Assessing multiplex tiling PCR sequencing approaches for detecting genomic variants of SARS-CoV-2 in municipal wastewater

Preparation of master mix

Prepare the following components in a 1.5 mL Eppendorf DNA LoBind tube for the number of samples that will be tested and dispense 4µL to 0.2mL each PCR tubes. Briefly spin down the 0.2 mL tubes containing Master Mix RT_1 and keep On ice

A	B
Component	Volume
50µM random hexamers	2 μL
10mM dNTPs mix (10mM each)	2 μL

Prepare the following components in a 1.5mL tube and keep the Master Mix RT_2 On ice .

A	B
Component	Volume (μL)
100 mM DTT	2
Ribonuclease Inhibitor (40 U/µL)	2
5x SSIV Buffer	8
SSIV Reverse transcriptase	2
Nuclease free H2O	10

This step should be conducted in the pre-PCR area (e.g. cleaned DNA hood). Keep all the Master Mix On icewhile doing this step.

Add 12µL to each 0.2 mL tube containing the Master Mix RT_1

Mix by pipetting or flicking the tube, spin down briefly.

Incubate the reaction mix in a thermocycler at 65°C for 0h 5m 0s , then spin down briefly, and incubate immediately On ice for at least 0h 1m 0s .

While on ice, add to each tube 24µL,

Gently mix by pipetting or flicking the tubes and briefly spin down.

Incubate the reaction mix in the thermocycler for:

A	B	C
Step	Temp	Time
1	42 °C	00:50:00
2	70 °C	00:10:00
3	4°C	hold

cDNA cleanup with (all centrifuge steps at 13,000xg)

Add 8µL and 8µL to the 40µL .

Incubate the reaction mix in a thermocycler at 65°C for 0h 15m 0s to hydrolyze RNA.

Transfer the hydrolysis reaction mix from the last step to a new 1.5 mL Eppendorf DNA LoBind tube and add 392µL to 56µL. Briefly vortex to mix, and pulse spin to collect thesample.

Transfer mixture to a provided Zymo-Spin™ Column in a Collection Tube.

Centrifuge for 0h 0m 30s and discard the flow-through.

Add 200µL to the column. Centrifuge for 0h 0m 30s .

Repeat step 3.6.

Add to the column matrix and incubate at Room temperature for 0h 5m 0s .

A	B
Primer Scheme	Elute volume (μL)
Swift_150bp	14
ARTIC V3_400bp	18.5
Midnight_1200bp	25

Transfer the column to a 1.5 ml microcentrifuge tube, then centrifuge for 0h 0m 30s to elute the DNA and keep on ice.

Prepare the following PCR mastermixs and keep On ice .

Prepare PCR mastermix for Swift_150bp primer scheme.

A	B	C
Component	Reaction 1	Reaction 2
Q5 Hot Start High-Fidelity 2X Master Mix	12.5 µL	12.5 µL
Swift Pool 1 (15nM each primer)	6.25 μL	0
Swift Pool 2 (15nM each primer)	0	6.25 μL
Total	18.75 μL	18.75 μL

Prepare PCR mastermix for ARTIC V3_400bp primer scheme.

A	B	C
Component	Reaction 1	Reaction 2
Q5 Hot Start High-Fidelity 2X Master Mix	12.5 µL	12.5 µL
V3 Pool 1 (15nM each primer)	4 μL	0
V3 Pool 2 (15nM each primer)	0	4 μL
Total	16.5 μL	16.5 μL

Prepare PCR mastermix for midnight_1200bp primer scheme.

A	B	C
Component	Reaction 1	Reaction 2
Q5 Hot Start High-Fidelity 2X Master Mix	12.5 µL	12.5 µL
midnight Pool 1 (15nM each primer)	1.1 μL	0
midnight Pool 2 (15nM each primer)	0	1.1 μL
Total	13.6 μL	13.6 μL

Add the corresponding amount of cDNA to each of the PCR reactions, mix by pipetting or flicking the tube and spin down.

A	B
Primer Scheme	Volume per reaction (μL)
Swift_150bp	6.25
ARTIC V3_400bp	8.5
Midnight_1200bp	11.4

Run in a thermal cycler using the following program:

Swift_150bp primer scheme

A	B	C	D
Step	Temperature	Time	Cycles
Initial denaturation	98 °C	30 s	1
Denaturation Annealing/Extension	98°C 65°C	15 s 2 min	35
Hold	4 °C	∞	1

OR

ARTIC V3 and midnight primer scheme

A	B	C	D
Step	Temperature	Time	Cycles
Initial denaturation	98 °C	30 s	1
Denaturation Annealing/Extension	98°C 65°C	15 s 5 min	35
Hold	4 °C	∞	1

Combine the 25µL PCR reaction mixtrues for the two pools per sample into new 1.5 mL Eppendorf tubes, one per sample.

