TITLE:  GenSwab Assay (SARS-CoV-2)
AUTHORS: huiyi.chen , Sid Roy

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The GenapSys SARS-CoV-2 GenSwab Assay is a reverse transcription sequencing (RT-Seq) test. The assay uses primers and probes that were first designed by OctantBio to detect RNA from SARS-CoV-2 in respiratory and saliva specimens from patients. This is the workflow for purified RNA samples, and starts with purified samples. Our protocol will accept RNA purified from any kit.</div></div>

[STEPS]
?. [Pre-PCR: S2 Spike-in Dilution]
Start with a stock solution of 1% Tween-20 in TE. Dilute 10x in TE to obtain 0.1% Tween-20 in TE.
?. [Pre-PCR: S2 Spike-in Dilution]
Retrieve S2 spike-in RNA from -80C freezer. S2 spike-in RNA is provided at 1e10 copies/ul. Perform three 100x dilutions, followed by a 10x dilution to obtain 1000 copies/ul. Dilutions should be done in Lo-Bind DNA tubes in 0.1% Tween-20 in TE.
?. [Pre-PCR: S2 Spike-in Dilution]
Add 198ul to 0.1% Tween-20 in TE to the tube containing the S2 spike-in RNA. This is the Dilution 1.
?. [Pre-PCR: S2 Spike-in Dilution]
Remove 2ul of Dilution 1 to 198ul of 0.1% Tween-20 in TE. This is Dilution 2.
?. [Pre-PCR: S2 Spike-in Dilution]
Remove 2ul of Dilution 2 to 198ul of 0.1% Tween-20 in TE. This is Dilution 3.
?. [Pre-PCR: S2 Spike-in Dilution]
Make a final 10x dilution by adding 10ul of Dilution 3 to 90ul of 0.1% Tween-20 in TE. Keep on ice until use.
?. [Pre-PCR: Setting up RT-PCR]
Each batch of reactions should be accompanied by 1 positive control and 1 no-template control, with a minimum of 1 set of controls per 96-well plate.
?. [Pre-PCR: Setting up RT-PCR]
While the primer and sample mix incubates on ice, prepare the mastermix. The mastermix comprises the reagents in blue. Combine the TaqPath Master Mix, diluted S2 spike-in RNA and water in the necessary quantities, and mix well by pipetting.
?. [Pre-PCR: Setting up RT-PCR]
To the 2 ul of 10x Indexed Primer Mix in each well, add 5 ul of the sample (purified RNA).
?. [Pre-PCR: Setting up RT-PCR]
Dispense 13 ul of MasterMix into each well.
?. [Pre-PCR: Setting up RT-PCR]
Seal the plate. Spin down the plate at 1000xg for 1 min to collect the contents, then mix using a plate mixer at 2000 rpm for 2 min (96-well plate). Load into thermocycler.
?. [Pre-PCR: Setting up RT-PCR]
Run the following PCR program:
?. [Post-PCR: Bead Clean Up]
Pool the samples. Remove 5ul from each reaction to be combined to a fresh tube or sterile reservoir (if using a multi-channel pipettor).
?. [Post-PCR: Bead Clean Up]
Transfer the reaction mix from the reservoir to a tube of appropriate size. Vortex to mix. Transfer 100ul to a fresh Lo-Bind 1.5ml tube.
?. [Post-PCR: Bead Clean Up]
Clean up using a dual-sided purification.Add  of AmpureXP beads. Vortex to mix, and incubate at room temperature for .
50 µl
?. [Post-PCR: Bead Clean Up]
Use a magnetic rack to collect the beads for at least 1 minute.
?. [Post-PCR: Bead Clean Up]
Transfer supernatant to a new tube. Add  of AmpureXP beads. Vortex and incubate at room temperature for .
130 µl
?. [Post-PCR: Bead Clean Up]
Use magnetic rack to collect the beads for at least 1 minute.
?. [Post-PCR: Bead Clean Up]
Remove and discard supernatant.
?. [Post-PCR: Bead Clean Up]
Wash the beads twice with of freshly made 80% ethanol.
500 µl
?. [Post-PCR: Bead Clean Up]
After the final wash, leave the lid open for 3 minutes to let the residual ethanol evaporate.
?. [Post-PCR: Bead Clean Up]
Elute in  of TE buffer. Vortex to mix and incubate for 2 minutes. Spin down briefly to collect.
40 µl
?. [Post-PCR: Bead Clean Up]
Use magnetic rack to collect the beads for at least 1 minute. Collect 35ul of the eluate.
?. [Post-PCR: Quantification of library]
Dilute the eluted library 1:10 in TE buffer: add 5ul of library to 45ul TE buffer.
?. [Post-PCR: Quantification of library]
Quantify the 1:10 dilution using the High Sensitivity DNA Qubit kit.
?. [Post-PCR: Quantification of library]
Convert the Qubit measurement to molarity assuming a 120bp amplicon.For convenience, an online tool like NEB's NEBioCalculator can provide the conversion.
?. [Post-PCR: Quantification of library]
Optional: qPCR measurement of the library would provide a more accurate concentration, and is recommended for initial tests. Subsequent tests can extrapolate from the conversion between Qubit and qPCR measurements.
?. [Post-PCR: Quantification of library]
Dilute the eluted library with TE to 33pM in Lo-bind tubes.
?. [Post-PCR: Quantification of library]
Proceed to clonal amplification. The suggested starting point for clonal amplification is 4ul of the 33pM library.
?. [Pre-PCR: Setting up RT-PCR]
Incubate the primer and sample mix at   for 5 min, then place on ice for at least 2 min.
65 °C