Pathogen-Oriented Low-cost Assembly & Re-sequencing on Opentrons (POLARtron): An automation-friendly highly sensitive and high-throughput SARS-CoV-2 diagnostic based on whole genome sequencing

For each saliva sample recieved add equal volume saliva and 2X DNA/RNA Shield from the Quick-DNA/RNA Viral magbead kit and vortex. Centrifuge the samples at 500rpm to bring down debris.

Without disturbing the pellet, transfer 25µL of saliva sample and 1X DNA/RNA to the bottom of a well in a 96-well deep-plate of each sample to a new tube

Add 25µL 1X DNA/RNA Shield supplemented with 2.5µL of Proteinase K (20mg/mL) to each sample.

Briefly mix by using a plate shaker at 1300rpm,25°C,0h 0m 0s for 0h 0m 10s and incubate 0h 15m 0s at Room temperature.

Equipment

Value	Label
ThermoMixer® C	NAME
Eppendorf	BRAND
Catalog No. 2231000680	SKU

Remove 5µL of MagBinding Beads per sample of the stock and place on a magnet stand. Incubate for 0h 2m 0s or until the beads have pelleted and the supernatant is completly clea. Then while avoiding the bead pellet, carefully remove the clear supernatnat. Resuspend the beads in 100µL of Viral DNA/RNA Buffer (2-Mercaptoethanol 0.5% (v/v)) per sample and vortex to fully resuspend

Add 100µL of the Viral DNA/RNA Buffer (2-Mercaptoethanol 0.5% (v/v)) and MagBinding Beads misture to each 50µL sample in 1X DNA/RNA Shield. Mix by using a plate shaker at 1300rpm,25°C,0h 0m 0s for 0h 10m 0s.

Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for 0h 2m 0s or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.

Equipment

Value	Label
SPRIPlate 96R Ring Super Magnet Plate	NAME
96-well Magnet Plate	TYPE
Agencourt	BRAND
A32782	SKU

Add 100µL MagBead DNA/RNA Wash 1 and mix by using a plate shaker at 1300rpm,25°C,0h 0m 0s for 0h 2m 0s.

Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for 0h 2m 0s or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.

Add 100µL MagBead DNA/RNA Wash 2 and mix by using a plate shaker at 1300rpm,25°C,0h 0m 0s for 0h 2m 0s.

Equipment

Value	Label
ThermoMixer® C	NAME
Eppendorf	BRAND
Catalog No. 2231000680	SKU

Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for 0h 2m 0s or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.

Equipment

Value	Label
SPRIPlate 96R Ring Super Magnet Plate	NAME
96-well Magnet Plate	TYPE
Agencourt	BRAND
A32782	SKU

Add 150µL 95-100% ethanol and mix by using a plate shaker at 1300rpm,25°C,0h 0m 0s for 0h 2m 0s.

Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for 0h 2m 0s or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.

Equipment

Value	Label
SPRIPlate 96R Ring Super Magnet Plate	NAME
96-well Magnet Plate	TYPE
Agencourt	BRAND
A32782	SKU

and repeat once.

To elute DNA/RNA from the beads, add 9µL DNase/RNase-Free Water and mix using a plate shaker at

1300rpm,25°C,0h 0m 0s for 0h 1m 0s

Equipment

Value	Label
ThermoMixer® C	NAME
Eppendorf	BRAND
Catalog No. 2231000680	SKU

Pellet the beads and transfer 7µL of supernatant into a new tube. The eluted DNA/RNA can be used immediately or stored frozen at -80°C.

Combine the following components into a thin-walled PCR tube.

10µL Luna Universal Probe One-Step Reaction Mix (2X)

1µL Luna WarmStart RT Enzyme Mix (20X)

1µL hCoV-2019/nCoV-2019 (V3) Primer Set mix (Primer pool 1 & 2) (100µm)

1µL Random Hexamers (100 µM)

7µL Viral RNA/DNA extract

Add 20µL of mineral oil to each RT-PCR reaction to avoid evaporation and subsequent reaction failure.

Set up and run the following RT-PCR program.

1. Reverse transcription: 0h 10m 0s at 55°C

2. Initial PCR activation: 0h 1m 0s at 95°C

3. 2-step PCR cycling (25X):

   Denaturation: 0h 0m 15s at 95°C

   Annealing & Extension: 0h 3m 0s at 63°C

4. Hold: 4°C

Equipment

Value	Label
Veriti 96-Well Thermal Cycler	NAME
Applied Biosystems	BRAND
4375786	SKU

Add 0.7x volume of sparQ PureMag beads to each sample well in RT-PCR plate and mix gently by either flicking or pipetting. For example, add 14µL sparQ PureMag beads to a 20µL reaction. Note that the volume of oil is not taken into consideration given the oil is effectively inert. Then pulse centrifuge to collect all liquid at the bottom of the tube.

Incubate for 0h 5m 0s at 4Room temperature.

Pellet the beads onto the side of the sample tube using a magnet stand. Incubate for 0h 2m 0s or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.

Equipment

Value	Label
SPRIPlate 96R Ring Super Magnet Plate	NAME
96-well Magnet Plate	TYPE
Agencourt	BRAND
A32782	SKU

Keeping on the magnetic plate, add 150µL of 4Room temperature freshly made 80% (v/v) ethanol to the side of the wall opposite to the pellet and let sit for 0h 0m 30s.