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

Remove and retain 15µL of eluate containing the DNA library per tube into new 1.5 mL Eppendorf tubes.

Add 50µL of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down.

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

Prepare fresh 80% ethanol in nuclease-free water that enough for 400µL per sample.

Pellet the beads on a magnet rack for 0h 5m 0s . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Keep the tubes on the magnet and wash the beads with 200µL of freshly prepared 80% ethanol without disturbing the pellet. Incubate for 0h 0m 30s and pipette off the ethanol.

Repeat the previous step (step 7.5).

Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about 0h 0m 30s , but do not dry the pellet to the point of cracking.

Remove the tubes from the magnetic rack and resuspended each pellet in 15µL nuclease-free water. Incubate for 0h 5m 0s at Room temperature .

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Quantify 1µL of each eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.

Thaw the NEBNext Ultra II End repair / dA-tailing Module reagents on ice.

Determine the volume of the cleaned-up PCR reaction that yields 200fmol (50ng) of DNA per sample and aliquot in new 0.5 mL PCR tubes.

Make up each sample per tube to 12.5µL using nuclease-free water.

Prepare end-prep mastermix, mix by pipetting at least 10 times or flicking the tube, spin down and place On ice .

A	B
Component	Volume
Ultra II End-prep reaction buffer	1.75 μl
Ultra II End-prep enzyme mix	0.75 μl
Total	2.5 μl

Add 2.5µL end-prep mastermix to each tube, mix by pipetting or flicking the tube.

Using a thermal cycler, incubate at 20°C for 0h 5m 0s and 65°C for 0h 5m 0s

Thaw the native barcodes at room temperature, enough for one barcode per sample. Individually mix the barcodes by pipetting, and place them On ice .

Thaw the tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place On ice .

Add the reagents in the following order per tube, mixing by flicking the tube between each sequential addition (one barcode per sample):

A	B
Component	Volume
Nuclease-free water	6 μl
End-prepped DNA	1.5 μl
Native Barcode	2.5 μl
Blunt/TA Ligase Master Mix	10 μl
Total	20 μl

Mix contents thoroughly by pipetting or flcking the tube and spin down briefly.

Using a thermal cycler, incubate at 20°C for 0h 20m 0s and at 65°C for 0h 10m 0s .

Pool each barcoded library into a new 1.5 Eppendorf DNA LoBind tube.

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

Remove the tubes from the magnetic rack and resuspended each pellet in 35µL nuclease-free water. Incubate for 0h 2m 0s at Room temperature .

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Remove and retain 35µL of eluate into a new 1.5 mL Eppendorf tubes.

Add 0.4x volume of pooled barcoded reaction of resuspended Mag-Bind® TotalPure NGS beads to the pooled barcoded reaction. Briefly vortex to mix and spin down.

Incubate on a Hula mixer (rotator mixer) at Room temperature for 0h 10m 0s .

Prepare 500µL fresh 80% ethanol in nuclease-free water.

Spin down and pellet the beads on a magnet rack for 0h 5m 0s . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Wash the beads by adding 700µL Short Fragment Buffer (SFB). Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.

Repeat the previous step (step 11.6).

Keep the tubes on the magnet and wash the beads with 500µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about 0h 0m 30s , but do not dry the pellet to the point of cracking.

Quantify 1µL of eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.

Thaw the Elution Buffer (EB), Short Fragment Buffer (SFB), and NEBNext Quick Ligation Reaction Buffer (5x) at Room temperature , mix by vortexing, spin down and place On ice . Check the contents of each tube are clear of any precipitate.

Spin down the T4 Ligase and the Adapter Mix II (AMII), and place On ice.

Taking the pooled and barcoded DNA, perform adapter ligation as follows, mixing by flicking the tube between each sequential addition.

A	B
Component	Volume
Pooled barcoded sample	30 μl
Adapter Mix II (AMII)	5 μl
NEBNext Quick Ligation Reaction Buffer (5X)	10 μl
Quick T4 DNA Ligase	5 μl
Total	50 μl

Mix gently by flicking the tube, and spin down.

Incubate the reaction for 0h 20m 0s at room temperature.

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

Add 20µL (0.4x volume) of resuspended Mag-Bind® TotalPure NGS beads to the reaction. Briefly vortex to mix and spin down.

