2. Dilute the libraries to a concentration of ~100pM (50 µL total volume for each chip to be loaded) for using Ion 510/520/530 chip loading. 

 

 

SECTION: Option 1: Library prep using Ion AmpliSeq Library Kit Plus (Manual library prep method)
8.3. Proceed to section creating chip plan on Ion Chef 
SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9. Perform the RT step as described in section 5.1 to 5.4.
SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.1. Thaw all the Ion AmpliSeq™ Kit for ChefDL8 reagent (in -20ᵒC), DL8 solution cartridge (in 4ᵒC) and primers (WGAG19041_PRD_CFPv01 CFP_with_SARS_2 pool 1 and 2) (in -20ᵒC) after putting the RT reaction in the thermocycler.
SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.2. Make the sample plan in Torrent suite software (TSS) in http://10.160.129.7/ after logging in to the server using credentials. Make sure to put the Ioncode PCR plate serial number and the sample numbers correctly. Preferably, the control form number should be marked. The sample numbers should be marked correct with the corresponding Ioncode barcodes. All the optional menus can be left as such in the plan. Mark the plan name with the ongoing library name (e.g.: COVID lib 51A).
SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.3. Make up the volume of cDNA from step 2.4 to 15 µL before adding to the Ioncode plate in the next step.

SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.4. Add 15 µL of the cDNA into the A-H (Fig 1) well in the Ioncode plates according to the plan made in the TSS.

 Position 1(A-H): Positions for adding 15µL of cDNA. 
Position 6 (A-H): Positions of lyophilized precoated Ioncode barcodes. 

SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.5. Add 150µL of each primer pool 1 and 2 to the first and second tubes, respectively, in the DL8 reagent cartridge. Vortex and spin down the primer pairs before placing them on the cartridge. 
SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.6. Take out the DL8 chef supplies.
SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.7. In the Ionchef, go to set up a Run> Prepare library> and Open the chef door. Keep all the chef supplies and solution/reagent cartridges in the correct position, as shown in Figure 2. Close the door of the Ion Chef and start with the deck check. 
  

 

SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.8. After the deck check, see for the server name and the saved sample plan name (e.g.: COVID lib 51 A). if the plan name is not coming on the display, refresh the screen. It may go up to 2-3 refresh.
SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.9. Check the prompted boxes showing number of primer (2), number of cycle (27) and extension time (4 min) if shown correct.
SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.10. The program is set, and the chef will start in under 1 minute time
SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.11. The program will generally go for above 7 hours according to the length and number of the cycle.

 

Take out all the consumables from the Ion Chef and go for clean the instrument.

 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.12. After completion of library prep, 700µL of pooled library at 100pM is obtained in the 4th tube in the reagent cartridge.
SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.13.    according to the number of samples

 

A total of 50 µL from step 10.12 is used to load the Ion Chip in the chip loading steps, and equal volumes of multiple barcoded libraries are used to load the Ion chip in the following steps (25 µL each if two sets of libraries are mixed together in a single chip)
SECTION: Option 2: Library prep using Ion AmpliSeq™ Kit for Chef DL8 (Automated library prep method)
9.14. Proceed to section creating chip plan on Ion Chef 
SECTION: Creating chip plan on Ion Chef
10. Create a chip plan on Ion Chef
SECTION: Creating chip plan on Ion Chef
10.2. Keep the Ion 510, 520, & 530 Reagent cartridge stored at -20ºC at room temperature for at least 30 minutes.
SECTION: Creating chip plan on Ion Chef
10.1. Sign-in into the TSS using credentials (http://10.160.129.7/).
SECTION: Creating chip plan on Ion Chef
10.3. In the Plan tab, click on Plan New Run.Select Ion Reporter Account as "None" and Sample Grouping as "Other" and click on Next.
SECTION: Creating chip plan on Ion Chef
10.4. Click on "DNA" in the Research Application and AmpliSeq DNA in Target Technique and click on Next.
SECTION: Creating chip plan on Ion Chef
10.5. Set the instrument type (e.g., Ion GeneStudioTM S5 System), Library kit (e.g.: Ion AmpliSeq Library kit plus) used accordingly and select Ion Chef.
SECTION: Creating chip plan on Ion Chef
10.6. In the Template Kit, select Ion 510, Ion 520 & Ion 530 Kit-Chef, and in Sequencing Kit, select Ion S5 Sequencing Kit from the drop-down menu.
SECTION: Creating chip plan on Ion Chef
10.7. In the Chip Type drop-down menu, select the chip accordingly to the number of different samples/barcodes used.
SECTION: Creating chip plan on Ion Chef
10.8. Barcode Set can be selected optionally depending on the type of barcode used
SECTION: Creating chip plan on Ion Chef
10.9. In the Flow tab, fill in 500 and click on Next. 
SECTION: Creating chip plan on Ion Chef
10.10. Select the previously uploaded Plugins for analysis, including Assembler SPAdes, and File exporter, and click Next.
SECTION: Creating chip plan on Ion Chef
10.11. Optionally, for saving results under a specific project, a project name can be created and selected in the Projects window or click on Next.
SECTION: Creating chip plan on Ion Chef
10.12. Fill in the Run Plan Name and already uploaded Reference Library- canine_feline_combo_with_SARS_2 and Target region- WGAG19041_PRD_CFPv01_edit.bed from the drop-down menu.
SECTION: Creating chip plan on Ion Chef
10.13. Fill in the required Number of barcodes and Chip Barcode (9-digit alphanumeric code).
SECTION: Creating chip plan on Ion Chef
10.14. Fill in the corresponding Barcode and Sample Name in the following table and click on Plan Run.
SECTION: Creating chip plan on Ion Chef
10.15. Repeat step 11.3. to create a plan for the second chip. 
SECTION: Creating chip plan on Ion Chef
10.16. After creating plans for one or both the chips, proceed to section 12.
SECTION: Chip loading on Ion Chef
11.  Load chip on Ion Chef
SECTION: Chip loading on Ion Chef
11.1. At least 30 min after keeping the Ion 510, 520, & 530 Reagent cartridge at room temperature, proceed with the following steps