Avoid disturbing the bead pellet, carefully remove and discard ethanol. Wait for 0h 0m 10s then remove any remaining ethanol.

and repeat once.

Add 11 µl of 10mM Tris-HCl (Ph 8.0) and pipette to mix well. Incubate for 0h 1m 0s at 37°C.

Equipment

Value	Label
ThermoMixer® C	NAME
Eppendorf	BRAND
Catalog No. 2231000680	SKU

Separate beads on the Agencourt SPRIPlate Super Magnet Plate for 0h 2m 0s or until the beads have pelleted.

Equipment

Value	Label
SPRIPlate 96R Ring Super Magnet Plate	NAME
96-well Magnet Plate	TYPE
Agencourt	BRAND
A32782	SKU

Pellet the beads and transfer 10 µl of supernatant containing SARS-CoV-2 amplicons into a new tube. The eluted DNA can be used immediately or stored frozen at -20°C.

Combine the following components into a thin-walled PCR tube.

4µL 5X Hacklfex Buffer (20mM Tris, 20 mM MgCl, 50% DMF)

5.5µL Nuclease-Free Water

0.5µL Enrichment Bead-Linked Transposomes (eBLT)

10µL SARS-CoV-2 amplicons

Set up and run the following thermocycler program.

1. Tagmentation: 0h 5m 0s at 55°C

2. Hold: 10°C

Equipment

Value	Label
ThermoMixer® C	NAME
Eppendorf	BRAND
Catalog No. 2231000680	SKU

Add 5µL of Hackflex Stop Buffer (0.2% SDS) to the sample.

Set up and run the following thermocycler program.

1. Tagmentation: 0h 5m 0s at 25°C

2. Hold: 10°C

Equipment

Value	Label
ThermoMixer® C	NAME
Eppendorf	BRAND
Catalog No. 2231000680	SKU

Place the plates on the Agencourt SPRIPlate Super Magnet Plate and incubate for 0h 5m 0s or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.

Equipment

Value	Label
SPRIPlate 96R Ring Super Magnet Plate	NAME
96-well Magnet Plate	TYPE
Agencourt	BRAND
A32782	SKU

Keeping on the magnetic plate, add 30µL of 4Room temperature Hackflex Wash Buffer (10% PEG 8000, 0.25 M NaCl, 10mM Tris-HCL pH 8.0, 0.1mM EDTA) gently mix and then let sit for 0h 0m 30s.

Separate beads on the Agencourt SPRIPlate Super Magnet Plate for 0h 2m 0s or until the beads have pelleted.

Equipment

Value	Label
SPRIPlate 96R Ring Super Magnet Plate	NAME
96-well Magnet Plate	TYPE
Agencourt	BRAND
A32782	SKU

Avoid disturbing the bead pellet, carefully remove and discard HWB (10% PEG 8000, 0.25 M NaCl, 10mM Tris-HCL pH 8.0, 0.1mM EDTA).

and repeat once.

Resuspend beads in the following 20µL Indexing-PCR Master Mix

10µL NEBNext Q5U Master Mix (2X)

1µL IDT for Illumina DNA/RNA UD Indexes

9µL Nuclease-free Water

Set up and run the following Indexing-PCR program.

1. Initial Denaturation: 0h 1m 0s at 98°C

2. 3-step PCR cycling (6X):

   Denaturation: 0h 0m 15s at 98°C

   Annealing: 0h 0m 30s at 62°C

   Extension: 0h 0m 30s at 65°C

3. Final Extension: 0h 1m 0s at 65°C

4. Hold: 4°C

Equipment

Value	Label
Veriti 96-Well Thermal Cycler	NAME
Applied Biosystems	BRAND
4375786	SKU

After PCR, pool 10µLof each sample into a single tube and vortex to mix.

Add an equal volume (1:1) of sparQ PureMag beads to the library pool and mix gently by either flicking or pipetting. For example, add 25µL sparQ PureMag beads to a 25µL reaction. Then pulse centrifuge to collect all liquid at the bottom of the tube.

Incubate for 0h 5m 0s at 4Room temperature.

Place the pool on a magnet and incubate for 0h 2m 0s or until the beads have pelleted and the supernatant is completely clear. Then, while avoiding the bead pellet, carefully remove the clear supernatant.

Equipment

Value	Label
Magnetic Stand	NAME
Magnetic Stand	TYPE
Thermo Scientific	BRAND
MR02	SKU
Any magnetic rack that fits your tubes will suffice.	SPECIFICATIONS

Keeping the pool on the magnet, add 200µL of 4Room temperature freshly made 80% (v/v) ethanol to the side of the wall opposite to the pellet and let sit for 0h 0m 30s.

Avoid disturbing the bead pellet, carefully remove and discard ethanol. Wait for 0h 0m 10s then remove any remaining ethanol.

and repeat once.

Add 25 µl of 10mM TE Buffer (10mM Tris-HCL pH 8.0, 0.1mM EDTA) and pipette to mix well. Incubate for 0h 1m 0s at 37°C.

Quantify final pool with adapted libraries. Load onto sequencer using platform appropriate dilutions.

Equipment

Value	Label
MiSeq	NAME
Sequencer	TYPE
illumina	BRAND
SY-410-1003	SKU