Incubate on a Hula mixer (rotator mixer) at Room temperature for 0h 10m 0s .

Spin down and pellet the beads on a magnet rack for 0h 5m 0s . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Wash the beads by adding 125µL Short Fragment Buffer (SFB). Flick the beads to resuspend, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.

Repeat the previous step (step 14.4).

Spin down and place the tubes on the magnet. Pipette off any residual supernatant.

Remove the tubes from the magnetic rack and resuspended each pellet in 15µL Elution Buffer (EB). Incubate for 0h 5m 0s at Room temperature .

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Remove and retain 15µL of eluate into a new 1.5 mL Eppendorf tubes.

Quantify 1µL of eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.

Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at Room temperature.

Mix the Sequencing Buffer (SQB), Flush Tether (FLT) and Flush Buffer (FB) tubes by vortexing and spin down at Room temperature .

Open the MinION Mk1B lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact.

Mix the prepared library gently by pipetting up and down just prior to loading.

Add 75µL of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.

Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION Mk1B lid.

Slide the priming port cover clockwise to open the priming port.

After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few μl).

To prepare the flow cell priming mix, add 30µL of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at Room temperature .

Load 800µL of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 0h 5m 0s . During this time, prepare the library for loading by following the steps below.

Thoroughly mix the contents of the Loading Beads (LB) by pipetting.

In a new tube, prepare the library for loading as follows:

A	B
Reagent	Volume
Sequencing Buffer (SQB)	37.5 μl
Loading Beads (LB), mixed immediately before use	25.5 μl
DNA library (50 fmol)	12 μl
Total	75 μl

Gently lift the SpotON sample port cover to make the SpotON sample port accessible.

Load 200µL of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.

Combine the 25µL PCR reaction mixtures for the two pools per sample into new 1.5 mL Eppendorf tubes, one per sample.

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

Remove the tubes from the magnetic rack and resuspended each pellet in 15µL nuclease-free water. Incubate for 0h 5m 0s at Room temperature .

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Remove and retain 15µL of eluate containing the DNA library per tube into new 1.5 mL Eppendorf tubes.

Add 42.5µL of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at Room temperature for 0h 5m 0s .

Prepare fresh 80% ethanol in nuclease-free water that enough for 400µL per sample.

Pellet the beads on a magnet rack for 0h 5m 0s . Keep the tubes on the magnet until the eluate is clear and colourless, pipette 92.5µL supernatant to new 1.5 mL Eppendorf tubes (keep supernatant).

Add 47.5µL of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at Room temperature for 0h 15m 0s .

Pellet the beads on a magnet rack for 0h 5m 0s . Keep the tubes on the magnet until the eluate is clear and colourless, pipette off the supernatant (keep beads).

Keep the tubes on the magnet and wash the beads with 200µL of freshly prepared 80% ethanol without disturbing the pellet. Incubate for 0h 0m 30s and pipette off the ethanol.

Repeat the previous step (step 17.7).

Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about 0h 0m 30s , but do not dry the pellet to the point of cracking.

Quantify 1µL of each eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.

Thaw the NEBNext Ultra II End repair / dA-tailing Module reagents on ice.

Determine the volume of the cleaned-up PCR reaction that yields 100ng of DNA per sample and aliquot in new 0.5 mL PCR tubes.

Make up each sample per tube to 20µL using nuclease-free water.

Prepare end-prep mastermix, mix by pipetting at least 10 times or flicking the tube, spin down and place On ice .

A	B
Component	Volume
Ultra II End-prep reaction buffer	2.8 μl
Ultra II End-prep enzyme mix	1.2 μl
Total	4 μl

Add 4µL end-prep mastermix to each tube, mix by pipetting or flicking the tube.

Using a thermal cycler, incubate at 20°C for 0h 30m 0s and 65°C for 0h 30m 0s , then hold at 4°C .

Dilute the NEBNext Adaptor for Illumina 10x in Tris/NaCl, pH 7.5-8.0.

Prepare adapter ligation mastermix, mix by pipetting at least 10 times or flicking the tube, spin down and place On ice

A	B
Component	Volume (µl)
NEBNext Adaptor for Illumina	1
NEBNext Ultra II Ligation Master Mix	12
NEBNext Ligation Enhancer	0.4
Total Volume	13.4

Add 13.4µL 1adapter ligation mastermix directly to the 24µL End Prep Reaction Mixture, mix by pipetting or flicking the tubes and spin down.

Incubate at 20°C for 0h 15m 0s in a thermocycler with the heated lid off (or open lid).