SECTION: Chip loading on Ion Chef
11.2. Add the diluted barcoded library pools from step 9.2 or step 10.12 into positions 1 and 2 of the Ion S5 Reagent cartridge. Position 1 of the Ion 510, 520, & 530 Chef Reagents cartridge corresponds to the libraries planned in Chip 1, and position 2 corresponds to those samples planned in Chip 2. (Figure. 3).
  

SECTION: Chip loading on Ion Chef
11.3. Click on the open symbol on the Ion Chef screen to open the door. Keep all the reagents and consumables as shown in Figure 4 following the on-screen prompt on Ion Chef.
  

SECTION: Chip loading on Ion Chef
11.4. Uncap all the tubes in the Ion 510, 520, & 530 Chef Reagents cartridge.
SECTION: Chip loading on Ion Chef
11.5. Close the door after keeping the reagents and other consumables and proceed for Deck Scan.

SECTION: Chip loading on Ion Chef
11.6. After completing Deck Scan successfully, click Nextand select the specific plans created in section D for each Chip; Chip 1 and Chip 2. In case of no plans are shown on the screen, click Refresh Plansand select the plans once they are shown. Click Next once the plan is selected.
SECTION: Chip loading on Ion Chef
11.7. On the next screen, click Timer and select the time to end the Chip loading function on Ion Chef. On completion, the chip will be stored in Ion Chef at 4ºC for a maximum of 24 hours. Otherwise, when the run is complete, unload the Ion Chef™ Instrument. Once taken out from the Ion Chef, and if two chips were loaded at the same time, the first chip goes directly into the sequencer for sequencing, and the second chip can be stored in a chip container at 4ºC until use.
SECTION: Chip loading on Ion Chef
11.8. After unloading the used consumables except for the empty pipette tip holder (which will be moved from position 3 to position 1 as in Figure 4) from the Ion Chef, close the door of the Ion Chef and proceed with the “Clean instrument” function when prompted. 
SECTION: Sequencing using Ion GeneStudio S5
12. Sequence using Ion GeneStudio S5
SECTION: Sequencing using Ion GeneStudio S5
12.1. Each initialization with the Ion S5 sequencing kit is suitable for sequencing two chips (two sequencing runs). The Ion S5 sequencing cartridge should be brought to room temperature at least 2 hours prior, and initialization should start at least 1 hour before the end of Chip loading described in step 12.7.
SECTION: Sequencing using Ion GeneStudio S5
12.2. To start the initialization of the sequencer, click on Initialize on the home screen of the Ion S5 sequencer.
SECTION: Sequencing using Ion GeneStudio S5
12.3. Remove the used Ion S5 wash solution bottle and empty the wash buffer waste tank.

SECTION: Sequencing using Ion GeneStudio S5
12.4. Place a new Ion S5 wash solution and an Ion S5 cleaning solution in the designated positions.
SECTION: Sequencing using Ion GeneStudio S5
12.5. Place the thawed Ion S5 Sequencing cartridge into the position in the sequencer.
SECTION: Sequencing using Ion GeneStudio S5
12.6. Place a used chip on the chip holder, close the sequencer door, and start initialization.
SECTION: Sequencing using Ion GeneStudio S5
12.7. Initialization will take approx. 45 minutes, and when completed, click on Home to open the Run option.
SECTION: Sequencing using Ion GeneStudio S5
12.8. Click Run and proceed as prompted on the screen of the Ion S5 sequencer. Replace the chip on the sequencer with the new Chip (Chip 1) from step 8.7, close the door of the sequencer and proceed as prompted.
SECTION: Sequencing using Ion GeneStudio S5
12.9. Click on the correct plan from the drop-down menu, uncheck the Enable post-run clean, and tap Review.
SECTION: Sequencing using Ion GeneStudio S5
12.10. Tap Start if the plan is shown correctly for the selected chip. 
SECTION: Sequencing using Ion GeneStudio S5
12.11. First chip sequencing will take 2h 30 mins and keep the second chip at room temperature at least 30 minutes prior to the end of first chip sequencing. 
SECTION: Sequencing using Ion GeneStudio S5
12.12. When the first run is finished, click on Run complete to open the dialogue window for the next run.
SECTION: Analysis using Torrent suite software and Geneious prime
13. Analysis 
SECTION: Analysis using Torrent suite software and Geneious prime
13.1. Open the Torrent Suite Software (TSS) (http://10.160.129.7/) and in the Data tab, click on the plan name described in section 3.15, which was created for running the sequencing.
SECTION: Analysis using Torrent suite software and Geneious prime
13.2. Aligned, trimmed files available as BAM files from the TSS can be downloaded into the local drive and can be opened in Geneious prime software (https://www.geneious.com/).

SECTION: Analysis using Torrent suite software and Geneious prime
13.3. Based on the alignment to reference sequences in the TSS, sequence reads can be viewed in the Geneious prime software.
SECTION: Analysis using Torrent suite software and Geneious prime
13.4. For additional confirmation, each sequence can be subjected to a BLAST analysis at https://blast.ncbi.nlm.nih.gov/ for the percent identity with SARS-CoV-2 sequences.