Add 1.2µL 11.2µl of USER^® Enzyme to the ligation mixture.

Mix well and incubate in a thermocycler at 37°C for 0h 15m 0s.

Transfer the 36µL ligation reaction mix into new 1.5 mL Eppendorf tubes, one per sample.

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

Remove and retain 15µL of eluate containing the DNA library per tube into new 0.5 mL PCR tubes.

Add 43.2µL of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at Room temperature for 0h 5m 0s .

Prepare fresh 80% ethanol in nuclease-free water that enough for 400µL per sample.

Pellet the beads on a magnet rack for 0h 5m 0s . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Keep the tubes on the magnet and wash the beads with 200µL of freshly prepared 80% ethanol without disturbing the pellet. Incubate for 0h 0m 30s and pipette off the ethanol.

Repeat the previous step (step 21.5).

Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about 0h 0m 30s , but do not dry the pellet to the point of cracking.

Remove the tubes from the magnetic rack and resuspended each pellet in 15µL nuclease-free water. Incubate for 0h 5m 0s at Room temperature .

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Prepare barcoding mastermix, mix by pipetting or flicking the tube, spin down and place On ice

A	B
Component	Volume (µl)
NEBNext Ultra II Q5 Master Mix	25
Index Primer (one per sample)	5
Universal PCR Primer	5
Total Volume	35

Add 35µL barcoding mastermix to 15µL cleaned adapter ligation mixture, mix well by pipetting or flicking the tubes.

Run in a thermal cycler using the following program:

A	B	C	D
Step	Temperature	Time	Cycles
Initial denaturation	98 °C	30 s	1
Denaturation Annealing/Extension	98 °C 65 °C	10 s 75 s	4
Final extension	65 °C	5 min	1
Hold	4 °C	∞	1

Transfer 20µL barcoded library into new 1.5 mL Eppendorf tubes, one per sample. Add 30µL nuclease-free water to each tube.

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

Remove the tubes from the magnetic rack and resuspended each pellet in 20µL nuclease-free water. Incubate for 0h 5m 0s at Room temperature .

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Remove and retain 20µL of eluate containing the DNA library per tube into new 1.5 mL Eppendorf tubes.

Add 35µL of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at Room temperature for 0h 5m 0s .

Prepare fresh 80% ethanol in nuclease-free water that enough for 400µL per sample.

Pellet the beads on a magnet rack for 0h 5m 0s . Keep the tubes on the magnet until the eluate is clear and colourless, pipette 85µL supernatant to new 1.5 mL Eppendorf tubes (keep supernatant).

Add 15µL of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down. Incubate at Room temperature for 0h 15m 0s .

Pellet the beads on a magnet rack for 0h 5m 0s . Keep the tubes on the magnet until the eluate is clear and colourless, pipette off the supernatant (keep beads).

Keep the tubes on the magnet and wash the beads with 200µL of freshly prepared 80% ethanol without disturbing the pellet. Incubate for 0h 0m 30s and pipette off the ethanol.

Repeat the previous step (step 23.7).

Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about 0h 0m 30s , but do not dry the pellet to the point of cracking.

Quantify 1µL of each eluted sample using a Qubit fluorometer with Qubit dsDNA HS Assay Kit.

Pool equal mass of each barcoded library to form a pooled barcoded library.

Resuspend the Mag-Bind® TotalPure NGS beads by vortexing.

Pellet the beads on a magnetic rack until the eluate is clear and colourless.

Remove and retain 20µL of eluate containing the DNA library per tube into new 0.5 mL PCR tubes.

Add 1.0x volume of pooled barcoded reaction of resuspended Mag-Bind® TotalPure NGS beads to each tube. Briefly vortex to mix and spin down.

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

Prepare fresh 80% ethanol in nuclease-free water that enough for 400µL per sample.

Pellet the beads on a magnet rack for 0h 5m 0s . Keep the tubes on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Keep the tubes on the magnet and wash the beads with 200µL of freshly prepared 80% ethanol without disturbing the pellet. Incubate for 0h 0m 30s and pipette off the ethanol.

Repeat the previous step (step 25.6).

Spin down and place the tubes on the magnet. Pipette off any residual ethanol. Allow to air dry for about 0h 0m 30s , but do not dry the pellet to the point of cracking.

Remove the tubes from the magnetic rack and resuspended each pellet in 20µL nuclease-free water. Incubate for 0h 5m 0s at Room temperature .

Quantify 1µL of pooled barcoded library using a Qubit fluorometer with Qubit dsDNA HS Assay Kit. This sequencing library is ready for submission.